Tropheryma whipplei, the agent of Whipple's disease, is an intracellular pathogen that replicates in macrophages. The phagocytic and cellular processes leading to the formation of T. whipplei replicative vacuole remain poorly understood. Macrophage microbicidal activity is largely related to macro/autophagy which is also essential for cell homeostasis. Here, we show that T. whipplei uptake by macrophages involved LC3-associated phagocytosis (LAP). Bacteria then escaped into the cytosol from where they were recaptured by xenophagy. We also demonstrate that T. whipplei blocked the autophagic flux to build its replicative compartment. Inhibition of LAP resulted in the decrease of interleukin (IL)-10 secretion and the restoration of the autophagy flux, suggesting that modulation of autophagy during infection alters immune response and promote persistence. Our results provide new insight in the intracellular fate of the bacteria during macrophage infection and suggest the possible involvement of previously unknown virulence factors in T. whipplei infection.
© 2025 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
Product Citations: 221
In Autophagy Rep on 21 May 2025 by Reyne, E., Arrindell, J., et al.
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Immunology and Microbiology
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Low extracellular pH protects cancer cells from ammonia toxicity.
In Cell Death Discovery on 3 April 2025 by Dravecka, M., Mikkola, I., et al.
Ammonia is a natural waste product of cellular metabolism which, through its lysosomotropic ability, can have detrimental effects on various cellular functions. Increased levels of ammonia were recently detected in the interstitial fluid of various tumours, substantiating that high ammonia concentrations are a pathophysiological condition in the tumour microenvironment, alongside hypoxia and acidosis. Since little is known about how cancer cells respond to elevated levels of ammonia in the tumour microenvironment, we investigated how a panel of cancer cell lines derived from solid tumours behaved when exposed to increasing concentrations of ammonia. We found that ammonia represses cell growth, induces genome instability, and inhibits lysosome-mediated proteolysis in a dose-dependent manner. Unexpectedly, we also found that small fluctuations in the pH of the extracellular environment, had a significant impact on the cytotoxic effects of ammonia. In summary, our data show that the balance of pH and ammonia within the interstitial fluids of cancerous tumours significantly impacts the behaviour and fate of cells residing in the tumour microenvironment.
© 2025. The Author(s).
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Cancer Research
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In Journal of Neurology on 29 March 2025 by Coysh, T., Jaunmuktane, Z., et al.
Inherited prion diseases (IPDs) are phenotypically diverse neurodegenerative conditions caused by mutations in the prion protein gene (PRNP). We describe IPD due to a novel PRNP E146G mutation in a 50-year-old man presenting with slowly progressive dysarthria, prominent myoclonus especially in the lower limbs, and less prominent gait ataxia, pyramidal and extrapyramidal signs. Cognitive impairment was not overt at disease onset. MRI revealed cerebellar atrophy and white matter hyperintensities. His 46-year-old sister carries the mutation and has subtle gait ataxia and dysarthria. Both patients exhibit a distinctive fluid biomarker profile: in CSF S100B is > twofold upper limit of normal, total tau is moderately elevated, and neurofilament light chain, 14-3-3 and RT-QuIC are negative; in plasma there is marked elevation of GFAP but repeatedly normal neurofilament light chain. The proband's father died aged 55 following an 8-year dementing illness with similar presentation. Post-mortem revealed cerebellar cortical atrophy and profuse large PrP amyloid plaques across cerebral and cerebellar grey matter. Immunoblotting identified low molecular weight protease-resistant PrP fragments. E146G mutation IPD broadly fits into the historical Gerstmann-Sträussler-Scheinker disease spectrum but, based on deep clinical phenotyping of this initial pedigree, we highlight some distinctive features, which may aid in identification of this disease.
© 2025. The Author(s).
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In International Journal of Molecular Sciences on 10 January 2025 by Romano, R., Cordella, P., et al.
Peripherin belongs to heterogeneous class III of intermediate filaments, and it is the only intermediate filament protein selectively expressed in the neurons of the peripheral nervous system. It has been previously discovered that peripherin interacts with proteins important for the endo-lysosomal system and for the transport to late endosomes and lysosomes, such as RAB7A and AP-3, although little is known about its role in the endocytic pathway. Here, we show that peripherin silencing affects lysosomal abundance but also positioning, causing the redistribution of lysosomes from the perinuclear area to the cell periphery. Moreover, peripherin silencing affects lysosomal activity, inhibiting EGFR degradation and the degradation of a fluorogenic substrate for proteases. Furthermore, we demonstrate that peripherin silencing affects lysosomal biogenesis by reducing the TFEB and TFE3 contents. Finally, in peripherin-depleted cells, the autophagic flux is strongly inhibited. Therefore, these data indicate that peripherin has an important role in regulating lysosomal biogenesis, and positioning and functions of lysosomes, affecting both the endocytic and autophagic pathways. Considering that peripherin is the most abundant intermediate filament protein of peripheral neurons, its dysregulation, affecting its functions, could be involved in the onset of several neurodegenerative diseases of the peripheral nervous system characterized by alterations in the endocytic and/or autophagic pathways.
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Cell Biology
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Vacuolar myopathy with monoclonal gammopathy and stiffness (VAMMGAS).
In European Journal of Neurology : the Official Journal of the European Federation of Neurological Societies on 1 January 2025 by Staedler, K., Allenbach, Y., et al.
Monoclonal gammopathy (MG) has been reported in association with numerous neurological disorders but the spectrum of MG-associated myopathies remains poorly described.
To report a newly acquired myopathy associated with MG.
Three adult patients with the same phenotype from two French referral centers were prospectively analyzed. Clinical, electrophysiological, muscle biopsy data, and patients' outcomes under treatment are reported.
The patients, aged 37, 46, and 56 years, presented progressive weakness with subacute worsening and stiffness, in the context of severe weight loss. The weakness mainly involved the proximal limbs and axial muscles. Creatine kinase levels were 1400-2900 IU/L and electromyography revealed a myopathic pattern with spontaneous complex repetitive discharges. Muscle biopsies showed vacuoles containing glycogen and autophagic material along with the presence of sarcolemmal complement membrane attack complex deposits. There was no evidence of a genetic glycogen metabolic disorder. IgGκ monoclonal gammopathy was identified in all cases, without signs of lymphoplasmocytic proliferation. All patients improved with a treatment combining corticosteroids, intravenous immunoglobulins, and immunosuppressants, and two patients recovered walking ability.
We report a new muscle disease defined by a vacuolar myopathy characterized by axial and proximal muscle weakness with prominent stiffness and high frequency discharges on electromyography associated with monoclonal gammopathy, defined under the acronym VAMMGAS.
© 2025 The Author(s). European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.
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In Int J Mol Sci on 10 January 2025 by Romano, R., Cordella, P., et al.
Fig.2.C
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Peripherin silencing affects lysosomal functionality. (A) Lysates of peripherin-silenced or control Neuro2a cells, as indicated, were subjected to Western blot analysis using antibodies against LAMP1, RAB7A, beclin 1, RILP, V1G1, peripherin, and Hsp90. Relative protein abundance was quantified by...
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Peripherin silencing affects lysosomal functionality. (A) Lysates of peripherin-silenced or control Neuro2a cells, as indicated, were subjected to Western blot analysis using antibodies against LAMP1, RAB7A, beclin 1, RILP, V1G1, peripherin, and Hsp90. Relative protein abundance was quantified by densitometric analysis normalizing against Hsp90. (B) Peripherin-silenced or control Neuro2A cells, as indicated, were subjected to immunofluorescence analysis using anti-TFEB antibody followed by Alexa488-conjugated secondary antibody, while nuclei were stained with DAPI. Bar = 10 µM. TFEB CTCF was calculated using ImageJ software. To check silencing efficiency, Western blot analysis was performed on same samples using antibodies against peripherin and Hsp90 as loading control. Samples of control and silenced cells were also subjected to Western blot analysis using antibodies against TFEB and TFE3. Relative protein abundance was quantified by densitometric analysis normalizing against Hsp90. (C) Lysates of control or peripherin-depleted Neuro2a cells were subjected to Western blot analysis using antibodies against RAB4, RAB5, RAB9, RAB11, peripherin, or Hsp90 as loading control. Relative protein abundance was quantified by densitometric analysis normalizing against Hsp90. (D) Peripherin-silenced or control Neuro2A cells were incubated with DQ-Red BSA for 8 h and then fixed and immunolabeled using anti-peripherin antibody followed by Alexa488-conjugated secondary antibody. Nuclei were stained with DAPI. Bar = 10 µM. DQ-BSA CTCF was calculated using ImageJ. (E) Peripherin-silenced or control Neuro2A cells were treated with cycloheximide for 1 h and then stimulated with EGF for 15 min and 3 h. Lysates were then subjected to Western blot analysis using antibodies against EGFR, peripherin, and β-actin as loading control. Percentage of degraded EGFR was calculated. (F) Lysates of control or peripherin-silenced Neuro2a cells were subjected to Western blot analysis using antibodies against p62, V0D1, peripherin, or Hsp90 as loading control. Relative protein abundance was quantified by densitometric analysis normalizing against Hsp90. * = p < 0.05; ** = p < 0.01; *** = p < 0.001.
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Cytoprotective Role of Autophagy in CDIP1 Expression-Induced Apoptosis in MCF-7 Breast Cancer Cells.
In Int J Mol Sci on 13 June 2024 by Inukai, R., Mori, K., et al.
Fig.3.B
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CDIP1 expressed in MCF-7 Tet-On CDIP1 cells is degraded via the lysosomal pathway. (A) MCF-7 Tet-On CDIP1 cells were cultured in a medium with Dox at the indicated concentrations for 24 h. Cell lysates were analyzed by Western blotting with the indicated antibodies. (B) MCF-7 Tet-On CDIP1 cells w...
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CDIP1 expressed in MCF-7 Tet-On CDIP1 cells is degraded via the lysosomal pathway. (A) MCF-7 Tet-On CDIP1 cells were cultured in a medium with Dox at the indicated concentrations for 24 h. Cell lysates were analyzed by Western blotting with the indicated antibodies. (B) MCF-7 Tet-On CDIP1 cells were cultured in a medium with 10 ng/mL Dox for 24 h, and then 50 μg/mL cycloheximide (CHX) was added for 6 h. Ten nM BafA1 was added 1 h before the addition of CHX. Cell lysates were analyzed by Western blotting with the indicated antibodies. The intensity of the CDIP1 band was normalized to α-tubulin, and then the remaining CDIP1 6 h after treatment with CHX was expressed relative to Time = 0 controls in the right graph. Data represent the mean ± SEM (n = 3). ** p < 0.01. The positions of LC3B-I and LC3B-II are indicated by I and II.
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Cytoprotective Role of Autophagy in CDIP1 Expression-Induced Apoptosis in MCF-7 Breast Cancer Cells.
In Int J Mol Sci on 13 June 2024 by Inukai, R., Mori, K., et al.
Fig.7.A
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Effects of the PIK3C3/VPS34 inhibitor SAR405 on autophagy and apoptosis in MCF-7 cells inducibly expressing CDIP1. (A) MCF-7 Tet-On GFP cells and MCF-7 Tet-On CDIP1 cells were cultured in a medium with 100 ng/mL Dox or 250 nM Torin1 in combination with the indicated concentrations of SAR405 for 2...
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Effects of the PIK3C3/VPS34 inhibitor SAR405 on autophagy and apoptosis in MCF-7 cells inducibly expressing CDIP1. (A) MCF-7 Tet-On GFP cells and MCF-7 Tet-On CDIP1 cells were cultured in a medium with 100 ng/mL Dox or 250 nM Torin1 in combination with the indicated concentrations of SAR405 for 24 h. Cell lysates were analyzed by Western blotting with the indicated antibodies. The positions of LC3B-I and LC3B-II are indicated by I and II. The positions of full−length and cleaved PARP1 are indicated by FL and Clvd. (B) Intensities of the bands were quantified. LC3B−II/α−tubulin ratio (top), LC3B-I/α-tubulin ratio (second), and p62/α-tubulin ratio (third) were expressed relative to the cells cultured in a medium without Dox. The percentage of cleaved PARP1 (bottom) was determined as the ratio of the cleaved band intensity to the total intensity. Data represent means ± SEM (n = 3). * p < 0.05, ** p < 0.01.
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Cytoprotective Role of Autophagy in CDIP1 Expression-Induced Apoptosis in MCF-7 Breast Cancer Cells.
In Int J Mol Sci on 13 June 2024 by Inukai, R., Mori, K., et al.
Fig.8.A
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Effects of the caspase inhibitor Z−VAD−FMK on autophagy and apoptosis in MCF-7 cells inducibly expressing CDIP1. (A) MCF-7 Tet-On CDIP1 cells were cultured in a medium with 100 ng/mL Dox in combination with or without 10 μM Z−VAD−FMK for the indicated time periods. Cell lysates were analyzed by W...
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Effects of the caspase inhibitor Z−VAD−FMK on autophagy and apoptosis in MCF-7 cells inducibly expressing CDIP1. (A) MCF-7 Tet-On CDIP1 cells were cultured in a medium with 100 ng/mL Dox in combination with or without 10 μM Z−VAD−FMK for the indicated time periods. Cell lysates were analyzed by Western blotting with the indicated antibodies. The positions of LC3B-I and LC3B-II are indicated by I and II. The positions of full−length and cleaved PARP1 are indicated by FL and Clvd. (B) Intensities of the LC3B-II band were normalized to α−tubulin, and the LC3B-II/α-tubulin ratio was expressed relative to Time = 0. (C) Intensities of the p62 band were normalized to α-tubulin, and the p62/α-tubulin ratio was expressed relative to Time = 0. (D) Intensities of cleaved and full−length PARP1 bands were quantified, and then the percentage of cleaved PARP1 was determined as the ratio of the cleaved band intensity to the total intensity. Data represent means ± SEM (n = 3). * p < 0.05.
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In Cell Death Dis on 8 February 2022 by Shi, D., Ding, J., et al.
Fig.3.H
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MYOCD activated the transcription of miR-30a by binding to the CArG box.The level of miR-30a-5p was examined in MYOCD overexpression (A) or knockdown (B) cells by RT-PCR (*P < 0.05, **P < 0.01, n = 3). C A schematic diagram of the miR-30a-5p promoter region represents wild-type and mutant CArG bo...
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MYOCD activated the transcription of miR-30a by binding to the CArG box.The level of miR-30a-5p was examined in MYOCD overexpression (A) or knockdown (B) cells by RT-PCR (*P < 0.05, **P < 0.01, n = 3). C A schematic diagram of the miR-30a-5p promoter region represents wild-type and mutant CArG boxes. D Luciferase reporter plasmids together with pcDNA3.1-MYOCD plasmids or vectors were cotransfected into HA-VSMCs, and 24 h after transfection, luciferase activity was assayed (**P < 0.01, n = 3). E HA-VSMCs were transfected with luciferase reporter plasmids together with shControl or shMYOCD plasmids for 24 h, and then luciferase activity was assayed (**P < 0.01, n = 3). F HA-VSMCs were transfected with luciferase reporter plasmids in the presence or absence of PDGF-BB for 24 h, and then luciferase activity was assayed (**P < 0.01, n = 3). G 48 h after transfection of pcDNA3.1-MYOCD-myc vector expression plasmids or vector plasmids, ChIP assays were performed using antibodies against myc (**P < 0.01, n = 3). H MiR-30a-5p mimics were transfected into MYOCD knockdown cells, and then western blotting was performed (*P < 0.05, **P < 0.01, n = 3).
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
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In Cell Death Dis on 8 February 2022 by Shi, D., Ding, J., et al.
Fig.1.F
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Knockdown of MYOCD triggered autophagy in HA-VSMCs.A The expression of MYOCD was detected in HA-VSMCs transfected with shRNA specifically targeting MYOCD by western blotting (*P < 0.05, **P < 0.01, n = 3). B The expression of contractile proteins ACTA2 and SM22 was detected in MYOCD-knockdown HA-...
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Knockdown of MYOCD triggered autophagy in HA-VSMCs.A The expression of MYOCD was detected in HA-VSMCs transfected with shRNA specifically targeting MYOCD by western blotting (*P < 0.05, **P < 0.01, n = 3). B The expression of contractile proteins ACTA2 and SM22 was detected in MYOCD-knockdown HA-VSMCs by western blotting (**P < 0.01, n = 3). C The expression of OPN and PCNA was tested in MYOCD-knockdown HA-VSMCs by western blotting (*P < 0.05, n = 3). D Cellular proliferation was evaluated in MYOCD-knockdown HA-VSMCs by EdU staining (**P < 0.01, n = 3). E The expression of beclin1, P62, and LC3 was tested in MYOCD-knockdown HA-VSMCs by western blotting (*P < 0.05, n = 3). F Bafilomycin A1 (0.1 μg/mL) was added to control or MYOCD-knockdown HA-VSMCs 1 h prior to harvest, and accumulation of P62 and LC3 was measured by western blotting (*P < 0.05,**P < 0.01, n = 3). G Representative images and quantification of GFP-LC3 puncta in MYOCD-knockdown HA-VSMCs transfected with GFP-LC3 vector in the presence or absence of bafilomycin A1 (*P < 0.05,**P < 0.01, n = 3).
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
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In Cell Death Dis on 8 February 2022 by Shi, D., Ding, J., et al.
Fig.4.F
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MiR-30a-5p decreased the expression of beclin1 by targeting the 3’UTR of beclin1.A HA-VSMCs were transfected with miR-30a-5p mimics, and then the expression of beclin1 was detected by western blotting (**P < 0.01, n = 3). B Alignment of the miR-30a-5p “seed region” with beclin1 3′-UTRs among diff...
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MiR-30a-5p decreased the expression of beclin1 by targeting the 3’UTR of beclin1.A HA-VSMCs were transfected with miR-30a-5p mimics, and then the expression of beclin1 was detected by western blotting (**P < 0.01, n = 3). B Alignment of the miR-30a-5p “seed region” with beclin1 3′-UTRs among different species. C HA-VSMCs were transfected with beclin1 3′-UTR or mutant beclin1 3′-UTR plasmids together with miR-30a-5p mimic or control for 24 h, followed by luciferase assays (**P < 0.01, n = 3). D and E HA-VSMCs were transfected with miR-30a-5p inhibitor or control for 24 h and then treated with bafilomycin A1 for 2 h. Cells were harvested and accumulation of P62 and LC3 was measured by western blotting (*P < 0.05, **P < 0.01, n = 3). F–I HA-VSMCs were transfected with miR-30a-5p inhibitor for 4 h and then treated with 5 mM 3-MA for another 48 h. Western blot images and quantification in each group are shown (*P < 0.05, **P < 0.01, n = 3). J and K Cellular proliferation was evaluated by EdU staining (**P < 0.01, n = 3).
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
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In Cell Death Dis on 8 February 2022 by Shi, D., Ding, J., et al.
Fig.5.H
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Inhibition of autophagy maintained the contractile phenotype in PDGF-BB-stimulated HA-VSMCs.A HA-VSMCs were starved overnight prior to stimulation using 40 ng/mL PDGF-BB for 24 h, and then the mRNA levels of OPN, PCNA, MYOCD, SM22α and ACTA2 were detected by qRT-PCR (*P < 0.05, **P < 0.01, n = 3)...
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Inhibition of autophagy maintained the contractile phenotype in PDGF-BB-stimulated HA-VSMCs.A HA-VSMCs were starved overnight prior to stimulation using 40 ng/mL PDGF-BB for 24 h, and then the mRNA levels of OPN, PCNA, MYOCD, SM22α and ACTA2 were detected by qRT-PCR (*P < 0.05, **P < 0.01, n = 3). B and C The protein levels of these proliferative markers and contractile genes were detected in HA-VSMCs treated with 40 ng/mL PDGF-BB for 48 h by western blotting (*P < 0.05, **P < 0.01, ***P < 0.001, n = 3). D and E Cellular proliferation was evaluated in PDGF-BB-stimulated HA-VSMCs by EdU staining (**P < 0.01, n = 3). F and G The expression of beclin1, P62 and LC3 was detected in PDGF-BB-stimulated HA-VSMCs by western blotting (*P < 0.05, **P < 0.01, n = 3). H and I HA-VSMCs were incubated with PDGF-BB for 24 h and then treated with bafilomycin A1 for 2 h. Cells were harvested and accumulation of P62 and LC3 was measured by western blotting (*P < 0.05,*P < 0.01, n = 3). J and K Serum-starved HA-VSMCs were pretreated with 5 mM 3-MA for half an hour and then incubated with 40 ng/mL PDGF-BB for another 48 h. Western blot images and quantification in each group are shown (*P < 0.05, **P < 0.01, n = 3).
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
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In Cell Death Dis on 8 February 2022 by Shi, D., Ding, J., et al.
Fig.5.F
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Inhibition of autophagy maintained the contractile phenotype in PDGF-BB-stimulated HA-VSMCs.A HA-VSMCs were starved overnight prior to stimulation using 40 ng/mL PDGF-BB for 24 h, and then the mRNA levels of OPN, PCNA, MYOCD, SM22α and ACTA2 were detected by qRT-PCR (*P < 0.05, **P < 0.01, n = 3)...
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Inhibition of autophagy maintained the contractile phenotype in PDGF-BB-stimulated HA-VSMCs.A HA-VSMCs were starved overnight prior to stimulation using 40 ng/mL PDGF-BB for 24 h, and then the mRNA levels of OPN, PCNA, MYOCD, SM22α and ACTA2 were detected by qRT-PCR (*P < 0.05, **P < 0.01, n = 3). B and C The protein levels of these proliferative markers and contractile genes were detected in HA-VSMCs treated with 40 ng/mL PDGF-BB for 48 h by western blotting (*P < 0.05, **P < 0.01, ***P < 0.001, n = 3). D and E Cellular proliferation was evaluated in PDGF-BB-stimulated HA-VSMCs by EdU staining (**P < 0.01, n = 3). F and G The expression of beclin1, P62 and LC3 was detected in PDGF-BB-stimulated HA-VSMCs by western blotting (*P < 0.05, **P < 0.01, n = 3). H and I HA-VSMCs were incubated with PDGF-BB for 24 h and then treated with bafilomycin A1 for 2 h. Cells were harvested and accumulation of P62 and LC3 was measured by western blotting (*P < 0.05,*P < 0.01, n = 3). J and K Serum-starved HA-VSMCs were pretreated with 5 mM 3-MA for half an hour and then incubated with 40 ng/mL PDGF-BB for another 48 h. Western blot images and quantification in each group are shown (*P < 0.05, **P < 0.01, n = 3).
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
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In Front Cell Dev Biol on 28 August 2020 by Friesen, E. L., Zhang, Y. T., et al.
Fig.4.E
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p62 and BAG5 modulate alpha-synuclein oligomerization. (A) Western blot illustrating the presence of the two alpha-synuclein constructs fused to either the N-terminus (syn-N) or the C-terminus (syn-C) of Gaussia princeps luciferase for the luciferase protein complementation assay (PCA) results pr...
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p62 and BAG5 modulate alpha-synuclein oligomerization. (A) Western blot illustrating the presence of the two alpha-synuclein constructs fused to either the N-terminus (syn-N) or the C-terminus (syn-C) of Gaussia princeps luciferase for the luciferase protein complementation assay (PCA) results presented in (B). (B) Luminescent signal is only generated when both syn-N and syn-C are present. (**p < 0.01, independent samples t-test, n = 3). (C) Western blot illustrating the levels of BAG5 and syn-N/syn-C for the PCA presented in (E). Representative of three independent studies. * indicates a non-specific band. (D) Densitometric quantification of alpha-synuclein protein in (C) [not significant (ns), independent samples t-test, n = 3]. (E) PCA illustrating the effect of small interfering RNA (siRNA)-mediated BAG5 knockdown (KD) on alpha-synuclein oligomer formation. (****p < 0.0001, independent samples t-test, n = 3). (F) Western blot illustrating the levels of p62, BAG5, and syn-N/syn-C for the PCA presented in (H). Representative of three independent studies. * indicates a non-specific band. (G) Densitometric quantification of alpha-synuclein protein in (F) (*p < 0.05, independent samples t-test, n = 3). (H) PCA illustrating the effect of siRNA-mediated p62 KD on alpha-synuclein oligomer formation. [not significant (ns), independent samples t-test, n = 3] (I) Western blot illustrating the levels of p62 and syn-N/syn-C for the PCA presented in (K). Representative of three independent studies. (J) Densitometric quantification of alpha-synuclein protein in (I) (*p < 0.05, independent samples t-test, n = 3). (K) PCA illustrating the effect of p62 overexpression on alpha-synuclein oligomer formation. (****p < 0.0001, independent samples t-test, n = 3). All graphs represent mean ± SEM.
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Collected and cropped from Front Cell Dev Biol by CiteAb, provided under a CC-BY license
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In Front Cell Dev Biol on 28 August 2020 by Friesen, E. L., Zhang, Y. T., et al.
Fig.3.A
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BAG5 regulates p62 protein levels. (A) Western blots of lysates from H4 cells treated with either siNTC or siBAG5. Proteins were sequentially probed with anti-p62, anti-BAG5, and anti-actin antibodies. Actin served as the loading control. * indicates a non-specific band. Three independent experim...
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BAG5 regulates p62 protein levels. (A) Western blots of lysates from H4 cells treated with either siNTC or siBAG5. Proteins were sequentially probed with anti-p62, anti-BAG5, and anti-actin antibodies. Actin served as the loading control. * indicates a non-specific band. Three independent experiments are included on the blot. (B) Densitometric quantification of the change in endogenous p62 elicited by BAG5 KD from the immunoblots in (A). p62 band intensity was normalized to actin and siNTC condition (***p < 0.001, independent samples t-test, n = 3). (C) Western blots of lysates from H4 cells transfected with either pcDNA control plasmid or FLAG-BAG5. Proteins were sequentially probed with anti-p62, anti-BAG5, and anti-actin antibodies. Actin served as the loading control. Three independent experiments are included on the blot. (D) Densitometric quantification of endogenous p62 from the immunoblots in (C). p62 band intensity was normalized to actin and siNTC condition (ns, no significance, independent samples t-test, n = 3). (E) Endogenous BAG5 (green) and p62 (red) were visualized in H4 cells using immunofluorescent staining. Cell nuclei were visualized with 4’,6-Diamidino-2-Phenylindole (DAPI) (blue). BAG5 demonstrated nuclear and cytoplasmic staining, while p62 was largely excluded from the nucleus and clustered into small puncta in the perinuclear space. BAG5 co-localized with most, but not all, of these puncta (yellow). Scale bar represents 20 μm. Results are representative of three independent experiments. (F) Endogenous p62 (red), including p62 in perinuclear puncta, was visualized using immunofluorescent staining in H4 cells treated with siNTC or siBAG5. Cell nuclei were visualized with DAPI (blue). Scale bar represents 50 μm. Representative image from three independent experiments. (G) Quantification of the percentage of cells with visible perinuclear p62-containing puncta (p62 puncta+) using the immunofluorescence images pictured in (F). A minimum of 200 cells were counted blinded. Data are representative of three independent experiments (*p < 0.05, independent samples t-test, n = 3). All graphs represent mean ± SEM.
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Collected and cropped from Front Cell Dev Biol by CiteAb, provided under a CC-BY license
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In Front Cell Dev Biol on 28 August 2020 by Friesen, E. L., Zhang, Y. T., et al.
Fig.3.C
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BAG5 regulates p62 protein levels. (A) Western blots of lysates from H4 cells treated with either siNTC or siBAG5. Proteins were sequentially probed with anti-p62, anti-BAG5, and anti-actin antibodies. Actin served as the loading control. * indicates a non-specific band. Three independent experim...
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BAG5 regulates p62 protein levels. (A) Western blots of lysates from H4 cells treated with either siNTC or siBAG5. Proteins were sequentially probed with anti-p62, anti-BAG5, and anti-actin antibodies. Actin served as the loading control. * indicates a non-specific band. Three independent experiments are included on the blot. (B) Densitometric quantification of the change in endogenous p62 elicited by BAG5 KD from the immunoblots in (A). p62 band intensity was normalized to actin and siNTC condition (***p < 0.001, independent samples t-test, n = 3). (C) Western blots of lysates from H4 cells transfected with either pcDNA control plasmid or FLAG-BAG5. Proteins were sequentially probed with anti-p62, anti-BAG5, and anti-actin antibodies. Actin served as the loading control. Three independent experiments are included on the blot. (D) Densitometric quantification of endogenous p62 from the immunoblots in (C). p62 band intensity was normalized to actin and siNTC condition (ns, no significance, independent samples t-test, n = 3). (E) Endogenous BAG5 (green) and p62 (red) were visualized in H4 cells using immunofluorescent staining. Cell nuclei were visualized with 4’,6-Diamidino-2-Phenylindole (DAPI) (blue). BAG5 demonstrated nuclear and cytoplasmic staining, while p62 was largely excluded from the nucleus and clustered into small puncta in the perinuclear space. BAG5 co-localized with most, but not all, of these puncta (yellow). Scale bar represents 20 μm. Results are representative of three independent experiments. (F) Endogenous p62 (red), including p62 in perinuclear puncta, was visualized using immunofluorescent staining in H4 cells treated with siNTC or siBAG5. Cell nuclei were visualized with DAPI (blue). Scale bar represents 50 μm. Representative image from three independent experiments. (G) Quantification of the percentage of cells with visible perinuclear p62-containing puncta (p62 puncta+) using the immunofluorescence images pictured in (F). A minimum of 200 cells were counted blinded. Data are representative of three independent experiments (*p < 0.05, independent samples t-test, n = 3). All graphs represent mean ± SEM.
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Collected and cropped from Front Cell Dev Biol by CiteAb, provided under a CC-BY license
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In PLoS One on 25 August 2020 by Kobylarz, M. J., Goodwin, J. M., et al.
Fig.6.D
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WB
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RAB7A KO partially rescues PIK-III dependency of RKO cells.(A) Characterization of RAB7A KO cells. Cell lysates of RKO CTRL and RAB7A KO cells were immunoblotted with the specified antibodies. (B) Knockout of RAB7A suppresses VPS34 vulnerability. RKO CTRL and RAB7A KO cells were treated with 1 μM...
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RAB7A KO partially rescues PIK-III dependency of RKO cells.(A) Characterization of RAB7A KO cells. Cell lysates of RKO CTRL and RAB7A KO cells were immunoblotted with the specified antibodies. (B) Knockout of RAB7A suppresses VPS34 vulnerability. RKO CTRL and RAB7A KO cells were treated with 1 μM PIK-III or vehicle control for 9 days and cell colonies were visualized with crystal violet staining. (C) RKO CTRL and RAB7A KO cells were treated with PIK-III in dose response for 5 days and cell viability was assessed using the CellTiter-Glo assay. The dose response was collected from three independent experiments and represented as the mean ± SD. Non-linear regression analysis with variable slope and least squares fit was performed using GraphPad Prism 8 to calculate the IC50 values. The IC50 inflection points for RKO CTRL and RAB7A KO are visualized by a dotted line and colored dark grey and red, respectively. (D) RAB7A KO cells were treated with the indicated concentrations of PIK-III for 1, 6, 12, or 24 hours and lysates were immunoblotted with the specified antibodies. (E) RKO RAB7A KO cells were treated with vehicle or 1 μM PIK-III for 24 hours followed by incubation with Transferrin Alexa Fluor 488 probe for 30 min at 37°C. Nuclei were labeled with Hoechst and live cell imaging was performed. The white bar represents 10 μm in length. (F) Transferrin Alexa Fluor 488 probe uptake in (E) was quantified as percent average intensity of total cells. Data in the form of technical replicates were averaged and presented as the mean ± SD from 6 wells. (G) RAB7A KO cells were treated with the indicated concentrations of PIK-III for 24 hours. Lysates were immunoblotted with the specified antibodies. (H) Mitochondrial respiration defect due to VPS34 inhibition is RAB7A-dependent. RKO CTRL or RAB7A KO cells were treated with the indicated concentrations of PIK-III for 24 hours and mitochondrial respiration was assessed by measuring OCR and ECR as described in Fig 5K. The OCR/ECAR ratio is presented as the mean ± SD (n = 7 or 8 wells). Individual OCR and ECAR graphs are shown in S5 Fig.
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Collected and cropped from PLoS One by CiteAb, provided under a CC-BY license
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In PLoS One on 25 August 2020 by Kobylarz, M. J., Goodwin, J. M., et al.
Fig.5.F
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WB
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VPS34 inhibition depletes intracellular iron by blocking iron uptake through enhanced degradation of TFR and excess iron rescues the growth defect in RKO cells.(A) Aconitase activity in RKO cells upon VPS34 inhibition. RKO CTRL cells were treated with 1 μM PIK-III, 30 μM deferasirox (DFX) or vehi...
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VPS34 inhibition depletes intracellular iron by blocking iron uptake through enhanced degradation of TFR and excess iron rescues the growth defect in RKO cells.(A) Aconitase activity in RKO cells upon VPS34 inhibition. RKO CTRL cells were treated with 1 μM PIK-III, 30 μM deferasirox (DFX) or vehicle control for 24 hours. The aconitase activity assay was used to quantify the isocitrate present in the cell extracts. All data was normalized to cells treated with vehicle, transformed to represent the percent isocitrate generated and averaged from three biological replicates ± SD; ****, p < 0.0001; NS, not significant (two-way ANOVA). (B) RKO CTRL cells were treated with vehicle or 0.5 μM PIK-III for 24 hours and lysates were immunoblotted with the indicated antibodies. (C) Quantitation of immunoblot in (B) as fold change versus vehicle treated cells. All data were normalized to the β-actin loading control. Data is averaged from three independent experiments ± SD, ****, p < 0.0001 (two-way ANOVA). (D) RKO.Cas9 cells were treated with vehicle or 1 μM PIK-III for 24 hours followed by incubation with Transferrin Alexa Fluor 488 probe for 30 min at 37°C. Nuclei were labeled with Hoechst and live cell imaging was performed. The white bar represents 10 μm in length. (E) Cellular uptake of Transferrin Alexa Fluor 488 probe from panel (D) was quantified as percent average intensity of total cells. Data were averaged and presented as the mean ± SD from 6 wells. (F) RKO CTRL cells were treated with the indicated concentrations of PIK-III for 1, 6, 12, or 24 hours and lysates were immunoblotted with the indicated antibodies. (G, H) RKO CTRL cells were treated with the indicated concentrations of PIK-III with or without 50 nM Bafilomycin A1 (G) or 30 μM chloroquine (H) for 12 and 24 hours and lysates were immunoblotted with the indicated antibodies. (I) RKO CTRL cells were treated with indicated concentrations of PIK-III along with vehicle, 1 μM or 50 μM Ferric Ammonium Citrate (FAC) for 5 days and cell viability was assessed using the CellTiter-Glo assay. Data is derived from three independent experiments and presented as mean ± SD, ***, p < 0.001; NS, not significant (two-way ANOVA). (J) RKO CTRL cells were treated with the indicated concentrations of PIK-III along with vehicle or 50 μM FAC for 24 hours. Lysates were immunoblotted with the specified antibodies. (K) Mitochondrial respiration defect due to PIK-III treatment. RKO CTRL cells were treated with the indicated concentrations of PIK-III along with vehicle for 24 hours with or without 50 μM FAC and mitochondrial respiration was assessed by measuring the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in response to the specified mitochondrial inhibitors using the Seahorse XFe96 analyzer. Oligomycin (Oligo) inhibits ATP synthase (Complex V) and decreases OCR; FCCP uncouples oxidative phosphorylation and increases OCR; and rotenone/antimycin A (ROT & AA) inhibits Complex I and III, respectively and decreases OCR. OCR and ECAR readouts from a single 96-well plate were normalized to the total DNA content measured in each well by DRAQ5 staining. Relative magnitude of mitochondrial oxidative phosphorylation and glycolysis is depicted as a ratio between the basal oxygen consumption rate and basal extracellular acidification rate (OCR/ECAR) and presented as the mean ± SD (n = 11 or 12 wells). Individual OCR and ECAR graphs are shown in S5 Fig.
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Collected and cropped from PLoS One by CiteAb, provided under a CC-BY license
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In PLoS One on 25 August 2020 by Kobylarz, M. J., Goodwin, J. M., et al.
Fig.4.D
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WB
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VPS34 inhibition decreases intracellular levels of cholesteryl-esters, blocks cholesterol uptake and enhances lysosomal degradation of LDLR.(A) Lipidomic analysis of RKO cells upon VPS34 inhibition. RKO CTRL cells were treated with 1 μM PIK-III for 24 hours and lipids were extracted and subjected...
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VPS34 inhibition decreases intracellular levels of cholesteryl-esters, blocks cholesterol uptake and enhances lysosomal degradation of LDLR.(A) Lipidomic analysis of RKO cells upon VPS34 inhibition. RKO CTRL cells were treated with 1 μM PIK-III for 24 hours and lipids were extracted and subjected to liquid chromatography mass spectrometry analysis. The table shows PIK-III-induced log2 fold change for aggregated and averaged lipid classes in RKO cells. The adjusted p-values were calculated between PIK-III and vehicle treatment group. Data represents the mean of three biological replicates and the entire dataset is reported in S4 Table. (B) RKO.Cas9 cells were treated with vehicle or 1 μM PIK-III for 24 hours followed by incubation with BODIPY FL LDL probe for 2 hours at 37°C. Nuclei were labeled with Hoechst and live cell imaging was performed. The white bar represents 10 μm in length. (C) BODIPY FL LDL probe cellular uptake in (B) was quantified as percent average intensity of total cells. Data in the form of technical replicates were averaged and presented as the mean ± SD from 6 wells. (D) RKO CTRL cells were treated with the indicated concentrations of PIK-III for 1, 6, 12, or 24 hours. (M) stands for mature protein and (P) stands for precursor protein. (E, F) RKO CTRL cells were treated with the indicated concentrations of PIK-III with or without 50 nM bafilomycin A1 (E) or 30 μM chloroquine (F) for 12 and 24 hours. Lysates were immunoblotted with the specified antibodies. (M) and (P) indicate the mature and precursor protein, respectively. (G) RKO CTRL cells were treated with indicated concentrations of PIK-III along with vehicle, 2 μg/ml and 10 μg/ml soluble cholesterol (Chol) for 5 days and cell viability was assessed using the CellTiter-Glo assay. Data presented is the mean from three independent experiments ± SD. NS, not significant (two-way ANOVA).
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Collected and cropped from PLoS One by CiteAb, provided under a CC-BY license
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In Elife on 14 August 2020 by Cendrowski, J., Kaczmarek, M., et al.
Fig.4.C
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WB
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BMP2K-L and BMP2K-S differentially regulate autophagic degradation and erythroid differentiation.(A) Western blots showing the effects of depletion of single (shBMP2K-L or shBMP2K-S) or all BMP2K splicing BMP2K variants on the levels of LC3B-I, LC3B-II and p62 proteins in K562 cells, as compared ...
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BMP2K-L and BMP2K-S differentially regulate autophagic degradation and erythroid differentiation.(A) Western blots showing the effects of depletion of single (shBMP2K-L or shBMP2K-S) or all BMP2K splicing BMP2K variants on the levels of LC3B-I, LC3B-II and p62 proteins in K562 cells, as compared to empty pLKO vector or non-targeting shRNA construct, shCtr. Graphs show densitometric analysis of western blotting bands for the indicated proteins using tubulin abundance for normalization (n = 4 +/- SEM). (B) p62 mRNA fold change levels in control cells or in cells with shRNA-mediated depletion of BMP2K variants, as in A (n = 5 +/- SEM). (C) The abundance of p62 protein in non-treated cells or in cells treated for 15 hr with 75 nM bafilomycin A1 (BafA1). Graph shows calculated fold increase of p62 protein levels induced by BafA1, obtained after densitometric analysis of western blotting bands using vinculin abundance for normalization (n = 1). (D) Percentage of benzidine-positive control cells or cells depleted of BMP2K variants, under basal growth conditions or after stimulation for 48 hr with 20 μM hemin (n = 5 +/- SEM). (E) Fold changes in mRNA levels of the indicated erythroid markers in control cells or in cells depleted of all BMP2K splicing variants using shRNA (n = 5 +/- SEM). *p<0.05, **p<0.01, ***p<0.001.Figure 4—source data 1.Numerical data for graphs in Figure 4.Numerical data for graphs in Figure 4.Benzidine staining of K562 cells depleted of BMP2K-L or -S.Representative images of experiments quantified in Figure 4D showing benzidine staining of non-treated (RPMI medium) or 20 µM hemin-treated (48 hr) K562 cells, control or depleted of single BMP2K splicing variants using shRNA. Dark grey/black cells contain hemoglobin.
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Collected and cropped from Elife by CiteAb, provided under a CC-BY license
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In Elife on 14 August 2020 by Cendrowski, J., Kaczmarek, M., et al.
Fig.4.A
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WB
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Mus musculus (House mouse)
BMP2K-L and BMP2K-S differentially regulate autophagic degradation and erythroid differentiation.(A) Western blots showing the effects of depletion of single (shBMP2K-L or shBMP2K-S) or all BMP2K splicing BMP2K variants on the levels of LC3B-I, LC3B-II and p62 proteins in K562 cells, as compared ...
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BMP2K-L and BMP2K-S differentially regulate autophagic degradation and erythroid differentiation.(A) Western blots showing the effects of depletion of single (shBMP2K-L or shBMP2K-S) or all BMP2K splicing BMP2K variants on the levels of LC3B-I, LC3B-II and p62 proteins in K562 cells, as compared to empty pLKO vector or non-targeting shRNA construct, shCtr. Graphs show densitometric analysis of western blotting bands for the indicated proteins using tubulin abundance for normalization (n = 4 +/- SEM). (B) p62 mRNA fold change levels in control cells or in cells with shRNA-mediated depletion of BMP2K variants, as in A (n = 5 +/- SEM). (C) The abundance of p62 protein in non-treated cells or in cells treated for 15 hr with 75 nM bafilomycin A1 (BafA1). Graph shows calculated fold increase of p62 protein levels induced by BafA1, obtained after densitometric analysis of western blotting bands using vinculin abundance for normalization (n = 1). (D) Percentage of benzidine-positive control cells or cells depleted of BMP2K variants, under basal growth conditions or after stimulation for 48 hr with 20 μM hemin (n = 5 +/- SEM). (E) Fold changes in mRNA levels of the indicated erythroid markers in control cells or in cells depleted of all BMP2K splicing variants using shRNA (n = 5 +/- SEM). *p<0.05, **p<0.01, ***p<0.001.Figure 4—source data 1.Numerical data for graphs in Figure 4.Numerical data for graphs in Figure 4.Benzidine staining of K562 cells depleted of BMP2K-L or -S.Representative images of experiments quantified in Figure 4D showing benzidine staining of non-treated (RPMI medium) or 20 µM hemin-treated (48 hr) K562 cells, control or depleted of single BMP2K splicing variants using shRNA. Dark grey/black cells contain hemoglobin.
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Collected and cropped from Elife by CiteAb, provided under a CC-BY license
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In Int J Mol Sci on 4 December 2019 by Lee, A. R., Seo, M. J., et al.
Fig.3.F
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WB
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Homo sapiens (Human)
Btz/Ler induces the cancer cell death accompanied by cytoplasmic vacuolation. (A–C) Cellular morphologies were observed by phase-contrast microscopy. Bars, 20 μm. (A) Cells were treated with 4 nM Btz and/or 10 μM for 24 h. (B) Cells were treated with 20 nM Btz and/or 10 μM for 24 h. (C) Cells wer...
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Btz/Ler induces the cancer cell death accompanied by cytoplasmic vacuolation. (A–C) Cellular morphologies were observed by phase-contrast microscopy. Bars, 20 μm. (A) Cells were treated with 4 nM Btz and/or 10 μM for 24 h. (B) Cells were treated with 20 nM Btz and/or 10 μM for 24 h. (C) Cells were treated with PIs (for MDA-MB 435S and MCF-10A cells, 20 nM Cfz, 100 nM Ixz, or 4 nM Btz; for SNU-475 and Chang cells, 20 nM Cfz, 100 nM Ixz, or 20 nM Btz) and/or 10 μM DHPs for 24 h. (D,F) MDA-MB 435S cells were treated with 5 μg/mL doxorubicin or 4 nM Btz and/or 10 μM Ler for 24 h. Western blotting of the indicated proteins was performed, with β-actin used as a loading control. (E,G) Cells were pretreated with the indicated inhibitors and further treated with the indicated concentrations of Btz/Ler for 24 h. (E) Cellular viability was assessed using the IncuCyte system. Data represent the means ± S.D. (n = 7). One-way ANOVA and Bonferroni’s post hoc test. * p < 0.001 vs. untreated cells. (G) Cellular morphologies were observed by phase-contrast microscopy. Bars, 20 μm.
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Collected and cropped from Int J Mol Sci by CiteAb, provided under a CC-BY license
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In Biomolecules on 12 September 2019 by Granato, M., Gilardini Montani, M. S., et al.
Fig.3.A
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WB
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Homo sapiens (Human)
The autophagic flux was blocked by EBV and restored by quercetin. (a) Sequestosome1/p62 (SQSTM1/p62) expression level was investigated following EBV infection of B cells treated or not with quercetin at 24 and 72 h post-infection. (b) Microtubule-associated protein 1 light chain 3-I/II (LC3-I/II)...
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The autophagic flux was blocked by EBV and restored by quercetin. (a) Sequestosome1/p62 (SQSTM1/p62) expression level was investigated following EBV infection of B cells treated or not with quercetin at 24 and 72 h post-infection. (b) Microtubule-associated protein 1 light chain 3-I/II (LC3-I/II) as evaluated by western blot analysis in EBV-infected B cells, treated or not with quercetin, in the presence or in the absence of Bafilomycin 1 (Baf), at 24 and 72 h post-infection. Densitometric analysis was performed using Image J software and the ratio of SQSTM1/p62 or LC3-II vs. b-actin was calculated. Histograms represent the mean ± SD of three independent experiments.
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Collected and cropped from Biomolecules by CiteAb, provided under a CC-BY license
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In Front Oncol on 10 August 2019 by Hwang, M., Jun, D. W., et al.
Fig.1.E
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WB
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Homo sapiens (Human)
Loss of EI24 expression in pancreatic cancer cells impairs autophagy. MIA PaCa-2 and Panc-1 cells were transfected with 10 nM control siRNA (siCtrl) or siRNA targeting EI24 or ATG5 for 48 h. (A) The mRNA levels of EI24, ATG5, and GAPDH were analyzed by reverse-transcription PCR. Values represent ...
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Loss of EI24 expression in pancreatic cancer cells impairs autophagy. MIA PaCa-2 and Panc-1 cells were transfected with 10 nM control siRNA (siCtrl) or siRNA targeting EI24 or ATG5 for 48 h. (A) The mRNA levels of EI24, ATG5, and GAPDH were analyzed by reverse-transcription PCR. Values represent the expression of EI24 or ATG5 relative to that of GAPDH (with siCtrl values set to 1). (B) The protein levels of EI24 ATG5 and β-actin were analyzed by western blotting. Representative data are shown. Values represent the ratio of the EI24 or ATG5 densitometry value to that of β-actin (with siCtrl values set to 1). (C) EI24 protein expression was observed by immunofluorescence staining using an anti-EI24 antibody. (D) LC3 puncta (autophagosome) were visualized by immunofluorescence staining using an anti-LC3 antibody. (E) Conversion of LC3-I to LC3-II and p62 accumulation were analyzed by western blotting using anti-LC3 and anti-p62 antibodies, respectively. Graphs represent the LC3-II or p62 densitometry value to that of β-actin (with siCtrl values set to 1) from five independent experiments. (F) Co-localization of the autophagosome and lysosome in EI24-knockdown MIA PaCa-2 and Panc-1 cells was analyzed by confocal microscopy using the ZEN 2012 program. Immunofluorescence staining with anti-LC3 (to detect autophagosomes; green dye) and anti-LAMP1 (to detect lysosomes; red dye) antibodies. Co-localization of LC3 with LAMP1 was analyzed by ZEN 2.6 (blue edition). The graph shows the Pearson's co-localization coefficient. (G) MIA PaCa-2 and Panc-1 cells were treated with 100 nM bafilomycin for 30 min, and their protein extracts were analyzed by western blotting using anti-LC3 and β-actin antibodies. Graphs represent the LC3-II densitometry value to that of β-actin (with siCtrl values set to 1) from three independent experiments. Data in (E–G) graphs represent the mean ± standard error of the means (SEM). Comparison were made using Student's t-test, *P < 0.05; **P < 0.01; n.s., not significant.
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Collected and cropped from Front Oncol by CiteAb, provided under a CC-BY license
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In Front Oncol on 10 August 2019 by Hwang, M., Jun, D. W., et al.
Fig.4.B
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WB
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Homo sapiens (Human)
Loss of EI24 expression using CRISPR-Cas9 in MIA PaCa-2 cells decreased cell proliferation. MIA PaCa-2 cells were transfected with CRISPR-Cas9 control (gRNA) and EI24 gRNA (gEI24) using a lentiviral system. (A) After 48 h of incubation, EI24 protein expression was observed by immunofluorescence s...
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Loss of EI24 expression using CRISPR-Cas9 in MIA PaCa-2 cells decreased cell proliferation. MIA PaCa-2 cells were transfected with CRISPR-Cas9 control (gRNA) and EI24 gRNA (gEI24) using a lentiviral system. (A) After 48 h of incubation, EI24 protein expression was observed by immunofluorescence staining using an anti-EI24 antibody. (B) After 48 h of incubation, the EI24 protein level was observed by western blotting using an anti-EI24 antibody. Conversion of LC3-I to LC3-II and p62 accumulation were analyzed by western blotting using anti-LC3 and anti-p62 antibodies, respectively. Graphs represent the mean ± SEM of EI24, LC3-II, and p62 densitometry value to that of β-actin (with gRNA values set to 1) from three independent experiments. Comparison were made using Student's t-test, *P < 0.05; **P < 0.01; ***P < 0.001. (C) After 24 h of incubation, cells were seeded into a 96-well plate (1,000 cells/well). Images acquired by the IncuCyte instrument at the indicated times were analyzed using the ZOOM 2016 program. Confluency was measured in triplicate wells for each sample. Values represent the means ± SEM (Student's t-test, *P < 0.05; **P < 0.01; ***P < 0.001). (D) Control and EI24 gRNA-transfected cells (5 × 106) were injected into both flanks of Balb/c nude mice. Tumor volume was measured on the indicated days. The y-axes of these graphs represent the fold change in tumor size relative to the initial tumor size. Values represent means ± SEM. (Student's t-test, n.s., not significant, control gRNA mice, n = 5; EI24 gRNA mice, n = 4).
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Collected and cropped from Front Oncol by CiteAb, provided under a CC-BY license
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In Front Oncol on 10 August 2019 by Hwang, M., Jun, D. W., et al.
Fig.3.B
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WB
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Homo sapiens (Human)
Loss of EI24 expression in HCT116 cells impairs autophagy but not cell proliferation. HCT116 cells were transfected with 10 nM control siRNA (siCtrl) or siRNA targeting EI24 or ATG5. (A) The mRNA levels of EI24, ATG5, and GAPDH were analyzed by reverse-transcription PCR. (B) The protein levels of...
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Loss of EI24 expression in HCT116 cells impairs autophagy but not cell proliferation. HCT116 cells were transfected with 10 nM control siRNA (siCtrl) or siRNA targeting EI24 or ATG5. (A) The mRNA levels of EI24, ATG5, and GAPDH were analyzed by reverse-transcription PCR. (B) The protein levels of EI24 ATG5 and β-actin were analyzed by western blotting. Representative data are shown. Values represent the ratio of the EI24 or ATG5 densitometry value to that of β-actin (with siCtrl values set to 1). Conversion of LC3-I to LC3-II and p62 accumulation were analyzed by western blotting using anti-LC3 and anti-p62 antibodies, respectively. Graphs represent mean ± SEM of the LC3-II and p62 densitometry value to that of β-actin (with siCtrl values set to 1) from three independent experiments. Comparison were made using Student's t-test, *P < 0.05; **P < 0.01. (C) After 24 h of transfection, cells were seeded into a 6-well plate and incubated for another 7 days. Cells were fixed and stained with crystal violet. (D) After 24 h of transfection, cells were seeded into a 96-well plate. Images acquired from the IncuCyte instrument at the indicated times were analyzed using the ZOOM 2016 program. Cell confluency was measured in triplicate wells for each sample. The plotted values represent means ± SEM. (E) Protein extracts from siRNA-transfected cells were analyzed for DNA fragmentation. The y-axes of the graphs indicate the extent of DNA fragmentation. Values plotted in the graphs represent means ± SEM (Student's t-test, n.s., not significant).
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Collected and cropped from Front Oncol by CiteAb, provided under a CC-BY license
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In Cell Death Dis on 7 August 2019 by Hill, C., Li, J., et al.
Fig.4.A
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ICC-IF
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Homo sapiens (Human)
Autophagy inhibition induces EMT via p62/SQSTM1-NF-κB-Snail2 pathway in ATII cells.a Immunofluorescence staining of p62/SQSTM1 (red) and p65/RELA (green) in ATII cells treated with hydroxychloroquine (HCQ, 25 μM) for 24 h. DAPI (blue) was used to stain nuclei. Scale bar: 40 μm. b Protein expressi...
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Autophagy inhibition induces EMT via p62/SQSTM1-NF-κB-Snail2 pathway in ATII cells.a Immunofluorescence staining of p62/SQSTM1 (red) and p65/RELA (green) in ATII cells treated with hydroxychloroquine (HCQ, 25 μM) for 24 h. DAPI (blue) was used to stain nuclei. Scale bar: 40 μm. b Protein expression of Snail2, p65/RELA and p62/SQSTM1 in ATII cells transfected with the indicated siRNAs followed by treatment of HCQ (25 μM) for 24 h. For protein expression of p62/SQSTM1, both short and long exposures (respectively) are shown. β-tubulin was used as a loading control
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
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In Cell Death Dis on 7 August 2019 by Hill, C., Li, J., et al.
Fig.3.C
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ICC-IF
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Homo sapiens (Human)
Autophagy inhibition by ATG5 depletion induces EMT and cell migration in ATII cells.a Protein expression of E-cadherin, p62/SQSTM and ATG12–ATG5 in ATII cells transfected with indicated siRNAs. β-tubulin was used as a loading control. b Fold change in the mRNA levels of CDH1 (E-cadherin), SNAI2 (...
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Autophagy inhibition by ATG5 depletion induces EMT and cell migration in ATII cells.a Protein expression of E-cadherin, p62/SQSTM and ATG12–ATG5 in ATII cells transfected with indicated siRNAs. β-tubulin was used as a loading control. b Fold change in the mRNA levels of CDH1 (E-cadherin), SNAI2 (Snail2) and VIM (Vimentin) in ATII cells transfected with indicated siRNAs. GAPDH-normalised mRNA levels in control cells were used to set the baseline value at unity. Data are mean ± s.d. n = 3 samples per group. **P < 0.01. c Immunofluorescence staining of p62/SQSTM (green) and F-actin (red) in ATII cells transfected with control or ATG5 siRNAs. Rhodamine-phalloidin was used to stain F-actin. DAPI (blue) was used to stain nuclei. Scale bar: 40 μm. d Scratch wound assay of ATII cells transfected with control or ATG5 siRNAs. Representative images of ATII cells with the indicated treatments at time 0 or 20 h after the scratch wound. Wounds have been artificially coloured red to aid visualisation. Scale bar: 200 μm. The graph shows the area of a wound evaluated with ImageJ, and data are mean ± s.d. n = 3. ***P < 0.001. e Transwell migration assays in control or ATG5-depleted ATII cells. Cells were stained with crystal violet. Scale bar: 100 μm. Data are mean ± s.d. n = 3. ***P < 0.001
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
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In Cell Death Dis on 25 March 2019 by Zhao, S., Wang, J. M., et al.
Fig.2.B
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WB
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Homo sapiens (Human)
BAG3 overexpression induces autophagy independent of Beclin 1 and PtdIns3K.a Control or BAG3-overexpressing HepG2 and MCF7 cells were transfected with scramble shRNA or shRNA specific against Beclin1 (shBeclin1), and Western blot analysis was performed using the indicated antibodies. b HepG2 or M...
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BAG3 overexpression induces autophagy independent of Beclin 1 and PtdIns3K.a Control or BAG3-overexpressing HepG2 and MCF7 cells were transfected with scramble shRNA or shRNA specific against Beclin1 (shBeclin1), and Western blot analysis was performed using the indicated antibodies. b HepG2 or MCF7 cells transduced with lentivirus containing empty or BAG3 construct were treated with vehicle, 3-MA or WM respectively, protein expression levels of LC3, p62 and BAG3 were analyzed using Western blot analysis. c HepG2 or MCF7 cells stably overexpressing EGFP-LC3B were infected with lentivirus containing empty or BAG3 construct. Then cells were treated with vehicle, 3-MA or WM, and the punctate distribution of EGFP-LC3B was visualized under the florescence microscopy. d Quantitative analysis of (c). Results shown represent mean ± SD from five representative microscopic fields and three independent experiments were performed. *P < 0.05. N.S. not significant. Error bars indicate means ± SD
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
Image 1 of 67