CiteAb

Fig.3.F showing Western Blotting from the publication: TRIM21-mediated PRMT1 degradation attenuates colorectal cancer malignant progression.

Fig.3.E showing Western Blotting from the publication: TRIM21-mediated PRMT1 degradation attenuates colorectal cancer malignant progression.

Fig.4.E showing Western Blotting from the publication: Chaperone-mediated autophagy modulates Snail protein stability: implications for breast cancer metastasis.

Fig.3.E showing Western Blotting from the publication: Defective N-glycosylation of IL6 induces metastasis and tyrosine kinase inhibitor resistance in lung cancer.

Fig.2.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: TAZ is involved in breast cancer cell migration via regulating actin dynamics.

Fig.3.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: TAZ is involved in breast cancer cell migration via regulating actin dynamics.

Fig.6.C showing Western Blotting from the publication: Investigating the Genetic Diversity of Hepatitis Delta Virus in Hepatocellular Carcinoma (HCC): Impact on Viral Evolution and Oncogenesis in HCC.

Fig.3.B showing Western Blotting from the publication: Corrigendum: FXYD5/Dysadherin, a biomarker of endometrial cancer myometrial invasion and aggressiveness: its relationship with TGF-β1 and NF-κB pathways.

Fig.4.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: Protein kinase B/AKT phosphorylates hypoxia-inducible factor-3α1 in response to insulin, promoting cell growth and migration.

Fig.5.D showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Housing conditions affect enterocyte death mode and turnover rate in mouse small intestine.

Fig.4.G showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Housing conditions affect enterocyte death mode and turnover rate in mouse small intestine.

Fig.4.A showing Immunohistochemistry in a Homo sapiens (Human) sample from the publication: Full thickness 3D in vitro conjunctiva model enables goblet cell differentiation.

Fig.4.C showing Western Blotting from the publication: ZHX2 deficiency enriches hybrid MET cells through regulating E-cadherin expression.

Fig.2.C showing Western Blotting from the publication: ZHX2 deficiency enriches hybrid MET cells through regulating E-cadherin expression.

Fig.2.D showing Western Blotting from the publication: ZHX2 deficiency enriches hybrid MET cells through regulating E-cadherin expression.

Fig.2.A showing Western Blotting from the publication: ZHX2 deficiency enriches hybrid MET cells through regulating E-cadherin expression.

Fig.2.B showing Western Blotting from the publication: ZHX2 deficiency enriches hybrid MET cells through regulating E-cadherin expression.

Fig.2.C showing Western Blotting in a Canis lupus familiaris (Domestic dog) sample from the publication: Septin 9 Orients the Apico-Basal Polarity Axis and Controls Plasticity Signals.

Fig.2.E showing Western Blotting in a Mus musculus (House mouse) sample from the publication: HAPLN1 potentiates peritoneal metastasis in pancreatic cancer.

Fig.3.A showing Western Blotting from the publication: Opposing USP19 splice variants in TGF-β signaling and TGF-β-induced epithelial-mesenchymal transition of breast cancer cells.

Fig.6.H showing Western Blotting from the publication: Opposing USP19 splice variants in TGF-β signaling and TGF-β-induced epithelial-mesenchymal transition of breast cancer cells.

Fig.2.A showing Western Blotting from the publication: Opposing USP19 splice variants in TGF-β signaling and TGF-β-induced epithelial-mesenchymal transition of breast cancer cells.

Fig.1.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: The essential role of YAP in ERα36-mediated proliferation and the epithelial-mesenchymal transition in MCF-7 breast cancer cells.

Fig.5.C showing Western Blotting from the publication: PDLIM2 is highly expressed in Breast Cancer tumour-associated macrophages and is required for M2 macrophage polarization.

Fig.6.B showing Western Blotting from the publication: Checkpoint Kinase 1 (CHK1) Functions as Both a Diagnostic Marker and a Regulator of Epithelial-to-Mesenchymal Transition (EMT) in Triple-Negative Breast Cancer.

401 images found

Force transmission is a master regulator of mechanical cell competition.

In Nature Materials on 1 June 2025 by Schoenit, A., Monfared, S., et al.

Cell competition is a tissue surveillance mechanism for eliminating unwanted cells, being indispensable in development, infection and tumourigenesis. Although studies have established the role of biochemical mechanisms in this process, due to challenges in measuring forces in these systems, how mechanical forces determine the competition outcome remains unclear. Here we report a form of cell competition that is regulated by differences in force transmission capabilities, selecting for cell types with stronger intercellular adhesion. Direct force measurements in ex vivo tissues and different cell lines reveal that there is an increased mechanical activity at the interface between two competing cell types, which can lead to large stress fluctuations resulting in upward forces and cell elimination. We show how a winning cell type endowed with a stronger intercellular adhesion exhibits higher resistance to elimination and benefiting from efficient force transmission to the neighbouring cells. This cell elimination mechanism could have broad implications for keeping the strong force transmission ability for maintaining tissue boundaries and cell invasion pathology.
© 2025. The Author(s).

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Host PIK3C3 promotes Shigella flexneri spread from cell to cell through vacuole formation.

In PLoS Pathogens on 16 May 2025 by Rolland, S. J., Lifschin, Z. J., et al.

Shigella flexneri is a human intracellular pathogen responsible for bacillary dysentery (bloody diarrhea). S. flexneri invades colonic epithelial cells and spreads from cell to cell, leading to massive epithelial cell fenestration, a critical determinant of pathogenesis. Cell-to-cell spread relies on actin-based motility, which leads to formation of membrane protrusions, as bacteria project into adjacent cells. Membrane protrusions resolve into intermediate structures termed vacuole-like protrusions (VLPs), which remain attached to the primary infected cell by a membranous tether. The resolution of the membranous tether leads to formation of double-membrane vacuoles (DMVs), from which S. flexneri escapes to gain access to the cytosol of adjacent cells. Here, we identify the class III PI3K family member PIK3C3 as a critical determinant of S. flexneri cell-to-cell spread. Inhibition of PIK3C3 decreased the size of infection foci formed by S. flexneri in HT-29 cells. Tracking experiments using live-fluorescence confocal microscopy showed that PIK3C3 is required for efficient resolution of VLPs into DMVs. PIK3C3-dependent accumulation of PtdIns(3)P at the VLP membrane in adjacent cells correlated with the transient recruitment of the membrane scission machinery component Dynamin 2 at the neck of VLPs at the time of DMV formation. By contrast, Listeria monocytogenes did not form VLPs and protrusions resolved directly into DMVs. However, PIK3C3 was also required for L. monocytogenes dissemination, but at the stage of vacuole escape. Finally, we showed that PIK3C3 inhibition decreased S. flexneri dissemination in the infant rabbit model of shigellosis. We propose a model of Shigella dissemination in which vacuole formation relies on the PIK3C3-dependent accumulation of PtdIns(3)P at the VLP stage of cell-to-cell spread, thereby supporting the resolution of VLPs into DMVs through recruitment of the membrane scission machinery component, DNM2.
Copyright: © 2025 Rolland et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Nephron progenitor fate is modulated by angiotensin type 1 receptor signaling in human kidney organoids.

In Stem Cells on 15 May 2025 by Chung, H., Rahmani, W., et al.

The renin-angiotensin system (RAS) is essential for normal kidney development. Dysregulation of the RAS during embryogenesis can result in kidney abnormalities. To explore how angiotensin type 1 receptor (AT1R) signaling modulates nephron progenitor (NP) fate specification, we used induced pluripotent stem cell (iPSC) derived human kidney organoids treated with angiotensin II (Ang II) or the AT1R blocker losartan during differentiation. Ang II promoted NP proliferation and differentiation preferentially toward a podocyte fate, depleted the podocyte precursor population, and accelerated glomerular maturation. By contrast, losartan expanded the podocyte precursor population, delayed podocyte differentiation, and regressed the transcriptional signature to a more immature fetal state. Overall, using various in silico approaches with validation by RNAscope, we identified a role for AT1R signaling in regulating NP fate during nephrogenesis in kidney organoids. Our work supports the use of RAS modulators to improve organoid maturation and suggests that RAS may be a determinant of nephron endowment in vivo.
© The Author(s) 2025. Published by Oxford University Press.

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DRP2 promotes EMT and serves as a potential therapeutic target for LUAD treatment.

In Scientific Reports on 13 May 2025 by Chen, Z., Shi, H., et al.

LUAD, a prevalent lung cancer with high mortality, has seen increased focus on molecular targeted therapies due to patient heterogeneity. Among these prospects, dystrophin-associated protein 2 (DRP2), a critical component of the dystrophin complex, underpins membrane-associated structures vital for intercellular interactions in vertebrates. Aberrations in DRP2 function have been linked to the occurrence and development of multiple diseases, prompting an inquiry into its potential link with LUAD progression. To delve into the potential roles of DRP2 in LUAD, we initiated a comprehensive investigation. First, we analyzed DRP2 expression patterns in LUAD using bioinformatics tools. This was subsequently validated through immunohistochemical staining, quantitative PCR, and Western blot analyses. Furthermore, we assessed the functional implications of DRP2 in LUAD cells, both in vitro and in vivo, utilizing assays such as cell cycle analysis, CCK-8 proliferation assay, Colony formation assay EdU incorporation, Transwell migration test, scratch wound healing assay, flow cytometry, and mouse models for tumor xenograft and metastasis. Results showed a strong correlation between high DRP2 expression in LUAD and poorer survival. Notably, DRP2 knockdown accelerated LUAD progression via the EMT pathway. These findings highlight DRP2's crucial role in LUAD and its potential as a therapeutic target.
© 2025. The Author(s).

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Extrusion of BMP2+ surface colonocytes promotes stromal remodeling and tissue regeneration.

In Nature Communications on 3 May 2025 by Heuberger, J., Liu, L., et al.

The colon epithelium frequently incurs damage through toxic influences. Repair is rapid, mediated by cellular plasticity and acquisition of the highly proliferative regenerative state. However, the mechanisms that promote the regenerative state are not well understood. Here, we reveal that upon injury and subsequent inflammatory response, IFN-γ drives widespread epithelial remodeling. IFN-γ promotes rapid apoptotic extrusion of fully differentiated surface colonocytes, while simultaneously causing differentiation of crypt-base stem and progenitor cells towards a colonocyte-like lineage. However, unlike homeostatic colonocytes, these IFN-γ-induced colonocytes neither respond to nor produce BMP-2 but retain regenerative capacity. The reduction of BMP-2-producing epithelial surface cells causes a remodeling of the surrounding mesenchymal niche, inducing high expression of HGF, which promotes proliferation of the IFN-γ-induced colonocytes. This mechanism of lineage replacement and subsequent remodeling of the mesenchymal niche enables tissue-wide adaptation to injury and efficient repair.
© 2025. The Author(s).

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Chaperone-mediated autophagy modulates Snail protein stability: implications for breast cancer metastasis.

In Mol Cancer on 11 October 2024 by Ryu, K. J., Lee, K. W., et al.

Fig.4.E

Fig.4.E showing Western Blotting from the publication: Chaperone-mediated autophagy modulates Snail protein stability: implications for breast cancer metastasis.

Evasion of CMA-mediated Snail degradation induces EMT and increases metastatic abilities in luminal-type breast cancer cells. (A) Immunoblot analysis of NL-treated WT-Snail- or 58AAAA-Snail-expressing MCF-7 cells (upper). Relative Snail levels were quantified using ImageJ (lower). *, P < 0.05: **... more

Evasion of CMA-mediated Snail degradation induces EMT and increases metastatic abilities in luminal-type breast cancer cells. (A) Immunoblot analysis of NL-treated WT-Snail- or 58AAAA-Snail-expressing MCF-7 cells (upper). Relative Snail levels were quantified using ImageJ (lower). *, P < 0.05: **, P < 0.01: ***, P < 0.001 as determined by t-test. ns, not significant. (B) Immunoblot analysis in LAMP2A-depleted WT-Snail- or 58AAAA-Snail-expressing MCF-7 cells (left). Relative Snail levels were quantified using ImageJ (right). **, P < 0.01 as determined by t-test. ns, not significant. (C) WT-Snail or 58AAAA-Snail-expressing MCF-7 cells were treated with CHX (100 µg/ml) for the indicated times before harvest. Cell lysates were immunoblotted with the indicated antibodies (left), and data quantified using ImageJ (right). Normalized to α-tubulin. **, P < 0.01: ***, P < 0.001: ****, P < 0.0001 as determined by t-test. ns, not significant. (D) Morphological changes of WT-Snail or 58AAAA-Snail-expressing MCF-7 cells. WT-Snail-, 58AAAA-Snail-expressing MCF-7 and control cells were stained with TRITC-conjugated phalloidin and visualized by confocal microscopy. Scale bars, 40 μm–20 μm. (E) Expression levels of EMT marker proteins in WT-Snail-, 58AAAA-Snail-expressing MCF-7 and control cells were analyzed by immunoblotting. (F) WT-Snail, 58AAAA-Snail-expressing MCF-7 and control cells were analyzed in wound-healing assays by visualizing wound closure via phase-contrast microcopy (left). Wound areas were measured using WimScratch software (Wimasis). The data shown represent the percentage of the wound area and are expressed as the means ± SD of three individual experiments (right). **, P < 0.01: ****, P < 0.0001 as determined by t-test. (G) WT-Snail-, 58AAAA-Snail-expressing MCF-7 and control cells were seeded onto Matrigel matrix-coated top chambers, and the fold-changes of invading cells were measured after 30 h. The data shown are expressed as the means ± SD of three individual experiments, each performed in triplicate. **, P < 0.01: ****, P < 0.0001 as determined by t-test. (H) The indicated cells were seeded in a 6-well plate at a concentration of 1 × 105 cells per well. After incubation for 1 to 4 days, the viable cells were counted with a hemocytometer after trypan blue staining. (I) About 2 × 106 of the MCF-7 cells stably expressing WT-Snail, 58AAAA-Snail, or control cells were injected into the nude mice by tail-vein injection. Representative pictures of HE staining of lung sections (left). Scale bars, 200 μm. The number of metastatic lung nodules in individual mice was quantified at 6 weeks after tail-vein injection (right). The data are shown as the means ± SD of 7 mice/group. **, P < 0.01: ***, P < 0.001 as determined by t-test less

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Defective N-glycosylation of IL6 induces metastasis and tyrosine kinase inhibitor resistance in lung cancer.

In Nat Commun on 9 September 2024 by Hung, C. H., Wu, S. Y., et al.

Fig.3.E

Fig.3.E showing Western Blotting from the publication: Defective N-glycosylation of IL6 induces metastasis and tyrosine kinase inhibitor resistance in lung cancer.

deNG-IL6 Induces EMT, Migration, and Metastasis in NSCLC.a IPA-identified “Disease and Functional Annotation” in the N73Q-IL6-CM-treated cells. A significant correlation was defined as p < 0.001, evaluated by a right-tailed Fisher’s Exact Test. b Hierarchical clustering of the IPA-enriched EMT/me... more

deNG-IL6 Induces EMT, Migration, and Metastasis in NSCLC.a IPA-identified “Disease and Functional Annotation” in the N73Q-IL6-CM-treated cells. A significant correlation was defined as p < 0.001, evaluated by a right-tailed Fisher’s Exact Test. b Hierarchical clustering of the IPA-enriched EMT/metastasis-related genes. c GSEA enrichment plot of EMT process-related genes in the AS2-Vec cells under WT-IL6-CM and N73Q-IL6-CM treatment. Nominal p-value was one-tailed test on the appropriate side of the null distribution. d The morphology of (bright field, left panel, scale bars, 200 μm) and F-ACTIN stress fibers (IF, right panel, scale bars, 20 μm) in AS2-Vec, Beas-2B, and NL-20 cells under WT-IL6-CM or N73Q-IL6-CM treatment (24 h) are shown. F-ACTIN was stained with phalloidin-TRITC (gray) and detected with an FV3000 microscope (60x). e Expression of EMT-associated proteins in AS2-Vec cells treated with WT-IL6-CM or N73Q-IL6-CM for 24 h. n = 3 independent experiments, mean ± SEM, two-tailed unpaired t-test, E-CADHERIN: ***p = 0.0005; CLAUDIN1: ***p < 0.0001; SLUG: ***p < 0.0001; N-CADHERIN: ***p < 0.0001; α-SMA: ***p < 0.0001; β-ACTIN: n.s. p = 0.9997. The samples derived from the same experiment but different gels for E-CADHERIN, CLAUDIN1, SLUG, N-CADHERIN, α-SMA, and β-ACTIN were processed in parallel. f Stress fibers in the AS2-Vec cells under N73Q-IL6-CM treatment (24 h) with or without α-GP130 neutralizing Ab pretreatment (2 h). Images are representative of three independent experiments. Scale bars, 20 μm. See also Supplementary Fig. 12. g Expression of mesenchymal proteins in AS2-Vec cells under N73Q-IL6-CM treatment (24 h) with α-GP80, α-IL6, and α-GP130 neutralizing Ab pretreatment for 2 h. n = 3 independent experiments, mean ± SEM, two-tailed unpaired t-test, SLUG: ***p < 0.0001, ***p < 0.0001, ***p < 0.0001; N-CADHERIN: ***p < 0.0001, ***p = 0.0001, ***p < 0.0001; α-SMA: ***p = 0.0001, ***p < 0.0001, ***p < 0.0001; β-ACTIN: n.s. p = 0.9926, n.s. p = 0.6517, n.s. p = 0.8847. The samples derived from the same experiment but different gels for SLUG, N-CADHERIN, α-SMA, and β-ACTIN were processed in parallel. h Migration of AS2-Vec, AS2-IL6-WT, and AS2-IL6- N73Q cells through Transwell membranes n = 3 independent experiments, mean ± SEM, two-tailed unpaired t-test, ***p = 0.0001. i Migration of CL1-0 cells in a Transwell coculture system cocultured with AS2-Vec, AS2-IL6-WT, and AS2-IL6-N73Q cells. n = 3 independent experiments, the percentage of migrated CL1-0 cells is presented as the mean ± SEM, two-tailed unpaired t-test, ***p < 0.0001. j In vivo metastasis of AS2-Vec, AS2-IL6-WT, and AS2-IL6-N73Q cells injected intravenously into nude mice (n = 3 per cell type, 3 repeated independent experiments; see also Supplementary Fig. 15) evaluated by gross anatomical examination (left) and TissueFAXS-imaged H&E staining (right). Each replicate experiment was terminated upon the death of the first animal; the necrotic lungs in these mice are indicated by dashed circles and were not used in further experiments. k The tumor area over the total lung area from (J), as quantified by HistoQuest. n = 3 independent animals for each glycoform in each experiment, 3 independent experiments, mean ± SEM, two-way ANOVA. l In vivo metastasis of AS2-IL6-WT-pLuc and AS2-IL6-N73Q-pLuc cells in nude mice after i.v. injection (n = 3 per cell type) detected with IVIS. Source data are provided as a Source Data file. less

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Corrigendum: FXYD5/Dysadherin, a biomarker of endometrial cancer myometrial invasion and aggressiveness: its relationship with TGF-β1 and NF-κB pathways.

In Front Oncol on 7 February 2024 by Besso, M. J., Rosso, M., et al.

Fig.3.B

Fig.3.B showing Western Blotting from the publication: Corrigendum: FXYD5/Dysadherin, a biomarker of endometrial cancer myometrial invasion and aggressiveness: its relationship with TGF-β1 and NF-κB pathways.

corrected. Modulation of FXYD5/Dys expression in Hec1a cells and changes in cell migration. (A) RT-qPCR analysis of E-cadherin mRNA expression in Hec1a cells transiently transfected with pcDNA3 empty plasmid (Hec1a pcDNA3 cells) or pcDNA3-FXYD5/Dys plasmid (Hec1a pcDNA3-FXYD5/Dys cells) (**P<0.01... more

corrected. Modulation of FXYD5/Dys expression in Hec1a cells and changes in cell migration. (A) RT-qPCR analysis of E-cadherin mRNA expression in Hec1a cells transiently transfected with pcDNA3 empty plasmid (Hec1a pcDNA3 cells) or pcDNA3-FXYD5/Dys plasmid (Hec1a pcDNA3-FXYD5/Dys cells) (**P<0.01, Wilcoxon Signed Rank Test). (B) Immunodetection of E-cadherin by Western immunoblotting of Hec1a pcDNA3 and Hec1a pcDNA3-FXYD5/Dys cell protein extracts using anti E-cadherin monoclonal antibody (610181, BD; 0.125 µg/mL). GAPDH (anti GAPDH monoclonal antibody 14C10; 1:1000) was used as loading control. C-E. RT-qPCR analysis of E-cadherin transcriptional repressors Snail (C) (*P<0.05), Slug (D) (**P<0.01) and Zeb1 (E) (P=0.055), Wilcoxon Signed Rank Test). (F) Left panel: Wound healing assay of Hec1a pcDNA3 and Hec1a pcDNA3-FXYD5/Dys cells. Representative images of cells at 0, 24 and 48 h post transfection are shown (magnification 40X). Right panel: Free-cell area was quantified using ImageJ software and the wound healing closure percentage was plotted at every time point (**P<0.01, ***P<0.0001, Two-way ANOVA, Bonferroni post-test). (G) Left panel: Hanging drop assay of Hec1a pcDNA3 and Hec1a pcDNA3-FXYD5/Dys cells. Representative images of cell aggregates at 48h are shown (magnification 100X). Right panel: Cell aggregates area was quantified using ImageJ software and mean cell aggregates areas (expressed as squared pixels) were plotted and compared (**P<0.01, Mann Whitney test). less

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Housing conditions affect enterocyte death mode and turnover rate in mouse small intestine.

In Sci Rep on 22 November 2023 by Matsuoka, Y. & Tsujimoto, Y.

Fig.5.D

Fig.5.D showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Housing conditions affect enterocyte death mode and turnover rate in mouse small intestine.

Enterocyte turnover and RIPK1-dependent enterocyte shedding were stimulated in TBK1-deficient mice. (A) The enterocyte turnover rates were higher in Ripk3−/− Tbk1−/− mice than in sex-matched littermate Ripk3−/− mice in three independent experiments. Distance was plotted; the horizontal line repre... more

Enterocyte turnover and RIPK1-dependent enterocyte shedding were stimulated in TBK1-deficient mice. (A) The enterocyte turnover rates were higher in Ripk3−/− Tbk1−/− mice than in sex-matched littermate Ripk3−/− mice in three independent experiments. Distance was plotted; the horizontal line represents the mean. (B, C) Enterocyte shedding was stimulated in RV culture from Ripk3−/− Tbk1−/− mice, and this stimulation was Nec-1s-sensitive. (B), Shed cells were counted; data are the mean ± SEM from three independent experiments. In (C), all plots of the three experiments are shown (Ripk3−/−: vehicle, n = 203, Nec-1s, n = 172, Ripk3−/− Tbk1−/−: vehicle, n = 165, Nec-1s, n = 141); the horizontal line represents the mean. For comparison, vehicle and Nec-1s were paired in each genotype and vehicles of each genotype were also paired. (D, E) Western blot showing the levels of S321-phosphorylated-RIPK1 (p-RIPK1 [S321]), total RIPK1 [RIPK1], and TBK1 in enterocytes of Ripk3−/− (n = 3) and Ripk3−/− Tbk1−/− mice (n = 3) (D). E-cadherin is a loading control. (E), Densitometry was performed to obtain the ratio of p-RIPK1 (S321) to total RIPK1. The mean ± SEM from three mice is shown. For (A, E), P values were calculated using two-tailed unpaired t-tests. For (B), P values were calculated using two-way ANOVA with Bonferroni post-tests. For (C), P values were calculated using Mann–Whitney U-tests. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. Uncropped Western blot data used in D are shown in supplementary Fig. S8. less

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Housing conditions affect enterocyte death mode and turnover rate in mouse small intestine.

In Sci Rep on 22 November 2023 by Matsuoka, Y. & Tsujimoto, Y.

Fig.4.G

Fig.4.G showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Housing conditions affect enterocyte death mode and turnover rate in mouse small intestine.

Inhibition of TBK1 results in stimulation of RIPK1-dependent enterocyte shedding. (A–F) TBK1i stimulated enterocyte shedding in RV culture, but TAK1i and MK2i did not. RV were treated with 2 µM TBK1i (MRT67307) (A, B), 100 or 300 nM TAK1i (5Z-7-Oxozeaenol) (C, D), or 1 or 10 µM MK2i (PF-3644022) ... more

Inhibition of TBK1 results in stimulation of RIPK1-dependent enterocyte shedding. (A–F) TBK1i stimulated enterocyte shedding in RV culture, but TAK1i and MK2i did not. RV were treated with 2 µM TBK1i (MRT67307) (A, B), 100 or 300 nM TAK1i (5Z-7-Oxozeaenol) (C, D), or 1 or 10 µM MK2i (PF-3644022) (E, F) for 6 h. In the case of TBK1i, RV were also pre-treated for 30 min with Nec-1s, and then 2 µM TBK1i was added for 6 h. Shed cells were counted; data are the mean ± SEM from three independent experiments (A, C, E). In (B, D, F), all plots of the three experiments are shown (B: vehicle, n = 159, TBK1i, n = 163, TBK1i + Nec-1s, n = 151, D: vehicle, n = 142 , TAK1i [100 nM], n = 127, TAK1i [300 nM], n = 130, F: vehicle, n = 181, MK2i [1 µM], n = 151, MK2i [10 µM], n = 167); the horizontal line represents the mean. (G–I) The amount of TBK1 and the S321-phosphorylation level of RIPK1 were lower in WT mice housed with αDri than in sex-matched littermate WT mice housed with Eco Chips. (G), Western blot analysis of the quantity of TBK1, S321-phosphorylated-RIPK1 (p-RIPK1 [S321]), and total RIPK1 (RIPK1) in the enterocytes of WT mice housed with αDri and sex-matched littermate WT mice housed with Eco Chips. E-cadherin was a loading control. (H), Densitometry of TBK1 was normalized to E-cadherin; then, the ratio (relative to a value of Eco Chips as 1) was calculated in each Eco Chips/ αDri pair. The mean ± SEM from three pairs is shown. (I), Densitometry was performed to obtain the ratio of p-RIPK1 (S321) to total RIPK1 (normalized to Eco Chips as 1) in each Eco Chips/αDri pair. The mean ± SEM from three pairs is shown. For (A–F), P values were calculated using one-way Anova with Dunnett’s multiple comparison tests. For (H, I), P values were calculated using one-sample t-tests. *P < 0.05, **P < 0.01, n.s., not significant. Uncropped Western blot data used in G, H, and I are shown in supplementary Fig. S7. less

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ZHX2 deficiency enriches hybrid MET cells through regulating E-cadherin expression.

In Cell Death Dis on 17 July 2023 by He, Y., Zhang, Q., et al.

Fig.4.C

Fig.4.C showing Western Blotting from the publication: ZHX2 deficiency enriches hybrid MET cells through regulating E-cadherin expression.

Loss of ZHX2 abrogates migration and invasion of TNBC cells through E-cadherin.A Representative scratch-wound images showing the healing ability in indicated MDA-MB-231 (upper) and SUM159 (lower) cells. Scale bar 100 μm. Error bars represent mean ± standard deviation (SD), one-way ANOVA. ****p < ... more

Loss of ZHX2 abrogates migration and invasion of TNBC cells through E-cadherin.A Representative scratch-wound images showing the healing ability in indicated MDA-MB-231 (upper) and SUM159 (lower) cells. Scale bar 100 μm. Error bars represent mean ± standard deviation (SD), one-way ANOVA. ****p < 0.0001. B The migration ability of MDA-MB-231 (upper) or SUM159 (lower) cells was determined by Transwell migration assay. Scale bar 100 μm. Error bars represent mean ± standard deviation (SD), one-way ANOVA. ****p < 0.0001. C Immunoblots from SUM159 cells infected with lentivirus encoding sg1-ZHX2 or control sgRNA, and then followed by knocking down E-cadherin. The expression of E-cadherin and Vimentin were examined by immunoblotting with antibodies as indicated. D Representative scratch-wound images showing the healing ability of indicated SUM159 cells. Scale bar 100 μm. Error bars represent mean ± standard deviation (SD), one-way ANOVA. ****p < 0.0001. E The migration ability of SUM159 cells, as described in C, was determined by Transwell migration assay. Scale bar 100 μm. Error bars represent mean ± standard deviation (SD), one-way ANOVA. ****p < 0.0001. F Phase contrast images of ZHX2 control and knock-out MDA-MB-231 organoids grown in 5 μg/ml Collagen I (2, 5, 9 days). The red arrow indicates branching and stellate structures. Scale bar 20 μm. G Representative phase contrast images of ZHX2 control and knockout MDA-MB-231 organoids. Quantitative analysis of spherical diameter is shown in the bar graphs. The white line indicates the spherical diameter. Scale bar 20 μm. Error bars represent mean ± standard deviation (SD), unpaired t-test. **p < 0.01. less

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Septin 9 Orients the Apico-Basal Polarity Axis and Controls Plasticity Signals.

In Cells on 9 July 2023 by Cai, T., Peng, J., et al.

Fig.2.C

Fig.2.C showing Western Blotting in a Canis lupus familiaris (Domestic dog) sample from the publication: Septin 9 Orients the Apico-Basal Polarity Axis and Controls Plasticity Signals.

Septin 9 regulates and impacts cell–cell adhesion and BM stability. (A) Schematic representation of the cell–cell adhesion structure. In the cytoplasm, the E-cadherin/catenin complex links to ZO-1, actin, and the actin-binding protein cortactin. (B) MDCK cells were transfected with scrambled siRN... more

Septin 9 regulates and impacts cell–cell adhesion and BM stability. (A) Schematic representation of the cell–cell adhesion structure. In the cytoplasm, the E-cadherin/catenin complex links to ZO-1, actin, and the actin-binding protein cortactin. (B) MDCK cells were transfected with scrambled siRNA (control) or with septin 9 siRNA for 24 h and plated on Matrigel for 4 days to form cysts and then stained for E-cadherin (green) and actin (red), β-catenin (green) and ZO-1 (red), and cortactin (green) and actin (red). A single confocal section through the middle of a cyst is shown. Quantification of the fluorescence intensity of each protein expression: E-cadherin (green), β-catenin (green), and ZO-1 (red). Scale bar 10 µm. (C) MDCK cells were transfected with scrambled siRNA (control) or with septin 9 siRNA (siSeptin9) for 24 h, then the medium was changed to continue grown on plates for 3 days. Cells were lysed and analyzed by Western blot to determine the expression of septin 9, E-cadherin, β-catenin, and cortactin proteins. (D) Another experiment; as in (C), qRT-PCR analysis of the expression of mRNA encoding septin 9, vimentin, N-cadherin, and ZEB1 in cells with control and septin 9 knock-down under 2D conditions. Data information: Data concern at least two replicates and cysts (n > 10) for 3D staining, three replicates for immunoblot and qRT-PCR. The statistical values are means ± s.e.m. Student’s t-test was used. * p < 0.05, ** p < 0.01, *** p < 0.001. less

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HAPLN1 potentiates peritoneal metastasis in pancreatic cancer.

In Nat Commun on 24 April 2023 by Wiedmann, L., De Angelis Rigotti, F., et al.

Fig.2.E

Fig.2.E showing Western Blotting in a Mus musculus (House mouse) sample from the publication: HAPLN1 potentiates peritoneal metastasis in pancreatic cancer.

HAPLN1 induces EMT and ECM remodeling in vitro.A ELISA-like assay for Hyaluronan (HA) in the serum-free supernatant of KPC and KPC-HAPLN1 cells. n = 4. B qRT-PCR analysis of HA-synthase (Has) expression levels. HAS1 & HAS3, n = 3: HAS2, n = 4. C HAS2 protein levels evaluated by Western blot. Left... more

HAPLN1 induces EMT and ECM remodeling in vitro.A ELISA-like assay for Hyaluronan (HA) in the serum-free supernatant of KPC and KPC-HAPLN1 cells. n = 4. B qRT-PCR analysis of HA-synthase (Has) expression levels. HAS1 & HAS3, n = 3: HAS2, n = 4. C HAS2 protein levels evaluated by Western blot. Left panel: representative image, right panel: quantification by normalization to house keeper β-actin. n = 6. D mRNA expression of epithelial and mesenchymal markers comparing KPC and KPC-HAPLN1 cells. Cdh1, n = 4; rest, n = 3. E Western blot analysis of epithelial and mesenchymal markers on protein lysates. Left panel: representative image, right panel: quantification by normalization to house keeper VCP. E-Cadherin, n = 3; rest, n = 6. F mRNA expression of stemness markers by qRT-PCR. Abcg1, n = 4; rest, n = 3. G Cells seeded in ultra-low attachment plates. Pictures taken after 48 h. Quantification of area as measure for proper spheroid formation. Scale bar: 100 µm. KPC, n = 11; KPC-HAPLN1, n = 12. H Flow cytometric analysis of DAPI− events using spheroids formed for 48 h. n = 3. I Schematic overview of HAPLN1 function as crosslinker of HA and proteoglycans aggrecan (ACAN) or versican (VCAN). J, K Gene expression analysis of Has2, n = 3 (J) and HAPLN1-associated proteoglycans (K) in spheroids. Vcan, n = 3; Acan, n = 4. Plots are shown as mean ± SD. Data points represent independent biological replicates. For panels A, B, D, F–J unpaired two-tailed T test was applied, for panels C, E paired two-tailed T test. less

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Opposing USP19 splice variants in TGF-β signaling and TGF-β-induced epithelial-mesenchymal transition of breast cancer cells.

In Cell Mol Life Sci on 17 January 2023 by Zhang, J., Van Dinther, M., et al.

Fig.3.A

Fig.3.A showing Western Blotting from the publication: Opposing USP19 splice variants in TGF-β signaling and TGF-β-induced epithelial-mesenchymal transition of breast cancer cells.

USP19-CY promotes TGF-β-induced EMT, cell migration and invasion. A Western blotting analysis of epithelial marker E-cadherin, mesenchymal markers N-cadherin, vimentin and SNAIL, and USP19 expression levels in A549-VIM-RFP cells without (pLKO-EV) or with USP19-CY knockdown that were treated with ... more

USP19-CY promotes TGF-β-induced EMT, cell migration and invasion. A Western blotting analysis of epithelial marker E-cadherin, mesenchymal markers N-cadherin, vimentin and SNAIL, and USP19 expression levels in A549-VIM-RFP cells without (pLKO-EV) or with USP19-CY knockdown that were treated with vehicle control or TGF-β (2.5 ng/mL) for 2 days. Tubulin was used as the loading control. B qRT‒PCR analysis of the expression of the EMT marker genes CDH1, CDH2, VIM and SNAI1 in A549-VIM-RFP cells infected with pLKO-EV or sh-CY in the absence or presence of TGF-β (2.5 ng/mL) for 2 days. The data are expressed as the mean ± SD, n = 3 (biological replicates). *P ≤ 0.05, based on unpaired Student’s t test. C Effect of USP19-CY depletion on vimentin expression in A549-VIM-RFP cells treated with or without TGF-β (2.5 ng/mL) for the indicated times. Time course of RFP-conjugated vimentin expression levels as measured by IncuCyte. Red staining intensity was normalized to the intensity at 0 h and expressed as the mean ± SD, n = 3 (biological replicates). *P ≤ 0.05, **P < 0.01, based on two-way ANOVA. D Representative images of RFP-vimentin expression in A549-VIM-RFP cells with pLKO-EV or sh-CY at the end time point (58 h). Scale bar = 400 μm. (E) Real-time scratch assay results, as analyzed by IncuCyte, in A549-VIM-RFP control cells (pLKO-EV) or with USP19-CY-knockdown cells treated with vehicle control or TGF-β (2.5 ng/mL) for the indicated times. The relative wound density (closure) is presented as the mean ± SD, n = 3 (biological replicates). *P ≤ 0.05, based on based on two-way ANOVA. F Representative images of scratch wounds in pLKO-EV control or USP19-CY-depleted A549-VIM-RFP cells that were treated with vehicle control or TGF-β (2.5 ng/mL) at the end time point. The area of the original scratch is indicated in white, and the open area of the scratch is indicated in purple. Scale bar = 400 μm. G mCherry-labeled MDA-MB-231 cells with pLKO-EV and sh-CY were injected into ducts of Cuvier of zebrafish embryos. Representative images with magnified pictures (outlined with a dashed square) of invasive cells were captured 4 days after injection by confocal microscopy. Scale bar = 300 μm or 150 μm. Extravasated cells in the avascular collagen-rich tail fin area are indicated with three arrows. H Quantification of the number of invasive cells in the tail fins of 25 embryos for each group. The data are expressed as the mean ± SD, n = 2 (biological replicates). ****P < 0.0001, based on unpaired Student’s t test less

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Opposing USP19 splice variants in TGF-β signaling and TGF-β-induced epithelial-mesenchymal transition of breast cancer cells.

In Cell Mol Life Sci on 17 January 2023 by Zhang, J., Van Dinther, M., et al.

Fig.6.H

Fig.6.H showing Western Blotting from the publication: Opposing USP19 splice variants in TGF-β signaling and TGF-β-induced epithelial-mesenchymal transition of breast cancer cells.

USP19-CY is highly expressed in breast cancer tissues, and USP19 mRNA splicing is regulated by herboxidiene. Representative images of USP19-CY (red) immunofluorescence staining in a human breast cancer tissue microarray containing 34 pairs of cancer adjacent tissues and cancer tissues (A) or canc... more

USP19-CY is highly expressed in breast cancer tissues, and USP19 mRNA splicing is regulated by herboxidiene. Representative images of USP19-CY (red) immunofluorescence staining in a human breast cancer tissue microarray containing 34 pairs of cancer adjacent tissues and cancer tissues (A) or cancer tissues of different stages (stage IIA, IIB, IIIA, IIIB) (B). Nuclei were counterstained with DAPI (blue). Large field and magnified pictures (outlined with a dotted square) are shown. Scale bar = 250 μm, 50 μm or 250 μm. C Quantification of the percent USP19-CY expression in pairs of breast tissues (adjacent and cancer tissues). Red lines indicate significant upregulation, and blue lines indicate downregulation of USP19-CY in cancer tissues compared to adjacent tissues; black lines indicate no significant change in USP19-CY in tissue pairs. The data are represented as the mean ± SD, tissue pairs, n = 34, **P < 0.01, based on a paired Student’s t test. D Quantification of percent USP19-CY expression in breast cancer adjacent tissues and cancer tissues of different stages. The data are expressed as the mean ± SD, adjacent tissues, n = 10; adenocarcinoma (stage IIA), n = 49; adenocarcinoma (stage IIB), n = 22; adenocarcinoma (stage IIIA), n = 16; adenocarcinoma (stage IIIB), n = 6; *P ≤ 0.05, ****P < 0.0001, based on unpaired Student’s t test. E qRT‒PCR analysis of the expression of the USP19, USP19-CY and USP19-ER in A549-VIM-RFP cells treated with 0.2 or 1 μM herboxidiene. The data are expressed as the mean ± SD, n = 3 (technical replicates). F MDA-MB-231 cells stably infected with pLV-EV or USP19-CY-wt were pretreated with 1 μM herboxidiene (Herbo) for 24 h and then, combined with vehicle control or TGF-β (2.5 ng/mL) for 1 h, followed by immunoblot analysis of the p-SMAD2 and t-SMAD2 expression levels. GAPDH: loading control. G HEK293T cells transfected with pRK5 or USP19-CY-wt were pretreated with 1 μM herboxidiene (Herbo) for 24 h and then, combined with vehicle control or TGF-β (2.5 ng/mL) overnight, followed by the analysis of CAGA12-luciferase transcriptional responses. The data were expressed as the mean ± SD, n = 3 (biological replicates). *P ≤ 0.05, **P < 0.01, ***P < 0.001, based on unpaired Student’s t test. H A549-VIM-RFP cells stably infected with pLV-EV or USP19-CY-wt were pretreated with 1 μM herboxidiene (Herbo) for 24 h and then, treated with vehicle control or TGF-β (2.5 ng/mL) for 48 h. Then, immunoblotting analysis of the expression of the epithelial marker E-cadherin and mesenchymal markers N-cadherin, vimentin and SNAIL was performed. GAPDH: loading control. I A549-VIM-RFP cells with pLV-EV and USP19-CY-wt plasmids were pretreated with 1 μM herboxidiene for 24 h and then, incubated with vehicle control or TGF-β (2.5 ng/mL) for the indicated times. The results of the scratch assay time course were analyzed by IncuCyte. The relative wound density (closure) is presented as the mean ± SD, n = 3 (biological replicates). *P ≤ 0.05, **P < 0.01, based on unpaired Student’ s t test less

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Opposing USP19 splice variants in TGF-β signaling and TGF-β-induced epithelial-mesenchymal transition of breast cancer cells.

In Cell Mol Life Sci on 17 January 2023 by Zhang, J., Van Dinther, M., et al.

Fig.2.A

Fig.2.A showing Western Blotting from the publication: Opposing USP19 splice variants in TGF-β signaling and TGF-β-induced epithelial-mesenchymal transition of breast cancer cells.

USP19-ER isoform inhibits TGF-β-induced EMT, and cell migration. A Immunoblotting analysis of the expression levels of the epithelial marker E-cadherin, mesenchymal markers N-cadherin, vimentin and SNAIL, and USP19 in USP19-ER-deficient A549-VIM-RFP cells that were treated with vehicle control or... more

USP19-ER isoform inhibits TGF-β-induced EMT, and cell migration. A Immunoblotting analysis of the expression levels of the epithelial marker E-cadherin, mesenchymal markers N-cadherin, vimentin and SNAIL, and USP19 in USP19-ER-deficient A549-VIM-RFP cells that were treated with vehicle control or TGF-β (2.5 ng/mL) for 2 days. Tubulin, was used as a loading control. B qRT‒PCR analysis of the expression of the EMT marker genes CDH1 (encodes the E-cadherin protein), CDH2 (encodes the N-cadherin protein), VIM (encodes the vimentin protein) and SNAI1 (encodes the SNAIL protein) in A549-VIM-RFP cells with pLKO-EV or USP19-ER shRNA in the presence of TGF-β (2.5 ng/mL) for 2 days. The data are expressed as the mean ± SD, n = 3 (biological replicates). *P ≤ 0.05, **P < 0.01, based on unpaired Student’s t test. C Effect of USP19-ER knockdown on vimentin expression in A549-VIM-RFP cells in response to TGF-β (2.5 ng/mL) treatment for the indicated times. The expression of RFP-conjugated vimentin expression over time was measured by IncuCyte. Red staining intensity was normalized to the intensity at 0 h and expressed as the mean ± SD, n = 3 (biological replicates). **P < 0.01, based on two-way ANOVA. D Representative images of RFP-vimentin expression at the end time point (58 h) in A549-VIM-RFP cells with pLKO-EV or sh-ER. Scale bar = 400 μm. E Real-time scratch assay results, as analyzed by IncuCyte, in pLKO-EV control or USP19-ER-depleted A549-VIM-RFP cells treated with vehicle control or TGF-β (2.5 ng/mL) for the indicated times. The relative wound density (closure) is presented as the mean ± SD, n = 3 (biological replicates). *P ≤ 0.05, based on two-way ANOVA. F Representative images of a scratch wound in pLKO-EV control or USP19-ER-deficient A549-VIM-RFP cells that were treated with vehicle control or TGF-β (2.5 ng/mL) at the end time point. The region of the original scratch is indicated in white, and the remaining scratch area is indicated in purple. Scale bar = 400 μm. G Crystal violet staining of MDA-MB-231 cells stably infected with the pLV-EV, ER-wt or ER-CS lentivirus after the Transwell migration assay. Scale bar = 300 μm. H Quantification of the migrated MDA-MB-231 cells stably expressing pLV-EV, ER-wt and ER-CS in the Transwell assay. The number of migrated cells per field is shown as the mean ± SD, n = 5 (biological replicates). ***P < 0.001, based on unpaired Student’s t test less

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