CiteAb

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

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

Fig.5.B showing Western Blotting from the publication: Auranofin as a Novel Anticancer Drug for Anaplastic Thyroid Cancer.

Fig.1.A showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Effects of cadherin mediated contact normalization on oncogenic Src kinase mediated gene expression and protein phosphorylation.

Fig.1.A showing Western Blotting from the publication: FBXO11 Mediates Ubiquitination of ZEB1 and Modulates Epithelial-to-Mesenchymal Transition in Lung Cancer Cells.

Fig.3.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Mitochondrial NME6 Influences Basic Cellular Processes in Tumor Cells In Vitro.

Fig.3.E showing Western Blotting in a Homo sapiens (Human) sample from the publication: Mitochondrial NME6 Influences Basic Cellular Processes in Tumor Cells In Vitro.

Fig.4.A showing Western Blotting from the publication: Presenilin Deficiency Results in Cellular Cholesterol Accumulation by Impairment of Protein Glycosylation and NPC1 Function.

Fig.6.C showing Western Blotting from the publication: N-cadherin directs the collective Schwann cell migration required for nerve regeneration through Slit2/3-mediated contact inhibition of locomotion.

Fig.3.A showing Western Blotting from the publication: Impact of KIF4A on Cancer Stem Cells and EMT in Lung Cancer and Glioma.

Fig.4.H showing Western Blotting from the publication: Impact of KIF4A on Cancer Stem Cells and EMT in Lung Cancer and Glioma.

Fig.5.D showing Western Blotting from the publication: Impact of KIF4A on Cancer Stem Cells and EMT in Lung Cancer and Glioma.

Fig.3.E showing Western Blotting from the publication: Downregulation of mitochondrial complex I induces ROS production in colorectal cancer subtypes that differently controls migration.

Fig.2.E showing Western Blotting from the publication: Downregulation of mitochondrial complex I induces ROS production in colorectal cancer subtypes that differently controls migration.

Fig.7.G showing Western Blotting from the publication: Isthmin-1 (Ism1) modulates renal branching morphogenesis and mesenchyme condensation during early kidney development.

Fig.5.I showing Western Blotting in a Mus musculus (House mouse) sample from the publication: RAB35 is required for murine hippocampal development and functions by regulating neuronal cell distribution.

Fig.5.E showing Immunocytochemistry-immunofluorescence in a Homo sapiens (Human) sample from the publication: RanBP1: A Potential Therapeutic Target for Cancer Stem Cells in Lung Cancer and Glioma.

Fig.3.C showing Immunocytochemistry-immunofluorescence in a Homo sapiens (Human) sample from the publication: RanBP1: A Potential Therapeutic Target for Cancer Stem Cells in Lung Cancer and Glioma.

Fig.5.B showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Altered expression of synaptic proteins and adhesion molecules in the hippocampus and cortex following the onset of diabetes in nonobese diabetic mice.

Fig.6.D showing Western Blotting from the publication: Antimetastatic Activity of Apoptolidin A by Upregulation of N-Myc Downstream-Regulated Gene 1 Expression in Human Colorectal Cancer Cells.

Fig.5.C showing Western Blotting from the publication: Antimetastatic Activity of Apoptolidin A by Upregulation of N-Myc Downstream-Regulated Gene 1 Expression in Human Colorectal Cancer Cells.

Fig.6.A showing Western Blotting from the publication: GRHL2 Regulation of Growth/Motility Balance in Luminal versus Basal Breast Cancer.

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.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.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.

155 images found

PKIB facilitates bladder cancer proliferation and metastasis through mediation of HSP27 phosphorylation by PKA.

In Cell Death & Disease on 1 July 2025 by Liu, X., Yin, X., et al.

Cyclic AMP-dependent protein kinase A (PKA) is recognized for its pivotal involvement in various cancer types, with Protein Kinase Inhibitor Beta (PKIB) serving as an endogenous inhibitor that curtails PKA activity. Despite the documented escalation of PKIB expression in several malignancies, a comprehensive understanding of its precise mechanistic implications in human cancers remains elusive. This investigation is centered on bladder cancer (BLCA), unveiling an augmented expression of PKIB concomitant with heightened BLCA cell proliferation, migration, and invasion in vitro and augmented tumorigenic potential in an in vivo model. Mechanistically, PKIB disrupts PKA kinase activity, thereby resulting in diminished phosphorylation of the substrate target protein HSP27 at serine 15, 78, and 82. Additionally, the transcription factor MYCN exhibits an affinity for the PKIB promoter, leading to its enhanced expression in the context of BLCA. These findings reveal the oncogenic proclivity of PKIB and introduce a novel signalling pathway in BLCA, providing valuable insights into potential therapeutic targets for precise intervention.
© 2025. The Author(s).

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Viscoelastic N‑cadherin-like interactions maintain neural progenitor cell stemness within 3D matrices.

In Nature Communications on 5 June 2025 by Huang, M. S., LeSavage, B. L., et al.

Neural progenitor cells (NPCs) hold immense potential as therapeutic candidates for neural regeneration, and materials-based strategies have emerged as attractive options for NPC expansion. However, maintaining NPC stemness has proven challenging in vitro, due to their propensity to form cell-dense neurospheres. While neurospheres promote cell-cell interactions required for NPC stem maintenance, they also restrict oxygen transport, leading to hypoxia and limited cell expansion. To overcome these limitations, we investigate two materials-based approaches to maintain NPC stemness: 1) physical matrix remodeling within a viscoelastic, stress-relaxing hydrogel and 2) matrix-induced N-cadherin-like signaling through a cell-instructive peptide. While viscoelasticity alone is sufficient to maintain NPC stemness compared to an elastic environment, NPCs still preferentially form neurospheres. The addition of N-cadherin-like peptides promotes a distributed culture of NPCs while maintaining their stemness through cadherin-mediated signaling, ultimately exhibiting improved long-term expansion and neural differentiation. Thus, our findings reveal matrix viscoelasticity and engineered N-cadherin-like interactions as having a synergistic effect on NPC expansion and differentiation within 3D matrices.
© 2025. The Author(s).

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Age-dependent differences in breast tumor microenvironment: challenges and opportunities for efficacy studies in preclinical models.

In Cell Death and Differentiation on 1 June 2025 by Falvo, P., Gruener, S., et al.

Immunity suffers a function deficit during aging, and the incidence of cancer is increased in the elderly. However, most cancer models employ young mice, which are poorly representative of adult cancer patients. We have previously reported that Triple-Therapy (TT), involving antigen-presenting-cell activation by vinorelbine and generation of TCF1+-stem-cell-like T cells (scTs) by cyclophosphamide significantly improved anti-PD-1 efficacy in anti-PD1-resistant models like Triple-Negative Breast Cancer (TNBC) and Non-Hodgkin's Lymphoma (NHL), due to T-cell-mediated tumor killing. Here, we describe the effect of TT on TNBC growth and on tumor-microenvironment (TME) of young (6-8w, representative of human puberty) versus adult (12 m, representative of 40y-humans) mice. TT-efficacy was similar in young and adults, as CD8+ scTs were only marginally reduced in adults. However, single-cell analyses revealed major differences in the TME: adults had fewer CD4+ scTs, B-naïve and NK-cells, and more memory-B-cells. Cancer-associated-fibroblasts (CAF) with an Extracellular Matrix (ECM) deposition-signature (Matrix-CAFs) were more common in young mice, while pro-inflammatory stromal populations and myofibroblasts were more represented in adults. Matrix-CAFs in adult mice displayed decreased ECM-remodeling abilities, reduced collagen deposition, and a different pattern of interactions with the other cells of the TME. Taken together, our results suggest that age-dependent differences in the TME should be considered when designing preclinical studies.
© 2025. The Author(s).

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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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Shc1 cooperates with Frs2 and Shp2 to recruit Grb2 in FGF-induced lens development.

In eLife on 6 May 2025 by Wang, Q., Li, H., et al.

Fibroblast growth factor (FGF) signaling elicits multiple downstream pathways, most notably the Ras/MAPK cascade facilitated by the adaptor protein Grb2. However, the mechanism by which Grb2 is recruited to the FGF signaling complex remains unresolved. Here, we showed that genetic ablation of FGF signaling prevented murine lens induction by disrupting transcriptional regulation and actin cytoskeletal arrangements, which could be reproduced by deleting the juxtamembrane region of the FGF receptor and rescued by Kras activation. Conversely, mutations affecting the Frs2-binding site on the FGF receptor or the deletion of Frs2 and Shp2 primarily impact later stages of lens vesicle development involving lens fiber cell differentiation. Our study further revealed that the loss of Grb2 abolished MAPK signaling, resulting in a profound arrest of lens development. However, removing Grb2's putative Shp2 dephosphorylation site (Y209) neither produced a detectable phenotype nor impaired MAPK signaling during lens development. Furthermore, the catalytically inactive Shp2 mutation (C459S) only modestly impaired FGF signaling, whereas replacing Shp2's C-terminal phosphorylation sites (Y542/Y580) previously implicated in Grb2 binding only caused placental defects, perinatal lethality, and reduced lacrimal gland branching without impacting lens development, suggesting that Shp2 only partially mediates Grb2 recruitment. In contrast, we observed that FGF signaling is required for the phosphorylation of the Grb2-binding sites on Shc1 and the deletion of Shc1 exacerbates the lens vesicle defect caused by Frs2 and Shp2 deletion. These findings establish Shc1 as a critical collaborator with Frs2 and Shp2 in targeting Grb2 during FGF signaling.
© 2024, Wang, Li, Mao et al.

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N-cadherin directs the collective Schwann cell migration required for nerve regeneration through Slit2/3-mediated contact inhibition of locomotion.

In Elife on 9 April 2024 by Hoving, J. J. A., Harford-Wright, E., et al.

Fig.6.C

Fig.6.C showing Western Blotting from the publication: N-cadherin directs the collective Schwann cell migration required for nerve regeneration through Slit2/3-mediated contact inhibition of locomotion.

The Slit-repulsive signal is required for the efficient collective migration of Schwann cells (SCs).(a) Representative stills from time-lapse microscopy (Figure 6—video 1) showing the collective migration of control or Slit2/Slit3 knockdown SCs seeded in chambers at the indicated time points and ... more

The Slit-repulsive signal is required for the efficient collective migration of Schwann cells (SCs).(a) Representative stills from time-lapse microscopy (Figure 6—video 1) showing the collective migration of control or Slit2/Slit3 knockdown SCs seeded in chambers at the indicated time points and quantified in Figure 6a (n = 3). The dashed lines indicate the leading edge of the migrating cells. Arrows indicate the direction of migration. Scale bar = 50 μm. (b) As in (a) but the SCs were treated with 2 μg/ml recombinant-Slit-2 (rSlit2) or PBS (n = 3), quantified in Figure 6d (Figure 6—video 3). Scale bar = 50 μm. (c) Representative western blot showing pTuner empty vector SCs or pTuner Sox2 SCs response to Shield treatment at 24 hr. Note that high Sox2 expression is only evident in the Shield-treated pTuner Sox2 SCs at 24 hr. N-cadherin (127 kDa), Sox2 (32 kDa) Vinculin (117 kDa), α-tubulin (50 kDa). (d) Diagram showing how nuclear alignment and cluster roundness were quantified. (e) Quantification of overall cluster roundness in Sox2 SCs treated with rSlit2 or (f) Slit2/3 siRNA (each dot represents a cluster from n = 3, red bars represent mean ± SEM). (g) Polar histograms representative of n = 3 experiments, showing the alignment of the nuclei within each cluster in control (blue) or Slit2/3 siRNA 1 and Slit2/3 siRNA2 (orange). p-values were calculated by t-test or one-way ANOVA as appropriate. **p<0.01, ***p<0.001.Figure 6—figure supplement 1—source data 1.Original gel for analysis of western blot showing pTuner empty vector SCs or pTuner Sox2 SCs response to Shield treatment at 24 hr in Figure 6—figure supplement 1 (N-cadherin).Figure 6—figure supplement 1—source data 2.Labelled gel for analysis of western blot showing pTuner empty vector SCs or pTuner Sox2 SCs response to Shield treatment at 24 hr in Figure 6—figure supplement 1c (N-cadherin).Figure 6—figure supplement 1—source data 3.Original gel for analysis of western blot showing pTuner empty vector SCs or pTuner Sox2 SCs response to Shield treatment at 24 hr in Figure 6—figure supplement 1c (Sox2).Figure 6—figure supplement 1—source data 4.Labelled gel for analysis of western blot showing pTuner empty vector SCs or pTuner Sox2 SCs response to Shield treatment at 24 hr in Figure 6—figure supplement 1c (Sox2).Figure 6—figure supplement 1—source data 5.Original gel for analysis of western blot showing loading controls for pTuner empty vector SCs or pTuner Sox2 SCs response to Shield treatment at 24 hr in Figure 6—figure supplement 1c (Vinculin and α-tubulin).Figure 6—figure supplement 1—source data 6.Labelled gel for analysis of western blot showing loading controls for pTuner empty vector SCs or pTuner Sox2 SCs response to Shield treatment at 24 hr in Figure 6—figure supplement 1 (Vinculin and α-tubulin).Figure 6—figure supplement 1—source data 7.Excel spreadsheet containing data used to generate graphs in Figure 6—figure supplement 1.Original gel for analysis of western blot showing pTuner empty vector SCs or pTuner Sox2 SCs response to Shield treatment at 24 hr in Figure 6—figure supplement 1 (N-cadherin).Labelled gel for analysis of western blot showing pTuner empty vector SCs or pTuner Sox2 SCs response to Shield treatment at 24 hr in Figure 6—figure supplement 1c (N-cadherin).Original gel for analysis of western blot showing pTuner empty vector SCs or pTuner Sox2 SCs response to Shield treatment at 24 hr in Figure 6—figure supplement 1c (Sox2).Labelled gel for analysis of western blot showing pTuner empty vector SCs or pTuner Sox2 SCs response to Shield treatment at 24 hr in Figure 6—figure supplement 1c (Sox2).Original gel for analysis of western blot showing loading controls for pTuner empty vector SCs or pTuner Sox2 SCs response to Shield treatment at 24 hr in Figure 6—figure supplement 1c (Vinculin and α-tubulin).Labelled gel for analysis of western blot showing loading controls for pTuner empty vector SCs or pTuner Sox2 SCs response to Shield treatment at 24 hr in Figure 6—figure supplement 1 (Vinculin and α-tubulin).Excel spreadsheet containing data used to generate graphs in Figure 6—figure supplement 1. less

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Impact of KIF4A on Cancer Stem Cells and EMT in Lung Cancer and Glioma.

In Cancers (Basel) on 22 November 2023 by Kahm, Y. J., Kim, I. G., et al.

Fig.4.H

Fig.4.H showing Western Blotting from the publication: Impact of KIF4A on Cancer Stem Cells and EMT in Lung Cancer and Glioma.

Regulatory role of KIF4A in cytokine expression within cancer stem cells. (A) The expression of KIF4A was suppressed using a cytokine array kit, and cytokines were screened. (B) RNA changes in cytokines were analyzed using qPCR. (C) After treating A549 cells with each neutralizing antibody, a sph... more

Regulatory role of KIF4A in cytokine expression within cancer stem cells. (A) The expression of KIF4A was suppressed using a cytokine array kit, and cytokines were screened. (B) RNA changes in cytokines were analyzed using qPCR. (C) After treating A549 cells with each neutralizing antibody, a sphere-forming assay was performed. (D) After each neutralizing antibody was treated with A549 cells, migration and invasion abilities were analyzed using a Boyden chamber. (E) After treatment with PAI-1 neutralizing antibody, the protein expression levels of KIF4A and PAI-1 were analyzed using WB. The numbers indicated beneath the blots represent the expression density of each protein, relative to β-actin. (F) After treatment with a PAI-1 neutralizing antibody, RNA expression levels of KIF4A and PAI-1 were analyzed using qPCR. (G) After treatment with a PAI-1 neutralizing antibody, expression levels of CSC marker proteins were analyzed by WB. The numbers indicated beneath the blots represent the expression density of each protein, relative to β-actin. (H) After treatment with a PAI-1 neutralizing antibody, expression levels of EMT marker proteins were analyzed by WB. The numbers indicated beneath the blots represent the expression density of each protein, relative to β-actin. Data are presented as the mean ± standard deviation of three independent experiments. Scale bar, 50 µm. * p ≤ 0.05. The original western blot figures can be found in Figure S7. less

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Impact of KIF4A on Cancer Stem Cells and EMT in Lung Cancer and Glioma.

In Cancers (Basel) on 22 November 2023 by Kahm, Y. J., Kim, I. G., et al.

Fig.5.D

Fig.5.D showing Western Blotting from the publication: Impact of KIF4A on Cancer Stem Cells and EMT in Lung Cancer and Glioma.

KIF4A regulates cancer stem cells, EMT, and cell behavior in glioma. (A) U87 cells were treated with siRNA, then cultured in sphere-forming media, and expression levels of CSC marker proteins (CD133, ALDH1A1, ALDH1A3) were compared and analyzed. The numbers indicated beneath the blots represent t... more

KIF4A regulates cancer stem cells, EMT, and cell behavior in glioma. (A) U87 cells were treated with siRNA, then cultured in sphere-forming media, and expression levels of CSC marker proteins (CD133, ALDH1A1, ALDH1A3) were compared and analyzed. The numbers indicated beneath the blots represent the expression density of each protein, relative to β-actin. (B) U87 cells were treated with siRNA and cultured in sphere-forming media, and the expression levels of CSC marker proteins were compared and analyzed using ICC. (C) The sphere-forming ability of U87 cells treated with siRNA was measured. (D) The expression of EMT marker proteins (E-cadherin, N-cadherin, and vimentin) in U87 cells treated with siRNA was compared and analyzed. The numbers indicated beneath the blots represent the expression density of each protein, relative to β-actin. (E) The expression of EMT marker proteins in siRNA-treated U87 cells was comparatively analyzed using ICC. (F) When the expression of KIF4A was inhibited in U87 cells, cell migration and invasion abilities were analyzed using a Boyden chamber. (G) After inhibiting the expression of KIF4A in U87 cells, the gene expression of PAI-1 was analyzed by qPCR. Data are presented as the mean ± standard deviation of three repeats. Scale bar, 50 µm. * p ≤ 0.05. The original western blot figures can be found in Figure S8. less

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Isthmin-1 (Ism1) modulates renal branching morphogenesis and mesenchyme condensation during early kidney development.

In Nat Commun on 25 April 2023 by Gao, G., Li, X., et al.

Fig.7.G

Fig.7.G showing Western Blotting from the publication: Isthmin-1 (Ism1) modulates renal branching morphogenesis and mesenchyme condensation during early kidney development.

Ism1 promotes mesenchyme cell aggregation through Integrin.a Immunostaining of N-cadherin and Six2 in E11.5 kidney sections from WT or Ism1−/− mice. Left panel scale bars, 50 μm. Right panel scale bars, 20 μm. b, c Bright field of hanging drop assay of CMMM-1 cells in the presence or absence of r... more

Ism1 promotes mesenchyme cell aggregation through Integrin.a Immunostaining of N-cadherin and Six2 in E11.5 kidney sections from WT or Ism1−/− mice. Left panel scale bars, 50 μm. Right panel scale bars, 20 μm. b, c Bright field of hanging drop assay of CMMM-1 cells in the presence or absence of rIsm1 for 24 h (b). Scale bars, 200 μm. Quantitative data are shown according to the bright field of hanging drop assay (c). Quantification of (b). Data from 3 independent experiments and each group contains at least 20 drops for analysis. d Crystal violet staining of aggregation assay in CMMM-1 cells. For the aggregation assay, CMMM-1 cells were pretreated with RGD peptide or saline and then subjected to rIsm1 or Npnt CM. CM conditioned medium. e Quantification of (d). Data from 3 independent experiments. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using two-way ANOVA, and corrections for multiple comparisons were performed with Sidak (adjusted p values are indicated in the figure). Source data are provided in Source Data file. f Immunostaining of N-cadherin in the aggregation assay of CMMM-1 cells. CMMM-1 cells treated with either Ism1 conditioned medium (CM) or rIsm1, in the presence or absence of AIIB2, the blocking antibody of Integrin β1. g CMMM-1 cells were serum starved with FreeStyle293 medium for 4 h followed by pretreatment with RGD peptide or saline. Cells were treated with either rIsm1 or Npnt conditioned medium for 30 min followed by Western blotting to detect N-cadherin protein and the phosphorylation of integrin downstream targets. h Immunostaining of active form of Integrin β1 and pFAK in E11.5 kidney sections from WT or Ism1−/− mice. Scale bars, 20 μm. i, j Phosphorylation of FAK at Y397 detected by Western Blotting (i) and Cdc42 expression detected by qPCR (j) in primary mesenchymal cells isolated from E11.5 Ism1−/− kidney rudiments, in the presence or absence of rIsm1 for 48 h. k Schematic model for Ism1-mediated Integrin regulation of cell-cell adhesion in renal mesenchyme. less

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RAB35 is required for murine hippocampal development and functions by regulating neuronal cell distribution.

In Commun Biol on 21 April 2023 by Maejima, I., Hara, T., et al.

Fig.5.I

Fig.5.I showing Western Blotting in a Mus musculus (House mouse) sample from the publication: RAB35 is required for murine hippocampal development and functions by regulating neuronal cell distribution.

Quantitative proteomics in Rab35 cKO P0 hippocampus.a Volcano plot of the TMT-based quantitative proteomes identifying the dysregulated proteins in Rab35 cKO hippocampus in comparison with the control hippocampus (n = 5 mice per genotype). b Number of proteins identified as significantly dysregul... more

Quantitative proteomics in Rab35 cKO P0 hippocampus.a Volcano plot of the TMT-based quantitative proteomes identifying the dysregulated proteins in Rab35 cKO hippocampus in comparison with the control hippocampus (n = 5 mice per genotype). b Number of proteins identified as significantly dysregulated and as either membrane traffic-related or neuronal migration-related. c, d Western blot analysis of control and Rab35 cKO P0 hippocampi using anti-contactin-2, anti-CHL1, and anti-actin antibodies. e, f Quantification of contactin-2 (c) and CHL1 (d) protein levels in control and Rab35 cKO P0 hippocampi. Band intensities of the indicated proteins were normalized to those of actin (n = 9 mice per genotype). Unpaired Student’s t-test; e, p = 0.0153; f, p = 0.0095. g, h Levels of contactin-2 (g) and CHL1 (h) were quantified by targeted MS using the PRM method (n = 5 mice per genotype). Unpaired Student’s t-test; gp = 0.0053; hp = 0.0229. i Western blot analysis of control and Rab35 cKO P0 hippocampus using anti-N-cadherin and anti-actin antibodies. j Quantification of N-cadherin protein levels in the control and Rab35 cKO P0 hippocampus (n = 9 mice per genotypes). Unpaired Student’s t-test, p = 0.9020. k Representative images of DIV 2 hippocampal primary neurons stained for contactin-2 (green), rhodamine-phalloidin (magenta) and DAPI (blue). Scale bar, 20 μm. l Quantification of contactin-2 intensity at the somatic plasma membrane in control (n = 4) and Rab35-deficient (n = 4) cells. Thirty neurons from four different cultures per genotype were analyzed. Mann–Whitney U-test, p = 0.0286. Data represent the mean ± SEM; n.s. not significant (p > 0.05); *p < 0.05; **p < 0.01. 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.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.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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