Journal: PLoS ONE

Article Title: MicroRNA-19a and microRNA-19b promote the malignancy of clear cell renal cell carcinoma through targeting the tumor suppressor RhoB

doi: 10.1371/journal.pone.0192790

Figure Lengend Snippet: (A) Flow cytometry results of Annexin-V5 analysis in 786-O cells transfected with miR-19a and miR-19b inhibitors. An inhibitor control was included as a negative control. Graphs showing changes of the apoptotic percentage of the cells (left); quantification of relative apoptosis (right). The data represent the means ± S.D. from three independent experiments performed in triplicate. *Significant differences from control oligo-transfected cells (P<0.05) (B) Western blot results showing the effect of miR-19a and miR-19b inhibitors on the expression of cleaved caspase-9 in 786-O cells. (C) Potential mechanism showing the effect of the negative regulation of RhoB by miR-19a/b on the proliferation, migration, invasion and apoptosis of ccRCC.

Article Snippet: The anti-RhoB antibody was obtained from Proteintech (Chicago, USA), anti-cleaved caspase-9 antibody was purchased from Epitomics (Burlingame, CA), and anti-β-actin antibody was obtained from ZSGB-BIO (Beijing, China).

Techniques: Flow Cytometry, Transfection, Control, Negative Control, Western Blot, Expressing, Migration

Journal: The FASEB Journal

Article Title: Cytotoxic necrotizing factor 1 promotes bladder cancer angiogenesis through activating RhoC

doi: 10.1096/fj.201903266rr

Figure Lengend Snippet: FIGURE 3 CNF1 modulates the expression of HIF1α and the secretion of VEGF by activating RhoC in bladder cancer cells. A, T24 cells were transfected with a scrambled control siRNA or those targeting respective Rho GTPase siRNAs for 24 hours, followed by stimulation with PBS or CNF1 (3 nmol/L) for another 24 hours under hypoxic conditions, and HIF1α expression was examined by western blotting. B, T24 cells were incubated with PBS or CNF1 for 3 to 36 hours and mobility-shifting was examined by electrophoresis. C, Western blotting analysis of activated RhoC in T24 cells treated with recombinant CNF1 protein (3 nmol/L) or PBS for 24 hours after immunoprecipitation with GTP pull-down assays using anti-RhoC antibody. D, Western blotting analysis of T24 cells transfected with vector, wild-type RhoC, or constitutively active RhoC (Q63E) under hypoxic conditions. E, T24 cells were transfected with vector, wild-type RhoC, or Q63E for 48 hours, and VEGF secretion in the culture medium was examined by ELISA (n = 3, three independent experiments). F, T24 cells were transfected with scrambled or RhoC siRNA for 24 hours followed by stimulation with CNF1 protein (3 nmol/L) or PBS for 24 hours, and VEGF secretion in culture medium was examined by ELISA (n = 3, three independent experiments). Data are the mean ± SD. *P < .05, **P < .01; one-way ANOVA (E, F)

Article Snippet: Sections (5 μm) were used for H&E and Immunohistochemistry (IHC) staining for RhoC (1:400, 10632-1-AP; Proteintech), HSP90α (1:300, 13171-1-AP; Proteintech), HIF1α (1:500, ab51608; Abcam), VEGF (1:300, 19003-1-AP; Proteintech).

Techniques: Expressing, Transfection, Control, Western Blot, Incubation, Electrophoresis, Recombinant, Immunoprecipitation, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: Cytotoxic necrotizing factor 1 promotes bladder cancer angiogenesis through activating RhoC

doi: 10.1096/fj.201903266rr

Figure Lengend Snippet: FIGURE 4 CNF1-induced RhoC activation modulates the HIF1α stabilization by upregulating HSF1- HSP90α interaction. A, Heatmap of the HIF1α degradation-related gene expression levels detected by RNA-seq in T24 cells stably expressing the RhoC constitutively active mutant Q63E or vector under hypoxic condition. B, qRT-PCR confirmation of the upregulated genes expression profile under hypoxic condition (n = 3, three independent experiments). C, Western blotting analysis of T24 cells transduced with vector and Q63E under hypoxic condition. D, Western blotting analysis of T24 cells treated with 1 nmol/L CNF1. E, T24 cells were transfected with combinations of MYC-HIF1α, HA-HSP90α, and FLAG-RhoC-Q63E under normoxic or hypoxic condition. Protein extracts from the transfected cells were subjected to IP with antibody against MYC and analyzed by immunoblotting with the indicated antibodies. F, Analysis of phosphorylated HSF1, total HSF1, HSP90α, and HIF1α in transduced T24 cells treated with the HSF1 inhibitor KRIBB11 (10 μmol/L), or with DMSO as the control under hypoxic condition. G and H, Western blotting analysis of transduced T24 cells (G) and 1 nmol/L CNF1 treated T24 cells (H) subject to siRNAs targeting HSF1 and scrambled non-targeting control siRNA under hypoxic conditions. I, T24 cells were transfected with scrambled or HSF1 siRNA for 24 hours followed by stimulation with CNF1 (3 nmol/L) or PBS for 24 hours under hypoxic condition, and VEGF secretion in culture medium was examined by ELISA (n = 3, three independent experiments). Data are the mean ± SD. **P < .01; one-way ANOVA (B, I)

Article Snippet: Sections (5 μm) were used for H&E and Immunohistochemistry (IHC) staining for RhoC (1:400, 10632-1-AP; Proteintech), HSP90α (1:300, 13171-1-AP; Proteintech), HIF1α (1:500, ab51608; Abcam), VEGF (1:300, 19003-1-AP; Proteintech).

Techniques: Activation Assay, Gene Expression, RNA Sequencing, Stable Transfection, Expressing, Mutagenesis, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Transduction, Transfection, Control, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: Cytotoxic necrotizing factor 1 promotes bladder cancer angiogenesis through activating RhoC

doi: 10.1096/fj.201903266rr

Figure Lengend Snippet: FIGURE 5 Active RhoC promotes the tumor-associated angiogenesis of bladder cancer in vivo. A, Morphological images of tumor xenografts resected from nude mice injected with T24 cells transduced with vector (VEC) or constitutively active mutant of RhoC (Q63E) after 5 weeks in each group. B, Tumor growth in nude mice with Q63E or VEC T24 cells subcutaneously injected into their flanks (n = 9, two independent experiments). Tumor volumes were determined by direct measurement using a caliper and calculated using the formula: (widest diameter × smallest diameter2)/2. Tumor volume (C) and weight (D) of xenograft nude mice injected with Q63E or VEC T24 cells in the xenograft model (n = 9, two independent experiments). E-H, Immunohistochemical analysis of RhoC, HSP90α, HIF1α, or VEGF expression in murine tumors. I, Immunofluorescence analysis of CD31+ blood vessels in murine tumors (n = 3, three independent experiments each with multiple fields). Data are the mean ± SD. *P < .05, **P < .01; non-parametric Mann-Whitney test (B, C, D and I). Scale bar = 50 μm (E-H) or 100 μm (I)

Article Snippet: Sections (5 μm) were used for H&E and Immunohistochemistry (IHC) staining for RhoC (1:400, 10632-1-AP; Proteintech), HSP90α (1:300, 13171-1-AP; Proteintech), HIF1α (1:500, ab51608; Abcam), VEGF (1:300, 19003-1-AP; Proteintech).

Techniques: In Vivo, Injection, Transduction, Plasmid Preparation, Mutagenesis, Immunohistochemical staining, Expressing, Immunofluorescence, MANN-WHITNEY

Journal: JACC: Basic to Translational Science

Article Title: Ubiquitin Specific Protease 9X Regulates the Activation of ARK5 and Promotes Progression of Fibrotic Remodeling

doi: 10.1016/j.jacbts.2025.02.014

Figure Lengend Snippet: ARK5 Facilitates TGF-β1–Induced Myofibroblast Transformation via Activating RhoA/MRTF-A Pathway (A) Fibroblasts were infected with USP9X siRNA, then subjected to TGF-β1 stimulation. Total RNA samples were collected and sequenced. The most significantly altered KEGG pathways were displayed. (B) RhoA activity was assessed using western blot analysis after transfecting CFs with ARK5 siRNA or siRNA-control. (C) CFs were infected with siRNA-control, siRNA-USP9X, Ad-GFP, or Ad-ARK5, and then tested for RhoA activity. (D) MRTF-A protein levels in the nuclear and cytosolic fractions were detected by western blot analysis. (E) Immunofluorescence staining was performed to detect the intensity of MRTF-A. Scale bar: 50 μm. (F) CFs were infected with siRNA-control, siRNA-USP9X, Ad-GFP, or Ad-ARK5, and then tested for MRTF-A protein levels. (G) Representative immunofluorescence staining of MRTF-A for each group. Scale bar: 50 μm. n = 3-5, Values represent the mean ± SEM. One-way analysis of variance followed by Tukey’s post hoc test was used for statistical analysis. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Abbreviations as in , , and .

Article Snippet: Antibodies against USP9X (55054-1-AP), α-SMA (14395-1-AP), mechanosensitive transcription factor myocardin-related transcription factor-A (MRTF-A) (21166-1-AP), Ubiquitin (10201-2-AP), glyceraldehyde 3-phosphate dehydrogenase (60004-1-Ig), PCNA (10205-2-AP), and RhoA (10749-1-AP) were purchased from Proteintech.

Techniques: Transformation Assay, Infection, Activity Assay, Western Blot, Control, Immunofluorescence, Staining