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: Pharmaceutics

Article Title: A Nanoliposome Platform Co-Delivery of Hydroxypinacolone Retinoate and Carnosine for Enhanced Epidermal/Dermal Delivery and Multi-Functional Anti-Aging Efficacy

doi: 10.3390/pharmaceutics18040454

Figure Lengend Snippet: In vitro skin permeability of Free-HC and HC-NLPs. ( A ) Cumulative skin permeation and ( B ) retention of CA over 24 h. ( C ) Cumulative skin retention of HPR. && p < 0.01 vs. Free-HC. Mean ± SD, n = 3. ( D ) Fluorescence microscopy images showing the penetration of RhoB (red) in porcine skin over 4 h. Scale bar: 200 μm.

Article Snippet: 4′,6-Diamidino-2-phenylindole (DAPI) and Ras Homolog Family Member B (RhoB) were purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA).

Techniques: In Vitro, Permeability, Fluorescence, Microscopy

Journal: Pharmaceutics

Article Title: A Nanoliposome Platform Co-Delivery of Hydroxypinacolone Retinoate and Carnosine for Enhanced Epidermal/Dermal Delivery and Multi-Functional Anti-Aging Efficacy

doi: 10.3390/pharmaceutics18040454

Figure Lengend Snippet: Cellular uptake of Free-RhoB and RhoB-NLPs in HDF cells. CLSM images showing intracellular RhoB fluorescence after 2 h and 4 h incubation. Scale bar: 50 μm.

Article Snippet: 4′,6-Diamidino-2-phenylindole (DAPI) and Ras Homolog Family Member B (RhoB) were purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA).

Techniques: Fluorescence, Incubation