Fig 1: ARHGAP5-AS1 recruited METTL3 for m6A modification of ARHGAP5 mRNA.a RMBase v2.0 was used to screen m6A modification sites of ARHGAP5 mRNA. Raw signal value for peak calling was derived from MeRIP-seq data. The scale ruler indicated normalized peak value. b The effect of ARHGAP5-AS1 downregulation on m6A modification of ARHGAP5 mRNA was assessed by RIP assay. c M6A methyltransferase levels in resistant cells after knocking down expression of ARHGAP5-AS1 were detected using Western Blotting. d RNA pull down assay using ARHGAP5-AS1 biotin-labeled probes was performed to analyze the interaction between ARHGAP5-AS1 and m6A methyltransferase. e, f The binding capacity of ARHGAP5-AS1 (e) or ARHGAP5 mRNA (f) with METTL3 were determined by RIP assay. g Interaction of ARHGAP5 mRNA with METTL3 after ARHGAP5-AS1 downregulation was assessed by RIP assay. h Effect of ARHGAP5-AS1 overexpression on m6A modification and METTL3 interaction of ARHGAP5 were detected by RIP. i The effect of METTL3 downregulation on m6A modification of ARHGAP5 mRNA was assessed by RIP assay
Fig 2: ARHGAP5-AS1 stabilized ARHGAP5 mRNA in the cytoplasm.a The subcellular localization of ARHGAP5-AS1 in SGC7901/SGC-R cells were determined by qRT-PCR after extracting RNAs from cytoplasmic and nuclear extracts. GAPDH/DANCR and U6/Malat1 were used as the cytoplasmic and nuclear RNA controls, respectively. b The separation of cytoplasmic and nuclear fractions was validated by Western Blotting. GAPDH and H3 were served as cytoplasmic and nuclear protein controls, respectively. c Biotin pull down assay was used to verify the interaction of ARHGAP5-AS1 with ARHGAP5 mRNA. NC meaned no probe; both sense and antisense probe were biotin labeled. The enrichment of ARHGAP5 mRNA was measured by qRT-PCR. The change of ARHGAP5 half-life after knocking down ARHGAP5-AS1 levels in SGC-R and BGC-R cells (d) or overexpressing ARHGAP5-AS1 in SGC7901 and BGC823 cells (e) were analyzed as in Fig. 2f. f The effect of ARHGAP5-AS1 downregulation on the interaction of ARHGAP5 mRNA with HuR protein was analyzed by RIP assay. g Change of ARHGAP5 half-life after knocking down HuR levels in SGC-R and BGC-R cells. h ARHGAP5 protein expression after knocking down HuR was determined by Western Blotting
Fig 3: Identification of expression profile and prognosis related to the Rho-GTPase-activating proteins (ARHGAP) family gene in bladder cancer (BCa). (A) ARHGAP mutation level in BCa according to the analysis of cBioPortal. (B) Expression level of ARHGAP5, ARHGAP17, and ARHGAP24 in several tumor and normal tissues and Expression, OS and DFS of ARHGAP5 in BCa.
Fig 4: Knockdown of ARHGAP5-, ARHGAP17-, and ARHGAP24-suppressed BC cell proliferation in vivo and in vitro. (A) System analysis of expression of ARHGAP family gene in BCa cancer cell lines in the CCLE database. (B) ARHGAP5, ARHGAP17, and ARHGAP24 expression in seven pairs of low-grade BCa and seven pairs of high-grade BCa; GAPDH serves as a loading control. (C) ARHGAP5, ARHGAP17, and ARHGAP24 expression in four bladder transitional cell carcinoma cell lines (RT4, UM-UC-3, T24, and 5637) compared with immortalized epithelium cell line (SV-HUC-1). (D,E) Cloning formation of T24, UMUC-3, and ARHGAP5, ARHGAP17, and ARHGAP24 knocking down T24, UMUC-3 (***p < 0.001 versus shNC). (F,G) 5-Ethynyl-2′-deoxyuridine (EdU) assay of T24 and shARHGAP5-, ARHGAP17-, and ARHGAP24-transfected T24 (***p < 0.001 versus shNC). (H) Tumor growth size of subcutaneous T24 tumors transfected with shNC or shARHGAP5, ARHGAP17, and ARHGAP24 21 days after tumor implantation. (I) Tumor growth curve of subcutaneous T24 tumors transfected with shNC or shARHGAP5, ARHGAP17, and ARHGAP24 in 21 days of tumor implantation. (J) Average weights of tumors from shNC and shARHGAP5, ARHGAP17, and ARHGAP24 are shown in the histogram. Error bars indicate SD.
Fig 5: Immunosuppressive tumor microenvironment was associated with ARHGAP family expression. (A,B) Representative immunohistochemistry staining of the ARHGAP family [(A); ARHGAP5, ARHGAP17, and ARHGAP24] and immune cells [(B); CD8 + T cells, CD4 + T cells, and Treg cells]. (C) Comparison of CD8 + T cell, CD4 + T cell, and Treg cell infiltration in bladder cancer according to ARHGAP5, ARHGAP17, and ARHGAP24 high/low groups. (D,E) Comparison of effector molecules [(D); IFN-γ, GZMB, and perforin] and immune checkpoint [(E); PD-1, Tim-3, and LAG-3] in bladder cancer according to ARHGAP5, ARHGAP17, and ARHGAP24 high/low groups.
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