Arhgap5

All experiments’ animal procedures were approved by the Animal Care Committee of Southern Medical University. For tumor metastatic studies in vivo, DU145 cells (1 × 10⁷ cells per mouse) transfected with NC or sh-has_circ_0003258 were injected through lateral tail vein of BALB/c nude male mice (n = 5 for each group). Lung tissues were collected and examined for metastasis. After 40 days, the tumors in vivo were evaluated by fluorescence imaging using the IVIS (PerkinElmer, USA). The presence of cancer cells was confirmed by H&E (hematoxylin and eosin) staining. At the same time, immunohistochemistry (IHC) staining was conducted using antibodies against ARHGAP5 (Abcam, USA, 1:100) and HDAC4 (Abcam, USA, 1:100).

Equal amounts of proteins were separated by 10% SDS-PAGE gels and blotted onto nitrocellulose membranes. The blots were incubated with primary antibodies against ARHGAP5 (1:1000; Abcam, Cambridge, USA) and β-Actin (1:5000; Santa Cruz Biotech, Santa Cruz, CA, USA) at 4°C overnight. The membranes were incubated for 2 h with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibody (1:5000; Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. Anti-β-Actin antibody (Santa Cruz Biotechnology, CA, USA) was used as a loading control.

Immunoblotting and IHC analysis were conducted with standard procedures, as previously described 15 (link), 16 (link). Blotting membranes were stripped and reprobed with anti-β-Actin antibody as a loading control. The degree of immunostaining of formalin-fixed, paraffin-embedded sections was reviewed and scored independently by two pathologists based on both the proportion of positively stained tumor cells and the intensity of the staining. The following antibodies were used for immunoblotting or IHC analysis: ARHGAP5 (#2562, WB, 1:1000), Vimentin (#5741; WB, 1:1000; IHC, 1:200), N-cadherin (#13116; WB, 1:1000; IHC, 1:100), E-cadherin (#14472; WB, 1:1000; IHC, 1:100), RhoA (#2171; WB, 1:1000), CREB1 (#9197; WB, 1:1000; IHC, 1:6000), β-Actin (#3700; WB, 1:3000) (all from Cell Signaling, Beverly, USA) and ARHGAP5 (#ab199160; IHC, 1:100) (Abcam, Cambridge, MA, USA).