Rhoa 2117

MTT was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Four synthetic, chemically modified short double-stranded RNA oligonucleotides (miR-650 mimics, mimics negative control, miR-650 inhibitor and inhibitor negative control) were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Primary antibodies against p38 (#9212), phosphorylated (p)-p38 (#4511), ERK1/2 (#4695), p-ERK1/2 (#4370) and RhoA (#2117) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA); active Rac1 (#26903) and active RhoA (#26904) from NewEast Biosciences (King of Prussia, PA, USA); E-cadherin (ab1416), β-catenin (ab32572), Rac1 (ab33186) and FITC-conjugated anti-rat (ab6717)/anti-mouse IgG (ab6785) secondary antibodies from Abcam (Cambridge, UK); α-smooth muscle actin (α-SMA, 55135–1-AP) from ProteinTech Group, Inc. (Chicago, IL, USA); vimentin (BM0135) from Wuhan Boster Biological Technology, Ltd. (Wuhan, China); and β-actin (sc-58673) and HRP conjugated goat anti-rabbit/goat anti-mouse secondary antibodies (sc-2004/sc-2005) from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The primary antibody ING4 (#40-7700) and the fluorescent dye Alexa Fluor 546 conjugated phalloidin (A22283, 1:5,000) were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Whole-cell lysates were prepared using RIPA lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 mmol/L EDTA), supplemented with complete EDTA-free protease inhibitor mixtures (4693116001, Roche, Indianapolis, IN, USA) and phosphatase inhibitors (04906845001, Roche). Protein samples were separated on SDS-polyacrylamide gels. For Western blot, separated proteins were transferred electrophoretically onto nitrocellulose membranes (ISEQ00010, Millipore, Sigma). The membranes were incubated overnight at 4 °C with antibodies against TIMP1 (16644-1-AP) from Proteintech (Rosemont, IL, USA); integrin β1 (sc-59829) and CD63 (sc-5275) from Santa Cruz Biotechnology (Dallas, TX, USA); VE-cadherin (ab33168), pFAK pY397 (ab81298) and total FAK (ab40794) from Abcam (Cambridge, UK); ZO-1 (40–2200), occludin (710192) and claudin-5 (35–2500) from Thermo Fisher Scientific; RhoA (2117) and Na/K ATPase (3010) from Cell Signaling Technology (Danvers, MA, USA). Following anti-mouse or anti-rabbit IgG-HRP (Thermo Fisher Scientific) were used as secondary antibodies for 1 h at room temperature, signals were detected using ImageQuant LAS 4000 (GE Healthcare).