Ab54835

Total protein was extracted using RIPA lysis buffer (Beyotime, Guangzhou, China), and the protein concentration was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Equal amounts of protein were separated by SDS‐PAGE, then transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). Membranes were subsequently incubated with primary antibodies against HNRNPC (1: 1000, Santa Cruz Biotechnology, sc‐32308, Dallas, TX, USA), RhoA (1: 3000, Abcam, ab54835, Cambridge, UK), ROCK2 (1: 1000, Abcam, ab71598), p‐γ‐H2AX (1: 1000, S139; Abcam, ab26350), α‐SMA (1: 500, Abcam, ab7817), FAP (1: 500, Santa Cruz Biotechnology, sc‐65398) and YAP (1: 1000, Abcam, ab56701), followed by incubation with secondary antibodies bound to horseradish peroxidase. Immunoreactivity was visualized using an ECL Western Blot system (Millipore, Burlington, MA, USA). β‐actin was used as an internal loading control.

The following materials were used: recombinant human transforming growth factor (TGF)‐β1, monocyte chemoattractant protein (MCP)‐1 and MCP‐1 ELISA kits with pre‐coated plates from R&D Systems; dexamethasone (DEX) from Sigma‐Aldrich; the Rho‐associated kinase (ROCK) inhibitor Y‐27632 from EMD Millipore; the murine monoclonal anti‐RhoA antibody ab54835 from Abcam; rhodamine phalloidin from Invitrogen; the Rho Activation Assay Biochem Kit from Cytoskeleton; goat anti‐mouse antibody (VectaFluor R.T.U. DyLight 488 anti‐mouse); and 4′,6‐diamidino‐2‐phenylindole (DAPI) (VECTASHIELD^® with DAPI) from Vector Laboratories.

Confluent HeLa cells grown with DMEM supplemented with 5% fetal bovine serum were intoxicated with cell-free supernatants from 24 h cultures in TYT. When a CPE was observed, the cells were lysed with 2% SDS and 20 µg of their proteins were resolved SDS-PAGE in 10% gels. The resulting gels were electro-transferred to PVDF membranes (Macherey-Nagel) that were probed with an anti-Rho monoclonal antibody that fails to recognize the glycosylated form of this small GTPase (ab54835, Abcam). Unintoxicated HeLa cells, as well as cells intoxicated with supernatant from a NAP1/027 strain, were assayed as controls.

Sections (3‐µm thick) were deparaffinized, rehydrated and endogenous peroxidase activity was blocked via incubation with 3% hydrogen peroxide for 15 min at room temperature. Antigens were retrieved in a sodium citrate buffer (pH 6.0) for 3 min using a pressure cooker. Sections were blocked with 5% bovine serum albumin (BSA) for 30 min at room temperature to remove non‐specific binding. Then, sections were incubated with primary antibodies against HNRNPC (1: 200, Santa Cruz Biotechnology, sc‐32308, Dallas, TX, USA) and RhoA (1: 200, Abcam, ab54835, Cambridge, UK) overnight at 4°C. Next, sections were incubated with the secondary antibody for 1 h at 37°C. Finally, a chromogenic reaction was developed with DAB, and sections were counterstained with haematoxylin.

Immunohistochemistry detection of Rho A, ROCK1, ROCK2, GLUT1, HK2, PDK1, PFK1, LDHA, OPN and RUNX2 in human aortic valve leaflets was performed. 4% PFA-fixed paraffin sections (5μm thick) were deparaffinized using dewaxed solution and hydrated using decreasing concentration of ethyl alcohol (100%, 90%, 80%, 70%, 60%), and then incubated at 65 °C for 20 min in antigen unmasking solution using PT Module-Lab Vision (Thermo Fisher Scientific) for antigenic retrieval. Following several washes (3 x 5 min) in PBS, paraffin sections were blocked for 30 min using 5% bovine serum albumin (BSA). After blocking, primary antibodies against Rho A (abcam, ab54835), ROCK1 (abcam, ab97592), ROCK2 (abcam, ab125025), GLUT1 (abcam, ab115730), HK2(abcam, ab209847), PDK1 (abcam, ab202468), PFK1 (CST, #8164), LDHA (abcam, ab52488), OPN (abcam, ab63856) and RUNX2 (abcam, ab192256) were diluted at optimized concentrations in 1% BSA and incubated with the sections in a wet box overnight at 4 °C. Subsequently, appropriate secondary antibodies were applied for 30 min at room temperature. Following secondary antibody incubation, the positive staining was detected using an UltraSensitive SP IHC Kit and DAB Kit, and hematoxylin staining was used for nuclear counterstaining. Images were visualized using a microscope (CKX53, Olympus), and Image J software was applied for data quantification.