Cell culture supplements

HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS, Bodinco DV), 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. BHK-21 cells were cultured in Glasgow’s minimal essential medium (GMEM, Gibco) supplemented with 8% (v/v) FCS, 10% (v/v) tryptose phosphate broth (TPB), 10 mM HEPES pH 7.4, 100 U/ml penicillin, and 100 mg/ml streptomycin. MARC-145 were cultured in DMEM supplemented with 8% (v/v) FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. DMEM and cell culture supplements were obtained from Lonza.
Primary antibodies that were used were mouse-α-β-actin (A5316, Sigma-Aldrich), rabbit-α-GFP [9 (link)], mouse-α-V5 (37–7500, Invitrogen), mouse-α-myc (9E10, Roche), mouse-α-HA (ab18181, Abcam), and mouse-α-FLAG (F1804, Sigma-Aldrich). As secondary antibodies, goat-α-mouse-HRP (P0447, Dako), swine-α-rabbit-HRP (P0217, Dako), goat-α-mouse-AlexaFluor488 (A11001, Life Technologies), and donkey-α-rabbit-Cy3 (711-165-152, Jackson). The antibody α-ORF7 (P3-05-A27, MSD Animal Health) was obtained using mouse antiserum. The antibody α-nsp2/3 LV was obtained using rabbit antiserum [13 (link)].

Primary rat aortic VSMCs, growth media, and cell culture supplements were purchased from Lonza (Basel, Switzerland). Fetal bovine serum for cell culture was supplied by Gibco Life Technologies (Darmstadt, Germany), and PDGF-BB was obtained from Bachem (Weilheim, Germany). Rat phaeochromocytoma cells (PC-12) cells were a kind gift from Prof. Harald Sitte (Medical University of Vienna). Horse serum and NGF were from Fisher Scientific (Vienna, Austria). The A10 cell line was purchased from LGC Standards (Teddington, UK). HEK293T cells were purchased from Merck (Darmstadt, Germany). Sprague Dawley rats (Charles River, MA, USA) were used for harvesting dorsal root ganglia. Biological material was only obtained after sacrificing the animal, which does not require explicit approval by an ethics committee under local jurisdiction. Capsaicin and BCTC were obtained from R&D Systems (Abingdon, UK) and Cayman Chemical (Ann Arbor, MI, USA), respectively. All other chemicals were purchased from Sigma-Aldrich (MO, USA).

Antibodies against human and mouse Runx2 were purchased from Cell Signaling, Inc. (Beverly, MA, USA). Antibodies against both human and mouse Osx were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). TGF-β1 and antibodies against ALP were purchased from R&D System (Minneapolis, MN, USA). Specific siRNAs for human Osx and Runx2 and lipofectamine 2000 were purchased from Life Technologies, Inc. (Grand Island, NY, USA). HiPerFect® transfection reagent, EndoFree Plasmid Maxi Kit, control miR, miR-204 mimics, and antagomirs were all obtained from Qiagen (Valencia, CA, USA). Lentivirus vector expressing miR-204 mimic or miR-204 antagomir, as well as Block-it lentiviral pol II miR RNAi expression plasmids were obtained from Invitrogen (Grand Island, NY, USA). TransDux transduction reagent was obtained from System Biosciences (Mountain View, CA, USA). Medium 199 and cell culture supplements were purchased from Lonza (Walkersville, MD, USA). All other chemicals and reagents were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA).