Antimycotic solution

The RBE and SSP25 cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The RBE and SSP25 were grown in RPMI-1640 basal medium with a supplementation of 10% fetal bovine serum and 1 × antibiotics- and 1 × antimycotic solution (Gibco). Cells were maintained in monolayer culture at 37°C in humidified air with 5% CO₂ in these growth media.

293 T cells (ATCC CRL-11268) and MDA-MB-231 cells (ATCC HTB-26) were cultured in Dulbecco’s modified Eagle’s medium (Corning, Lowell, MA) with 10% fetal bovine serum (Corning), 1% penicillin/streptomycin (Gibco, Carlsbad, CA), and 1% antimycotic solution (Gibco). The cells were incubated at 37°C with 5% CO₂ in a humidified atmosphere. Antibodies to Flag, caspase-3, Bcl-xL, p-Smad2 (Ser465/467), and Smad2 were purchased from Cell Signaling Technology (Beverly, MA). Antibodies to CKB, actin, β-tubulin, LDHA, LDHB, and Smad7 were from Santa Cruz Biotechnology. Sno antibody was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Doxorubicin was purchased from Calbiochem (Darmstadt, Germany). Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN). TGF-β type I receptor inhibitor (SB431542) was purchased from TOCRIS (Bristol, UK). 3TP-Lux and pCAGA12-Luc were kindly provided by Jae Youn Yi, Ph.D. (Korea Institute of Radiation and Medical Sciences, Seoul, Korea). NF-κB-Luc (Ju et al. 2011 (link)) and FHRE-Luc (Bianco et al. 2003 (link)) plasmids were previously described.

Dulbecco’s modified eagle medium, StemPro osteogenesis differentiation medium, StemPro chondrogenesis differentiation medium, fetal bovine serum, Hank’s balanced salt solution, antibiotic and antimycotic solution were products of Invitrogen (Carlsbad, CA). Human foreskin fibroblasts were obtained from American Type Culture Collection (CRL-2429, Manassas, VA). PC was synthesized according to the procedures described [23 (link)]. In all experiments, 1mm PC was used because it has been used in previous studies and shows no toxic effect on cells [15 (link), 19 (link)].

DNA oligos were purchased from Integrated DNA Technologies (IDT, India) MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide), trypsin-EDTA, propidium iodide, DMSO and protease inhibitor cocktail, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell growth media DMEM and RPMI were purchased from Sigma-Aldrich (St. Louis, MO, USA), FBS antibiotic and antimycotic solution were purchased from Invitrogen (Whitefield, Bangalore). Fluorescein Isothiocyanate (FITC)-labelled Annexin V (Annexin V-FITC) kit was obtained from BD Biosciences (San Diego, CA). Antibodies against human poly (ADP-ribose) polymerase (PARP), γH2AX, cleaved Caspase 3, Caspase 9, P⁵³, and MDM2 were purchased from Cell Signalling Technology (CST, Beverly, MA). PCNA and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO)