P21waf1

Immunoblotting was performed to assess protein expression in response to the different treatments, as previously described [31 (link)]. Briefly, control and treated HuREC were lysed using radioimmunoprecipitation assay buffer lysis buffer containing 1% phosphatase and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Fifty micrograms of each protein sample was electrophoresed on SDS-PAGE and transferred onto a Polyvinylidene difluoride (PVDF) membrane. The membrane was then blocked using 5% skim milk and incubated with the following primary antibodies: SIRT1 (1:1000; Cell Signaling) and p16^INK4a, TrxR2 (1:1000; Abcam, Cambridge, MA), p21^Waf1 and SOD2 (1:500; Santa Cruz Biotech, Dallas, TX, USA), and corresponding secondary horseradish-conjugated antibodies (GE Healthcare, Pittsburg, PA, USA). After immunoblotting, the membranes were stripped using stripping buffer (Bio-Rad) and re-probed with anti-β-actin antibody (1:3000; Sigma-Aldrich, St. Louis, MO, USA). Chemiluminescence-based assay was used for band detection (ThermoFisher, Waltham, MA, USA). Scanned images of blots were used to quantify protein expression using NIH ImageJ software (http://rsb.info.nih.gov/ij/).

Hydrangenol was purchased from Coresciences Co. (Seoul, South Korea). Antibodies against CDK2, CDK4, cyclin D1, cyclin E, p21^WAF1, p27^KIP1, p53, and GAPDH were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Total ERK, p38, JNK, AKT, phospho-ERK, phospho-p38, phospho-JNK, and phospho-AKT antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The polyclonal MMP-9 antibody was purchased from Chemicon (Temecula, CA, USA). The nuclear extract kit and the electrophoretic mobility shift assay (EMSA) gel shift kit were obtained from Panomics (Fremont, CA, USA). The p38-specific inhibitor SB203585 was purchased from Calbiochem (San Diego, CA, USA).

Cells were treated with adriamycin (Sigma-Aldrich) or etoposide (Sigma-Aldrich), and SDS protein samples were then prepared by directly lysing the cells in SDS sample buffer (Elpisbiotech. Inc., Daejeon, Korea) after washing with ice-cold phosphate-buffered saline (PBS).
Western blots were conducted using antibodies against MDM2 (Santa Cruz Biotechnology, Inc., #sc-965), p21^WAF1 (Santa Cruz Biotechnology, Inc., #sc-6246), p53 (Santa Cruz Biotechnology, Inc., #sc-126), p16^INK4A (Santa Cruz Biotechnology, Inc., #sc-1661), p14^ARF (Cell Signaling #74560), and GAPDH (Cell Signaling #2118).