Proliferating cell nuclear antigen (pcna)

If necessary, the dermis was separated from the epidermis manually using forceps. After cryogenic grinding, tissues were lysed with RIPA buffer containing a protease inhibitor cocktail (Roche, Bâle, Switzerland). Protein extracts (15µg) were then loaded onto a 12% reducing SDS-PAGE gel. After transfer on a nitrocellulose membrane (Bio-Rad, CA, USA), and blocking in TBS containing 0.1% Tween-10 and 5% nonfat milk for 1 hour, blots were incubated overnight at 4 °C with mouse primary antibodies: PCNA (Biolegend, CA, USA, 1:1000), or with rabbit primary antibodies: CD45 (Abcam, Cambridge, England, 1:1000), STAT1, and p-STAT1, (all Cell Signaling Technology, MA, USA, 1:1000), or with a goat anti-GAPDH (Bio-Rad, CA, USA, 1:20,000). Secondary antibodies used were 1:2500 dilution of goat anti-rabbit HRP labeled, goat anti-mouse HRP labeled (both Thermo Fisher Scientific, MA, USA), and rabbit anti-goat HRP labeled (Novex by Life Technologies, CA, USA). The proteins of interest were detected using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK). Quantification of immunoblots were performed by densitometry in ImageJ.

Chrysophanol and N-acetyl-L-cysteine were provided by Cayman Chemical (MI, USA). Cisplatin was purchased from Sigma (MO, USA). Dulbecco's Modified Eagle Medium, CellROX® Oxidative Stress Reagents, and Pierce BCA Protein Assay kit were provided by ThermoFisher Scientific (MA, USA). Fetal bovine serum was purchased from Corning (NY, USA). Penicillin/streptomycin was provided from Bioindustry (London). PhosSTOP and complete ULTRA tablets, the In Situ Cell Death Detection kit, and fluorescein were purchased from Roche (Germany). Z-VAD-FMK was provided from R&D systems (MN, USA). Anti-cyclin A2, E-cadherin, cyclin E1, cyclin D1, CDK4, cdc2, and CDK2 antibodies were purchased from Cell Signaling (MA, USA). The anti-EpCAM antibody and Apoptosis/Necrosis Assay kit were purchased from Abcam (UK). Anti-vimentin and PCNA were provided from BioLegend (CA, USA) and ABclonal (MA, USA), respectively. Anti-β actin antibody was purchased from Santa Cruz Biotechnology (TX, USA).