Cyclophilin a

WI38 cells were purchased from American Type Culture Collection. Cells were cultured in DMEM supplemented with 10% FBS (R&D Systems) and 1% Penicillin-Streptomycin (ThermoFisher Scientific). These cells were screened for mycoplasma every four months using the ATCC Universal Mycoplasma Detection Kit (Catalogue # 30–121 1012 K). Early passage primary MEFs were harvested and cultured from C57BL/6 mice as previously described [20 (link), 44 (link), 45 (link)]. β-gal staining was achieved using the Cellular Senescence Kit (Millipore). Recombinant proteins included Cyclophilin A (R&D) and S100A4 (Abcam). SN52 peptide (AAVALLPAVLLALLAPVQRKRRKALP) and SN52-mut peptide (AAVALLPAVLLALLAPVQRNGRKALP) were acquired form GenScript. Inhibitors used were: WP1066, BMS345541, G06976, Staurosporine, and KU60019 (all from Selleck), and Roscovitine (Sigma). The following siRNAs were used: siGENOME non-targeting siRNA#3 (Dharmacon) and si-STAT3 (sc-29,493, Santa Cruz). siRNA transfection was performed with Oligofectamine (Invitrogen).

10 μg lysate sample or 7.5 μg concentrated conditioned media sample was run on a gel, transferred by Trans-Blot® Turbo™ (Biorad) blotting to a PVDF membrane which was then blocked in 2.5% milk in TBST for 30 min. Blots were next incubated for 1 h with either Cat L (1:2000 dilution) or Cat B (1:1000 dilution) antibodies from R&D Systems. The bottom half of the blots were incubated with antibodies for loading controls; anti-galectin (R&D Systems) (1:2000 dilution) for the conditioned media samples and cyclophilin A (R&D Systems) (1:2000 dilution) for the lysate samples. All antibodies were diluted in 5% BSA in TBST. Following incubation, blots were washed with TBST, incubated with 1:1000 diluted anti-goat secondary antibody (Jackson ImmunoResearch) for 1 h, washed with TBST again and treated with ECL (Millipore). The blots were imaged using the Alpha Innotech Imaging system and the bands were quantified using the Alpha Innotech software.