Sc 15335

Cells were fixed in 4% paraformaldehyde (Millipore-Sigma, St. Louis, MO) for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 20 min at room temperature for nuclear markers. Cell fixation and permeabilization for cytoskeletal markers was done with cold (−20°C) methanol for 3–5 min. Samples were washed three times (5 min each time) with PBS between each step and blocked with 3% normal donkey serum (NDS; Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS for 30 min. Samples were incubated at 4°C with primary antibodies for: cardiac troponin T (TNNT2; rabbit; ab45932; Abcam, Cambridge, MA), α-actinin (ACTN1; mouse; sc-15335) and GATA4 (rabbit; sc-9053; both from Santa Cruz Biotechnology, Dallas, TX). Incubation with secondary antibodies was performed at room temperature for 1 h with donkey anti-rabbit or anti-mouse antibodies conjugated to DyLight 488 or 549 (Jackson ImmunoResearch Inc., West Grove, PA). Nuclear DNA was stained with DAPI (Sigma-Aldrich, St. Louis, MO). Controls were stained with IgG instead of with a primary antibody. Immunostaining was visualized with a Leica TCS SPE confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL).

Total proteins were extracted with RIPA lysis buffer (89900, Thermo Scientific) supplemented with 1X Protease Inhibitor—Complete ULTRA tablets mini (5892791001, Roche) and 1X Benzonase nuclease HC (712063, Millipore) for 1 h at 4°C. Equal amounts of total cellular proteins were resolved on NuPAGE® Novex® 4–12% Bis-Tris Protein Gels (NP0336BOX, Thermo Fisher Scientific) and transferred to nitrocellulose membranes (iBlot, Thermo Fisher Scientific) following the manufacturer’s instructions. Membranes were then blocked in Odyssey Blocking Buffer (927-4-0000, Li-Cor) for 1 h at room temperature. Incubation with primary antibodies was carried out at 4°C overnight in Odyssey Blocking Buffer. The following antibodies were used: rabbit anti-β-SG (dilution 1/100, HPA011422, Sigma) and rabbit anti-Actinin alpha (H-300) (dilution 1/1000, sc-15335, Santa Cruz). After 1 h incubation with donkey anti-rabbit 680 antibody (926–68073, EuroBio) at room temperature, proteins were detected by fluorescence (Odyssey, Li-Cor) following the manufacturer’s instructions. Western blot signal quantification was performed with the Plot Lanes Analysis tool of Image J software (NIH).

1.3 × 10⁵ senescent BJ cells were untreated or reverse transfected in a 6-well plate with control siRNA or p53 siRNA and harvested in RIPA buffer after 72 h. Equal amounts of protein were separated using SDS-PAGE and transferred to a nitrocellulose membrane. The p53 protein was detected using the anti-p53 (DO-1) mouse monoclonal antibody (sc-126, Santa Cruz Biotechnology), and the loading control alpha-actinin was detected using the rabbit anti-alpha-actinin (H-300) polyclonal antibody (sc-15335, Santa Cruz Biotechnology). Western blots were developed using appropriate secondary antibodies conjugated to HRP followed by ECL detection.