Figure 3. C/EBPβ isoform binds SASP promoters in senescent cells.

a. Treatment of 293T HEK cells with bleomycin induces a significant increase in senescence as indicated by increased senescence-associated β-galactosidase staining, n=3, *p<0.05. Scale bar=100 μm. b. Chromatin immunoprecipitation (ChIP) using a C/EBPβ antibody which recognizes all three C/EBPβ isoforms or a non-specific control (IgG) indicates that C/EBPβ significantly binds to the OPN and IL-6 promoters in both vehicle and bleomycin-treated 293Ts, and to the IL-8 promoter in vehicle treated cells, representative experiment, n=3, *p<0.05. “nspc.” indicates pulldown with C/EBPβ antibody at a distal site. c. Western blot using an anti-CEBPβ antibody indicating similar C/EBPβ expression in non-senescent and senescent ChIP treatments, n=1. d. ChIP in 293Ts transfected with a Flag-tagged activating C/EBPβ isoform (Flag-LAP2). An antibody recognizing all three C/EBPβ isoforms (CEBP) or an anti-Flag antibody was used to detect binding of the total C/EBPβ relative to exogenous Flag-LAP2 to the OPN, IL-6 and IL-8 promoters in vehicle (Non-Sen) and bleomycin-treated (Sen) 293Ts. While there was little change in total C/EBPβ bound to the OPN, IL-6, or IL-8 promoters, Flag-LAP2 binding to all three promoters was significantly increased in senescent cells, representative experiment, n=3, *p<0.05. e. Western blotting using an anti-Flag antibody indicated that Flag-LAP2 expression is significantly higher in non-senescent 293Ts than senescent 293Ts, n=3.