Figure 8. NOTCH1 controls the pro-inflammatory SASP through repression of C/EBPβ.

(a) ER:HRAS^G12V/TRE-N1ICD-FLAG IMR90 cells treated with or without 6 days of 4OHT and 3 days of doxycycline were analysed for expression of RELA and C/EBPβ in whole cell lysate and fractionated chromatin by immunoblotting. (b) Time series analysis of chromatin-bound N1ICD-FLAG and C/EBPβ after doxycycline treatment of TRE-N1ICD IMR90 cells with or without dnMAML1. (c and d) TRE-N1ICD IMR90 cells with or without ectopic C/EBPβ-LAP* and 3 days of doxycycline treatment were analysed for C/EBPβ, IL-8 (c) and IL1A (d) expression by immunoblot and qRT-PCR; n = 3 biologically independent experiments. (e) TRE-N1ICD IMR90 cells treated with or without doxycycline for 3 days, then with or without 100ng/ml TNF-α for 1 hour were analysed for expression and chromatin binding of indicated mRNA and proteins by qRT-PCR and immunoblot respectively; unpaired T-test; n = 3 biologically independent experiments; bars are means. (f) TRE-N1ICD IMR90 cells treated with or without doxycycline for 3 days, and 10ng/ml IL-1α for the final 24 hours were analysed by immunoblotting. (g) ER:HRAS^G12V- and TRE-N1ICD-FLAG-expressing IMR90 cells treated with or without 6 days of 4OHT and 3 days of doxycycline were subjected to chromatin immunoprecipitation of endogenous C/EBPβ and subsequent qPCR for proximal and distal sites at the IL1A locus (Supplemental Figure 8E and METHODS); n = 3 biologically independent experiments; One way ANOVA with Dunnett's multiple comparison test; values are mean ± SEM. (h) Model for NOTCH-mediated SASP switch during senescence. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Statistics source data for d, e & g are provided in Supplementary Table 2.