Supplementary Figure 8: NOTCH1 controls the pro-inflammatory SASP through repression of C/EBPβ.
From: NOTCH1 mediates a switch between two distinct secretomes during senescence
(a) IMR90 cells expressing HRASG12V, N1ICD-FLAG or both were analysed for expression of CEBPB by qRT-PCR; One-way ANOVA with Dunnett’s multiple comparison test; values are mean ± s.e.m.; n = 6 biologically independent experiments; ∗∗∗P ≤ 0.001. (b) Genes upregulated by C/EBPβ are highly enriched by geneset enrichment analysis amongst genes upregulated by HRASG12V (RIS) and downregulated by N1ICD (NIS) and the co-expression of N1ICD and HRASG12V (N+RIS); FDR, false discovery rate; NES, normalised enrichment score. (c) TRE-N1ICD IMR90 cells treated with or without doxycycline for 3 days, then with or without 100 ng ml−1 TNF-α for 1 h were analysed for expression and chromatin binding of indicated NF-κB family components by immunoblot. (d) Ectopic expression of N1ICD-FLAG in HaCaT immortalised human keratinocyte cells leads to a downregulation of C/EBPβ, but not cell-autonomous proliferative arrest; analysis by immunoblot and qRT-PCR; bars are means; unpaired t-test; n = 3 biologically independent experiments; ∗∗∗P ≤ 0.001. (e) Genome browser snapshot of the IL1A locus demonstrating ChIP-Seq binding profiles for C/EBPβ, key histone marks and accessibility of chromatin by DNAse-Seq (derived from ENCODE). There is a small C/EBPβ binding peak at the Gencode-annotated TSS (proximal), but much larger binding peaks at a distal enhancer region 8kb upstream (distal), recently demonstrated to be tethered to the IL1A core promoter by 3D chromatin interactome analysis. Representative RefSeq and Gencode annotated IL1A transcripts are shown. Solid vertical lines represent C/EBPβ ChIP-qPCR ‘target’ sites at the ‘Proximal’ and ‘Distal’ regions, whereas the dotted vertical line represents ‘non-specific’ site. (f) Chromatin immunoprecipitation of endogenous C/EBPβ (from experiment described in Fig. 8g) and subsequent qPCR for loci at IL6 and IL8 promoters; n = 3 biologically independent experiments; one way ANOVA with Dunnett’s multiple comparison test; values are mean ± s.e.m. ∗∗∗P ≤ 0.001. Statistics source data for a,d and f are provided in Supplementary Table 2.
