Figure 3. Paracrine senescence depends on the p16^INK4a/Rb and p53/p21^CIP1 tumour suppressor networks.

(a) IMR90-Cherry cells were co-cultured with IMR90 ER:RAS cells in the presence or absence of 200 nM 4OHT. 7 days after 4OHT treatment, cells were subjected to IF. The percentage of IMR90 ER:RAS (top, centre) or IMR90 Cherry cells (top, right) positive for each of the markers is shown. Data is a representative experiment. The source data for 2 independent experiments are provided in Supplementary Table S8. Representative pictures are shown in the bottom. Scale bar, 30 μm. (b) mRNA expression profiling of IMR90, IMR90 ER:RAS or IMR90 cells cultured in Transwells together with IMR90 vector or IMR90 ER:RAS cells during 7 days in the presence of 200 nM 4OHT and 0.5 % FBS. The plot shows the correlation between cells undergoing OIS and paracrine senescence. (c) Hierarchical clustering of genes changing more than 2-fold, centred in a cluster that defines the equivalence between OIS and paracrine senescence. (d) GSEA of a signature associated with senescence ¹⁸ in IMR90 cells undergoing paracrine senescence. (e) A signature derived from IMR90 cells undergoing paracrine senescence is found enriched in mouse PANIN ⁴⁴ and human serrated sessile adenomas (SSA) ³⁶. NES, normalized enrichment score; FDR, false discovery rate. (f) Paracrine senescence is dependent on the p53/p21^CIP1a and p16^INK4a pathways. IMR90 cells were transfected with the indicated siRNAs. The next day, CM from IMR90 ER:RAS or IMR90 vector cells was added. The proliferation of IMR90 ER:RAS cells was assessed by BrdU incorporation 2 days after CM addition (right).). Data are mean ± s.d., n = 3 independent experiments. The source data and statistics (Student’s t-test) are provided in Supplementary Table S8. IMR90 cells transfected with the different siRNAs were subjected to IF as a control for knockdown efficiency (centre).