Supplementary Figure 5: NOTCH1 drives juxtacrine senescence through JAG1-mediated lateral induction.

(a) IMR90 cells stably expressing N1ICD underwent transcriptional profiling by mRNA-seq; differential expression of canonical NOTCH ligands was then analysed. (b) Immunoblot for JAG1 and coomassie gel staining of whole cell lysate (WCL) and conditioned media (CM) from TRE-N1ICD IMR90 cells treated with or without doxycycline for 3 days. (c) RPE1 cells expressing ectopic N1ICD were analysed for markers of senescence; expression of the indicated proteins by immunoblotting; SA-β-gal and DNA synthesis by BrdU incorporation; unpaired t-test; values are mean ± s.e.m.; n = 6 biologically independent experiments; ^∗∗∗P ≤ 0.001 relative to vector control. Insets, magnified picture of dotted rectangular areas. Scale bar 200 μm. (d) Analysis of proliferation, by BrdU incorporation, and expression of p21 by immunofluorescence in mRFP-IMR90 cells when cocultured with TRE-N1ICD IMR90 cells with vector or shJAG1-4 and with or without 10 μM DAPT (See analogous experiment in Fig. 5bg). Bars are means of ≥200 mRFP-positive cells per condition; n = 3 biologically independent experiments; one-way ANOVA with Dunnett’s multiple comparisons test; ^∗∗P ≤ 0.01, ^∗∗∗P ≤ 0.001. (e) The proliferative ability of RPE1 cells expressing N1ICD or vector was analysed (upper). The proliferative ability of mRFP-IMR90 cells during co-culture with N1ICD or vector-expressing RPE1 cells, with and without DAPT, was analysed (lower) by proliferation assay; values are mean ± s.e.m.; representative of three independent experiments with similar results. (f) Model of NOTCH-induced lateral induction of NOTCH-signalling through JAG1 and TGF-β. How exactly the NOTCH activation induces JAG1 and TGF-β remains to be elucidated (dotted line). Activation of NOTCH signalling in the left-hand cell leads to the expression of both JAG1 and TGF-β. JAG1 acts in a cell-contact dependent fashion to induce activation of endogenous NOTCH in the right-hand cell and in concert with TGF-β to induce juxtacrine senescence. (g) Proliferation of mRFP1-IMR90 cells was analysed during co-culture with TRE-N1ICD cells treated with or without doxycycline and ER:RAS cells expressing dnMAML1-mVenus treated with or without 4OHT; representative of three independent experiments with similar results. Statistics source data for a,c and d are provided in Supplementary Table 2.