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. Author manuscript; available in PMC: 2014 Feb 1.
Published in final edited form as: Nat Cell Biol. 2013 Jun 16;15(8):978–990. doi: 10.1038/ncb2784

Figure 5. A role for TGFβ signalling on mediating paracrine senescence.

Figure 5

(a) Comparing the paracrine and autocrine screens. Autocrine screen is in Sup Fig S5A. (b) GSEA of TGFβ target genes during OIS and paracrine senescence.. (c) qRT-PCR for a set of TGFβ target genes in OIS and paracrine senescence. Data are mean ± s.d., n = 3 independent experiments (d) Induction of TGFβ ligands during OIS by qRT-PCR (bars). Induction in the SILAC experiments is in blue. Data are mean ± s.d., n = 3 independent experiments (e) IMR90 cells were treated with CM and 10 μg/ml of antibodies for 2 days. Growth was evaluated by BrdU incorporation (right). Percentage of cells positive for p-SMAD2/3 is presented (left). Data are mean ± s.d., n = 4 independent experiments. p was calculated using Student’s t-Test. pSMAD 2/3 statistics: ActivinA p=0.0037; TGFβ p =0.91; BMP2 p =0.52; ActivinA/TGFβ p =0.0029; ActivinA/TGFβ/BMP2 p =0.040; TGFβ inhibitor II p value 0.0007. For BrdU, statistics: ActivinA p=0.009905573; TGFβ p =0.282677043; BMP2 p =0.233112557; ActivinA/TGFβ p =0.000723521; ActivinA/TGFβ/BMP2 p =0.002428509; TGFβ inhibitor II p =4.44×10−5. Source data are in Supplementary Table S8. (f) TGFBR1 inhibitors partially prevent paracrine senescence. IF against phosphorylated SMAD2/3. Scale bar, 10 μm. Representative pictures (left). IMR90 cells were treated as indicated. INK4b and CDKN1a expression measured by qRT-PCR (right). Data are mean ± s.d., (n = 3 independent experiments, p, Student’s t-Test). pSMAS2/3 statistics: Inhibitor I p= 4.49×10−5; Inhibitor II 4.53×10−5. INK4B statistics: Inhibitor I p =0.00083; Inhibitor II p= 0.010. CDKN1A statistics: Inhibitor I p = 0.00054; Inhibitor II p = 0.00014. Source data are provided in Supplementary Table S8. (g) Alk5 deletion suppresses KrasG12D-driven OIS in PANIN. Diagram showing mice strains used (top left). GSEA shows TGFβ activation in PANIN (bottom left). Ki67 and SA-β-Gal staining (centre) and quantification (right) showing decreased senescence in pancreatic lesions of Pdx1-cre KrasG12D/+ TGFβR1fl/fl mice. Scale bar, 100 μm. Boxplot represent first and third quartiles (n=5 mice per condition). Inside lines shows median. Whiskers extend to highest or lowest observation. p= 0.0184 for both experiments calculated using Mann-Whitney.