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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 4. Multiple components of the SASP are involved on paracrine senescence.

Figure 4

(a) Diagram summarizing the proteomics approach. (b) Comparison between mRNA and protein expression for the secretome of cells undergoing OIS. Overall Pearson correlation is 0.64. A lower correlation was observed for proteins induced more than 4-fold (red line, Pearson correlation=0.15). This suggests post-trancriptional upregulation of SASP components (e.g. MMP7, IGFBP5, IGFBP6, THBS1, THBS2 and IL6ST). (c) Plot of 2 forward and reverse SILAC experiments. Significant changes in at least 2/3 experiments are coloured. (d) Screening for compounds inhibiting paracrine senescence. IMR90 fibroblasts were grown in the indicated CM in the presence of a collection of 78 drugs. 2 days later, BrdU incorporation was measured. −, IMR90 treated with DMSO and grown in CM of IMR90 ER:RAS − 4OHT; +, IMR90 treated with DMSO and grown in CM of IMR90 ER:RAS +4OHT. A gray area represents an arbitrary cut-off of 120 % the value of BrdU in the no drug control (+). Inhibitors over the cut-off targeting VEGFR2 and/or FLT3 (orange), CCR2 (green) or TGFBR1 (brown) are marked. Data are mean ± s.d., n = 3 independent screen plates. (e) IMR90 cells cultured 2 days with the indicated CM and drugs (concentrations 10 μM to 10 nM). Proliferation was evaluated by BrdU incorporation. Graph shows one representative experiment out of two independent experiments. (f) Infected IMR90 cells were treated with CM and growth evaluated by CV (top). Data are one representative experiment out of two independent experiments. IF against CCR2 is shown as a control for the efficiency of the shRNAs used (bottom). Scale bar, 10 μm. (g) Knockdown of receptors of the TGFβ family partially rescue paracrine senescence. IMR90 cells infected with the indicated vectors were treated with CM of senescent or control cells and senescence evaluated 10-14 days after by CV (top left) and SA-β-Gal staining (bottom left). Knock down efficiency was measured by qRT-PCR (right). Data are one representative experiment out of 2 independent experiments. The source data for 2 independent experiments are provided in Supplementary Table S8. Scale bar, 50 μm.