Fig. 3

IGFBP7 and Activin A. (A) The histogram shows the percentage of (P) cycling, (Q) quiescent, (St) stressed, and (Sen) senescent cells in MSCs cultures incubated with Activin A (ACV) and/or IGFBP7 and/or antibodies targeting Activin A receptors (AbαACVR1, AbαACVR1B). Data are expressed with standard deviation (n = 3 biological replicates). The symbol **p < 0.01 represents statistical significance between the control and treated samples. The symbols ###p < 0.001 and ##p < 0.01 represent statistical significance between the indicated samples. On the right, immunoprecipitation experiment performed on secretome of cells incubated with IGFBP7 and Activin A. The proteins were immunoprecipitated with anti-IGFBP7 antibody and western blot was performed with anti-Activin A antibody. Input: western blot performed on proteins present in culture medium; SUP and IP indicate the supernatant and the immunoprecipitated components of immunoreaction, respectively. (B) SMAD pathways. Representative western blot analysis of nuclear and cytoplasmic levels of phosphorylated-SMAD2/3 (pSMAD2/3), SMAD2/3, phosphorylated-SMAD1/5 (pSMAD1/5), SMAD1/5 in cells incubated with IGFBP7 and/or Activin A. GAPDH and Histone 4 (H4) were used as cytoplasmic and nuclear markers, respectively. The histograms show the cytoplasmic and nuclear pSMAD2-3/SMAD2-3 and pSMAD1-5/SMAD1-5 ratios in the different experimental conditions. Data are expressed with standard deviation (n = 3 biological replicates). In the histograms, the symbols on the white bars ***p < 0.001 and **p < 0.01 represent a statistical significance between the control and treated samples; the symbols on the blue bars ###p < 0.001 and ##p < 0.01 represent a statistical significance between the control and treated samples. (C) On the left, representative immunoprecipitation experiment performed on secretome of cells incubated with IGFBP7. The proteins were immunoprecipitated with anti-IGFBP7 antibody and western blot was performed with anti-ACVR1 and anti-ACVR1B antibodies. Input: western blot performed on proteins present in secretome; SUP and IP indicate the supernatant and the immunoprecipitated components of immunoreaction, respectively. On the right, representative images of Duolink assay to identify physical proximity between IGFBP7 and ACVR1 and ACVR1B. The red dots represent a close interaction between IGFBP7 and ACVR1 or ACVR1B. The nuclei were DAPI stained with (blue). The scale bar corresponds to 100 microns