Fig. 2

IGFBP7 and its molecular partners. (A) Insulin and IGFBP7. The histogram shows the percentage of (P) cycling, (Q) quiescent, (St) stressed, and (Sen) senescent cells in MSCs cultures incubated with Insulin (INS) and/or IGFBP7 and/or antibodies targeting Insulin receptors (AbαINSR). 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 < 0.01 and #p < 0.05 represent statistical significance between the indicated samples. On the right, immunoprecipitation experiment performed on culture medium containing both IGFBP7 and Insulin. The proteins were immunoprecipitated with anti-IGFBP7 antibody and western blot was performed with anti-Insulin antibody. Input: western blot performed on proteins present in medium; SUP and IP indicate the supernatant and the immunoprecipitated components of immunoreaction, respectively. (B) IGFII and IGFBP7. The histogram shows the percentage of (P) cycling, (Q) quiescent, (St) stressed, and (Sen) senescent cells in MSC cultures incubated with IGFII and/or IGFBP7 and/or antibodies targeting IGFII receptors (AbαIGFR1, AbαIGFR2). Data are expressed with standard deviation (n = 3 biological replicates). The symbols ***p < 0.001, **p < 0.01 and *p < 0.05 represent statistical significance between the control and treated samples. The symbols ##p < 0.01 and #p < 0.05 represent statistical significance between the indicated samples. On the right, 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-IGFR1 antibody. Input: western blot performed on proteins present in secretome; SUP and IP indicate the supernatant and the immunoprecipitated components of immunoreaction, respectively. (C) ERK and AKT pathways. The histograms show the percentage of senescent cells in MSCs cultures incubated with IGFBP7 and/or Insulin (INS), and/or IGFII, and/or U0126, a MEK inhibitor (UO), and/or GSK-690,693, an AKT inhibitor (GSK). Data are expressed with standard deviation (n = 3 biological replicates). In the two histograms, the symbols on the white bars **p < 0.01 and *p < 0.05 represent a statistical significance between the control and treated samples. In the U0 treated samples (grey bars): the symbol ###p < 0.001 represents a statistical difference between the samples incubated with IGFBP7 with/without U0; the symbol #p < 0.05 represents a statistical difference between samples incubated with IGFBP7 and insulin with/without U0; the symbol ¶¶¶p < 0.001 represents a statistical difference between the samples incubated with IGFII with/without U0; the symbol °°°p < 0.001 represents a statistical difference between the samples incubated with IGFBP7 and IGFII with/without U0. In the GSK treated samples (blue bars): the symbol ###p < 0.001 represents a statistical difference between the samples incubated with IGFBP7 with/without GSK; the symbol °°p < 0.01 represents a statistical difference between the samples incubated with IGFBP7 and IGFII with/without GSK; the symbol ¶¶¶p < 0.001 represents a statistical difference between the samples incubated with Insulin with/without GSK. (D) ERK and AKT pathways. Representative western blot analysis of nuclear and cytoplasmic levels of phosphorylated-ERK (pERK), ERK, phosphorylated-AKT (pAKT), AKT in cells incubated with IGFBP7 and/or Insulin and/or IGFII. GAPDH and Histone 4 (H4) were used as cytoplasmic and nuclear markers, respectively. The histograms show the cytoplasmic and nuclear pERK/ERK and pAKT/AKT 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