Fig. 1

IGFBP7 and senescence. (A) On the left, the pictures show representative images of senescent (Ki-67-; β-galactosidase+) in healthy control samples (CTRL) and induced senescence (H₂O₂) samples. Cells were stained to identify nuclei (DAPI in blue), Ki67 (red), to evaluate β-galactosidase activity (dark gray). We used a Zeiss Axioscope 5 microscope, equipped with an Axicam 305 digital camera. The β-galactosidase activity was detected as a gray stain using this configuration. This method allowed us to identify cells that exhibited a visible light signal β-galactosidase along with others expressing fluorescent signals within the same cell. The black bar corresponds to 100 microns. The accompanying histogram shows the percentage of (P) cycling (Ki67+; β-galactosidase-), (Q) quiescent (Ki67; β-galactosidase-), (St) stressed (Ki67+; β-galactosidase+), and (Sen) senescent (Ki67-; β-galactosidase+) cells three days following treatment with H₂O₂. Data are expressed with standard deviation (n = 3 biological replicates). The symbol **p < 0.01 indicate statistical significance between the control (CTRL) and H₂O₂ treated samples. At the top on the right, the picture shows a representative western blot analysis of IGBP7 in MSCs secretome collected 72 h following H₂O₂ treatment and in controls. The histogram shows the protein levels expressed in arbitrary units (A.U.). Data are expressed with standard deviation (n = 3 biological replicates). The symbol ***p < 0.001 indicate statistical significance between the control and H₂O₂ treated samples. (B) On the left, the histogram shows the percentage of (P) cycling (Ki67+; β-galactosidase-), (Q) quiescent (Ki67; β-galactosidase-), (St) stressed (Ki67+; β-galactosidase+), and (Sen) senescent (Ki67-; β-galactosidase+) in MSCs incubated for 72 h either with secretome of healthy cells (CM-CTRL) or with secretome of H₂O₂ treated cells with and without supplementation of anti-IGFBP7 neutralizing antibodies (AbαIGFBP7). Data are expressed with standard deviation (n = 3 biological replicates). The symbols **p < 0.01 and *p < 0.05 indicate statistical significance between the control (CTRL) and treated samples. The symbol #p < 0.05 indicates statistical significance between the CM-H₂O₂ sample, chosen as a reference, and CM-H₂O₂ + AbαIGFBP7. On the middle, the pictures show representative images of senescent (Ki-67-; β-galactosidase+) in healthy control samples (CTRL) and samples incubated with IGFBP7. Cells were stained to identify nuclei (DAPI in blue), Ki67 (red), to evaluate β-galactosidase activity (dark gray). The black bar corresponds to 100 microns. The accompanying histogram shows the percentage of (P) cycling, (Q) quiescent, (St) stressed, and (Sen) senescent. Data are expressed with standard deviation (n = 3 biological replicates). The symbols **p < 0.01 and *p < 0.05 indicate statistical significance between the control (CTRL) and treated samples. (C) On the left, Flow cytometry chart of Annexin V assay on healthy MSCs (CTRL) and in cells incubated with IGFBP7. The percentage of detected apoptotic cells is reported in the histogram. On the right, Cell cycle plots of healthy (CTRL) and IGFBP7 treated MSCs. Data are expressed with standard deviation (n = 3 biological replicates). For every cell cycle phase, the symbols *p < 0.05 and **p < 0.01 indicate the statistical difference between the CTRL samples and those treated with IGFBP7. (D) On the left, representative western blot analysis carried out on healthy (CTRL) and IGFBP7 treated MSCs. The accompanying histogram shows the protein levels expressed in arbitrary units (A.U.). GAPDH was used to normalize protein expression levels. Data are expressed with standard deviation (n = 3 biological replicates).The symbols **p < 0.01 and *p < 0.05 indicate statistical significance between the control (CTRL) and treated samples. On the right, the histogram shows the mRNA levels of the indicated genes, which were expressed in arbitrary units (A.U.). GAPDH was used to normalize mRNA expression levels. Data are expressed with standard deviation (n = 3 biological replicates). The symbols **p < 0.01 and *p < 0.05 indicate statistical significance between the control (CTRL) and treated samples. (E) On the left, the histogram shows the percentage of (P) cycling, (Q) quiescent, (St) stressed, and (Sen) senescent cells three days following H₂O₂ treatment with/without Anti-oxidant supplement (Anti-Ox) or with/without Parecoxib (PXB). Data are expressed with standard deviation (n = 3 biological replicates). The symbols **p < 0.01 and *p < 0.05 indicate statistical significance between the control and treated samples. The symbol ##p < 0.01 represents statistical significance between the indicated samples. On the right, IGBP7 expression level in the secretome of cells treated as above reported. At the top of histogram, a representative western blot image of IGFBP7 protein expression. 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 symbol ##p < 0.01 represents statistical significance between the indicated samples