Fig. 1.

Release of IGFBP5 in SASP and its effect on senescence. A Representative micrographs of MSCs stained to identify nuclei (DAPI), Ki67 (red), and to evaluate β-galactosidase activity (dark gray). The white arrows indicate senescent cells, which are β-galactosidase positive (β-gal +) and Ki67 negative (Ki67-). We employed a Leica CTR500 microscope, which was equipped with a DCF3000G digital monochrome camera. The β-galactosidase activity was captured as a gray-stain using this configuration. This experimental method allowed us to identify cells that exhibited a visible light signal β-galactosidase along with others expressing fluorescent signals within the same cell. CT: untreated cells; CM-IR: cells treated with conditioned medium (CM) collected from irradiated (IR) cells; CM-IR + ab αIGFBP5: cells treated with CM in the presence of IGFBP5 neutralizing antibodies; rIGFBP5: cells incubated with recombinant IGFBP5. The scale bar corresponds to 100 microns. The graph shows the percentage of senescent cells under different experimental conditions. The symbols *** p < 0.001 and * p < 0.05 indicate statistical significance between the control (CT) and treated samples. The symbol ## p < 0.01 indicates statistical significance between the CM-IR sample, chosen as a reference, and CM-IR + ab αIGFBP5. B The graph shows the level of IGFBP5 in CM of MSCs 24 h following irradiation (IR). IR: irradiated MSCs; Anti-oxi: the irradiated MSCs were treated with an anti-oxidant mixture; PXB: irradiated cells were incubated with a drug inhibiting COX2 activity. The symbol ** p < 0.01 indicates statistical significance between the control (CT) and irradiated samples. The symbols ## p < 0.01 and # p < 0.05 indicate statistical significance between the IR sample (second column), chosen as a reference, and IR + Anti-oxi or IR + PXB. The western blot under the graph shows a representative image of IGFBP5 immunodetection. C The graph shows the percentage of senescent cells (β-galactosidase positive and Ki67 negative) under different experimental conditions. rIGFBP5: cells incubated with recombinant IGFBP5; IGFII: cell treated with IGFII either in presence or absence of antibody against IGFIIR (abα IGFIIR); siLRP1: siRNA targeting LRP1 mRNA; siCAV1: siRNA targeting CAVEOLIN-1; siCTR: control siRNA. The symbol ** p < 0.01 indicates statistical significance between the control (first column) and samples (from second to fourth columns) treated with rIGFBP5 and/or IGFII. The symbol °°° p < 0.001 indicates statistical significance between cells treated with abα IGFIIR alone (chosen as reference, see fifth column) and the other samples treated with the antibody in presence of rIGFBP5 and/or IGFII (from sixth to eight columns). The symbols §§§ p < 0.001 indicate and § p < 0.05 indicate statistical significance between the sample treated with siCT (chosen as the reference, see ninth column) and the other siRNA treated samples, reported in columns from the 10th to 12th. The symbol ## p < 0.01 indicates the statistical difference between the sample treated with genistein (Gen) in presence of IGFBP5 and the one treated with IGFBP5 only (second column from left), chosen as reference