Fig. 5
Decreased expression of p16ink4a reduces the number of senescent microglia and initiates new cell divisions, reducing dysfunctional disease-associated microglia (DAM). A SA-β-gal activity (blue) and immunostaining with anti-Iba1 antibody (brown). Yellow arrows indicate senescent cells (blue) that merge with Iba1-stained cells (brown). The second images indicate the magnified area. Scale bar: (left) 300 µm, (right) 20 µm. The third images show scatter plot of each staining images. B Comparison of Pearson’s correlation coefficient for Iba1 channel and SA-β-gal activity channel in brain tissue. ***p < 0.005, versus scrambled siRNA NPs; unpaired Student’s t test. C Graph representing results of colocalization analysis the colocalization plugin from ImageJ program. Results are presented as the mean ± SEM. ****p < 0.001, versus p16ink4a siRNA NPs; unpaired Student’s t test (n = 6). D The mRNA levels of the cyclin-dependent kinase inhibitors, p21, and a panel of SASP factors were determined by RT-qPCR. ****p < 0.001, versus p16ink4a siRNA NPs; unpaired Student’s t test (n = 6). E Co-localization of Ki67 (proliferative marker) and Iba1 (microglia marker). White arrows indicate microglia merged with Ki67. Scale bar: 20 µm. ***p < 0.005, versus scrambled siRNA NPs; unpaired Student’s t test. F Co-localization of Lamp1 (Lysosomal activity marker) and Iba1 (microglia marker). White arrows indicate microglia merged with Lamp1. Scale bar: 20 µm. ****p < 0.001, versus scrambled siRNA NPs; unpaired Student’s t test. G Dot plots from a representative BV2 cells with transfected p16ink4a siRNA and scrambled siRNA are shown. The plot shows the increased expression of ki67 in the p16ink4a siRNA transfected BV2 cell group compared scrambled siRNA transfected cell group. All data were used for each experiment that was repeated three times. H Dot plots from a representative BV2 cells with transfected p16ink4a siRNA and scrambled siRNA are shown. The plot shows the increased expression of BrdU in the p16ink4a siRNA transfected BV2 cell group compared scrambled siRNA transfected cell group. 45 h after siRNA transfection, BrdU was treated for 3 h. All data were used for each experiment that was repeated three times. I Co-localization of TREM2 (Disease associated microglia marker) and Iba1 (microglia marker). Scale bar: 20 µm. ****p < 0.001, versus scrambled siRNA NPs; unpaired Student’s t test. J Co-localization of Clec7a (Disease associated microglia marker) and Iba1 (microglia marker). Scale bar: 20 µm. ****p < 0.001, versus scrambled siRNA NPs; unpaired Student’s t test