Fig. 3: A genetically induced nuclear barrier results in RS-like senescence.
From: Disruption of nucleocytoplasmic trafficking as a cellular senescence driver
a HDFs infected with lentivirus expressing CRM1 shRNA or importin-α1 shRNA for 9 days and treated with 1 μg/ml puromycin 2 days after infection. Representative images of cells stained for SA-β-gal activity by the cytochemical method. Scale bar, 50 μm. b Percentages of β-gal-positive cells in control and CRM1- or importin-α1-knockdown HDFs (n > 200 cells). Y and S, young and senescent HDFs, respectively. The β-gal assay was carried out 7 days after the start of puromycin selection. c Immunoblot of whole-cell lysates from HDFs infected with virus expressing CRM1 shRNA or importin-α1 shRNA. d Cell lysates immunoprecipitated with an anti-RanGTP antibody. As positive and negative controls, young cell lysates were used to pull down RanGTP after treatment with GDP or GTPγS. Immunoblotting of immunoprecipitates (IP) with an anti-Ran antibody. The right panel shows the quantitative analysis of RanGTP expression normalized to that of heavy chain. The results are representative of three independent experiments that are shown in the bar graph as the means ± SD. *, P < 0.05 (Student’s t-test). Y and S, young and senescent HDFs, respectively. e HDFs infected with lentivirus expressing RCC1 shRNA for 10 days. The cells were selected by treatment with 1 μg/ml puromycin 2 days after infection. Assays were performed 10 days after infection. Wide-field micrographs of cells stained for SA-β-gal activity by the cytochemical (X-gal) method. Bottom panels show 3× magnified versions of images as indicated by the dashed boxes on top. Scale bar, 50 μm. f Percentages of β-gal-positive cells among young, control shRNA-treated, RCC1-knockdown HDF, and RS HDF populations (Senescent) (n > 200 cells). Left (white) and right (red) bars indicate the percentages estimated by cytochemical and chemiluminescence assays, respectively. The data represent the means ± SD (n = 3); a.u., arbitrary unit. g Immunoblot using whole-cell lysates of HDFs infected with a lentivirus expressing control or RCC1 shRNA. h Comet assay with RCC1-knockdown HDFs and RS HDFs. The data represent the means ± SD (n > 50 cells).
