Fig. 2: A chemically induced nuclear barrier leads to RS-like senescence.

a HDFs stained for SA β-gal activity by the cytochemical (X-gal) method. Young HDFs were treated with 6 μg/ml wheat germ agglutinin (WGA) or 0.1 μg/ml leptomycin B (LMB) for 8 days and then cultured for an additional 4 days after WGA and LMB removal. Bottom panels show 3× magnified versions of images, as indicated by the dashed box on top. Scale bar, 50 μm. b Percentages of β-gal-positive cells (n > 200 cells). Left (white) and right (red) bars indicate the percentages estimated by cytochemical and chemiluminescence assays, respectively. The means ± SD (n = 3) are reported; a.u., arbitrary unit. c Immunoblot using whole-cell lysates of young HDFs (NT), young HDFs treated with WGA or LMB, or RS HDFs (S). d Immunocytochemical detection of DNA damage repair markers (53BP1 and γH2AX foci) in HDFs treated with WGA or LMB. Scale bar, 50 μm. Quantitative analysis of γH2AX and 53BP1 double-positive nuclear foci was performed using micrographs of cells labeled with DNA damage repair markers (n > 200 cells). e Comet assay of young HDFs, WGA- and LMB-treated young HDFs, and RS HDFs (n > 50 cells). f Volcano plots showing upregulated (red) and downregulated (blue) proteins in the nucleus (left) and cytoplasm (right). g Relationships between upregulated (top) or downregulated (bottom) proteins in the nucleus (left) and cytoplasm (right) of cells undergoing RS or LMB-NBIS. h Scatter plots showing significant (P < 0.05) Pearson correlations (ρ) of log₂-fold-changes for the differentially expressed sibling peptides with consistent alteration directions for all DEPs measured from the nucleus (left) and cytoplasm (right) between cells undergoing RS and LMB-NBIS. Red lines denote regression lines, and the regression equations are shown together with R² values. i Heat maps showing correlations of protein levels measured from individual samples of young, senescent (RS), and LMB-treated (LMB-NBIS) HDFs. Color bar, gradient of the Pearson correlation. j Heat map showing the gene enrichment patterns of GOBPs and KEGG pathways by upregulated and downregulated proteins in the nucleus or cytoplasm of cells undergoing RS or LMB-NBIS. Color bar, gradient of Z-score for the enrichment P value determined with DAVID software.