Fig. 1: Characteristics of young, mid-old, and old cells in vitro.

a mRNA (left panel) and protein (right panel) levels of representative senescence markers (p53, p21^Waf1, and p16^INK4A) in young, mid-old, and old cells are shown. b Relative distances represented as VST using count data from young, mid-old, and old samples are shown (n = 2, each). c GSEA using the “FRIDMAN: senescence up” gene set in old vs. young (left panel), old vs. mid-old (middle panel), and mid-old vs. young cells (right panel) (adjp = 0.0029, 0.016, and 0.53, respectively). d NES calculated from GSEA using gene sets representing fibroblast functions are presented. The gene sets include “CYCLE” representing self-replicative ability, “ECM” representing ECM productive ability, “TISSUE” representing tissue regeneration ability, and “INFLAM” representing inflammation-regulating ability. Used gene sets and related statistics for this analysis can be found in Supplementary Data 2. e, f GSEA of old vs. young, old vs. mid-old, and mid-old vs. young using the “HALLMARK: inflammatory response” gene set is shown (adjp = 0.0075, 0.0087 for (e), and 0.31 for (f), respectively). g GSEA of mid-old vs. young using the “REACTOME: interleukin 1 signaling pathway” gene set is shown (adjp = 0.018). h NES calculated from GSEA using gene sets representing the inflammatory pathway are shown. Used gene sets and related statistics in this analysis can be found in Supplementary Data 2. i The mRNA levels of multiple inflammatory pathways related genes and SAA1 protein level were analyzed by real time PCR and ELISA analysis. j A list of analyzed genes that act as anti-inflammatory chemokines and cytokines used in this study is shown (left panel). The mRNA levels of the listed genes are shown (middle panel). ELISA analysis of SLIT2 in young, mid-old, and old cells (right lower panel). k A schematic image illustrating the characteristics of young, mid-old, and old cells with representative gene expression. l GSEA using the “GOBP: nuclear transport” gene set in mid-old vs. young (left panel), old vs. young (middle panel) and old vs. mid-old (right panel) cells is shown (adjp =  0.085, 0.046, and 0.042, respectively). m The mRNA expression of nuclear transport-related genes in RNA-seq (upper panel). The figures in the box represent FPKM counts. S1-6 represents the subunits of the importin α. Real-time PCR results of nuclear transport-related genes are shown (lower panel). n Erk1/2 phosphorylation and nuclear translocation were analyzed after serum stimulation for 30 min (upper panel). Quantification of the number of cells with p-Erk1/2 nuclear translocation (lower left panel). p-Erk1/2 level was analyzed by western blot (lower right panel). o Investigation of p-Erk1/2 translocation dynamics. Schematic illustration of Erk1/2-KTR-mClover system and kinetic parameters (left upper and left lower). Young (n = 4), mid-old (n = 6), and old (n = 3) cells were assessed with the system, respectively. Three independent experiments were repeated. Representative images of Erk1/2-KTR-mClover translocation (right upper). Erk1/2-KTR-mClover translocation C/N curves for each cell type (middle). Cells were starved for 24 h with serum free media prior to experiments. Fluorescent images were acquired every 15 sec for 30 min after serum stimulation. Comparison of translocation kinetic parameters between cell types (lower panel). The p value in (a), (i), (j), and (m–o) was calculated using the one-tailed Mann–Whitney U-test. The p values in (c–h), and (i) are obtained using GSEA statistics in ‘fgsea’ package. ES enrichment score, NES normalized enrichment score, adjp = adjusted p value. The graph in (a), (i), (j), and (m–o) was represented as mean ± SD. A thousand times of permutations were performed for adjustment in (c–h) and (l).