Fig. 5: Young cell-driven factors reverse the phenotype of mid-old cells.
From: Mid-old cells are a potential target for anti-aging interventions in the elderly
a A schematic drawing of the stromal cell population according to the aging process is shown. b Mid-old cells are co-cultured with young or mid-old cells for 30 days, respectively. IF staining for Ki67 was performed (left upper panel). Quantification data for Ki67 is shown in the right upper panel. The cell size was compared and quantified (left lower panel). After 30 days of co-culture, cell growth rates were measured over a period of 10 days (right lower panel). c EdU incorporation assay. Mid-old cells co-cultured with young or mid-old cells for 30 days and then analyzed EdU incorporation. Quantification data is shown in the lower panel. d GSEA was performed in mid-old cells co-cultured with young and mid-old cells, respectively (n = 2, each). Functional gene sets of fibroblasts including “CYCLE” representing self-replicative ability, “ECM” representing ECM productive ability, “TISSUE” representing tissue regeneration ability, and “INFLAM” representing inflammation-regulating ability were analyzed. Used gene sets and related statistics for this analysis can be found in Supplementary Data 2. e GSEA was performed for “GOBP: mitotic cell cycle”, “GOBP: DNA replication”, “GOMF: extracellular matrix structural constituent”, “GOBP: tissue migration”, and “Hallmark: inflammatory response” (adjp = 0.038, 0.038, 0.038, 0.038, and 0.11). The p value and adjp are obtained using GSEA statistics in ‘fgsea’ package. ES: enrichment score; NES: normalized enrichment score; adjp: adjusted p value. f The mRNA expression of inflammatory, anti-inflammatory genes, and ECM-related molecules in mid-old cells co-cultured with young or mid-old cells (upper panel). ELISA analysis of IL1β and SAA1 in mid-old cells co-cultured with young or mid-old cells (lower panel). g The expression level of Lnc-RNAs was measured using real-time PCR in young, mid-old, and old cells (upper panel). Lnc-RNAs were isolated from multiple cellular compartments and shown using conventional real time-PCR (lower panel). h Mid-old cells were infected with lentivirus harboring Lnc-SBLC for 4 days. Cell morphology (upper panel) was observed and p53 and p21Waf1 expression were analyzed using real-time PCR (left lower panel) and western blot analysis (right lower panel). i The upper panel shows the cell growth rate, which was measured by counting the cells every 4 days. The lower panel displays the expression levels of inflammatory genes, which were measured using real-time PCR. j Mid-old cells were infected with lentiviruses carrying sh-p21Waf1 for 4 days. Cell morphology was examined and depicted in the left panel, while the cell growth rate was monitored by counting the cells every 4 days (right upper panel). The expression of various inflammatory genes was assessed using real-time PCR (right lower panel). The p values of (b) (right upper and lower), (c), and (f), (j) are obtained from the one-tailed Mann–Whitney U test. The p value of (b) (left lower) is obtained from two-tailed Student’s t test. The p values of (e) (left lower) are obtained using GSEA statistics in ‘fgsea’ package in R. The graphs in (b), (c), and (f–j) were represented as mean ± SD. A thousand times of permutations were performed for GSEA adjustment.
