Fig. 6: SLIT2 has an impact on mid-old cells.
From: Mid-old cells are a potential target for anti-aging interventions in the elderly
a Mid-old cells were treated for 24 h with rhSLIT2, and inflammatory gene expression was analyzed using real-time PCR (upper panel). IL1β and SAA1 protein level was analyzed by ELISA in rhSLIT2-treated mid-old cells (lower panel). b Mid-old cells were treated with rhSLIT2 for the indicated times and analyzed for Pyk2-NFκB signaling by western blot analysis (left panel). The number of nuclear p-NFκB-positive cells was quantified in mid-old cells using IF staining (right panel). c SLIT2-expressing lentivirus infected into mid-old fibroblasts, and inflammation related genes expression including SAA1 and IL1β were analyzed using real-time PCR (right lower panel). IL1β and SAA1 protein level was analyzed by ELISA in SLIT2-overexpressing mid-old cells (left lower panel). d The morphology and cell size of mid-old cells were analyzed after treating rhSLIT2 for 20 days. Cell growth rate was analyzed every 4 days for 20 days by counting the number of cells. e The expression of p53 and p21Waf1 were measured in mid-old cells after administration of rhSLIT2 at the indicated concentration for 2 days using real-time PCR (upper panel) and western blot analysis (lower panel). f The changes in gene expression of p53 and p21Waf1-regulating genes were evaluated after treating cells with rhSLIT2 using RNA-seq FPKM count (left panel). The mRNA expression of SOX2 and OCT4 were analyzed using real-time PCR (right upper panel) and western blot analysis (right lower panel) in rhSLIT2-treated mid-old cells. g Young cells were infected with shSLIT2 lentivirus and stabilized for 2 days, and subsequently cultured for 8 days. The mRNA expression level of SAA1 and IL1β was analyzed using real-time PCR (upper right panel), while the protein expression level was assessed using ELISA (lower right panel). h The cell growth rate was evaluated by counting the number of cells every 2 days over a period of 8 days (right upper panel). EdU incorporation assay. Same experiment scheme as (g) was applied for EdU incorporation study (left panel). i SLIT2 was knocked down in young cells infecting shSLIT2 lentivirus. The knockdown was then rescued by treating the cells with rhSLIT2 protein at a concentration of 100 ng/ml for 2 days. SAA1 and IL1β mRNA expression levels were analyzed with real-time PCR. j Mid-old cells were co-cultured with sh-control or sh-SLIT2 lentivirus infected young cells for 30 days. At the experimental endpoint, the morphology of mid-old fibroblasts was observed (left panel), and EdU incorporation assay (middle panel) and cell proliferation (right upper panel) was measured. The p values of (a–c), (d) (lower panel), and (e–j) are obtained from one-tailed Mann–Whitney U test. The p value of (d) (right upper panel) is obtained from two-tailed Student’s t test. The graphs in (a–j) were represented as mean ± SD.
