Fig. 4

IGF2 knockdown stimulates senescence and senescence-associated secretory phenotypes in d-gal and H₂O₂ treated AML12 cells. AML12 cells were transfected with sh-IGF2 or its corresponding negative control sh-NC treated with d-gal, H₂O₂ or the PBS control. Western blotting analysis (A) and RT‒qPCR analysis (B) for the expression of IGF2 protein and mRNA. C The senescence of AML12 cells detected by SA-β-gal staining. SA-β-gal staining area analysis was shown. Scale bar, 50 μm. D Immunofluorescence analysis of γ-H2AX expression in AML12 cells. Fluorescence intensity analysis was shown. Scale bar, 50 μm. E Western blot analysis to measure the senescence-associated genes P53, P21 and P16 in AML12 cells. The quantitative data are presented. F Cell viability of AML12 cells tested by CCK-8 assay. G The cell cycle distribution of AML12 cells analyzed by flow cytometry. H RT‒qPCR analysis of mRNA levels of senescence-associated secretory phenotype genes IL-6, IL-1β, TNF-α and NF-κB1. Data are shown as means ± SEM. One-way ANOVA was used for comparison among multiple groups. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. All experiments were repeated three times independently