Research Paper Volume 16, Issue 9 pp 7683—7703

Figure 2. Dox-induced cellular senescence increases Fe²⁺ accumulation and lipid peroxidation and alters the ferroptosis-related gene expression. (A) Representative live-cell fluorescence images of intracellular Fe²⁺ in proliferating and senescent cells. Fe²⁺ was detected by the probe of FerroOrange. Nuclei was stained with Hoechst 33342. Scale bars, 20 μm. (B) Representative fluorescent images of C11-BODIPY 561/591 staining shows the increase of lipid peroxidation in senescent HSkM cells. Red represents reduced BODIPY-C11 signal and Green represents oxidized BODIPY-C11 signal. Nuclei were stained with Hoechst 33342. Scale bars, 20 μm. (C) MDA detection assay. The MDA production assay was performed in both proliferation and senescent cells. P-values were calculated by two-tailed unpaired student’s t-test, ^*P < 0.05. Data represented as mean ± SD. And three biological experiment repeats have been performed. (D, E) Western blot analysis of ferroptosis-response protein expression in proliferating and senescent HSkM cells. (D) Immunoblot representative images and (E) quantification of ACSL4, GPX4, FTH1, FSP1, SLC7A11, SLC40A1, CD71/TFR1 and SLC11A2/DMT1 protein expression. Each protein band intensity was measured and normalized to GAPDH and compared between proliferating and senescent cells. P-values were calculated by two-tailed unpaired student’s t-test, ^*P < 0.05, ^**P < 0.01. Data represented as mean ± SD. And three independent biological experiment repeats have been performed.