Research Paper Volume 16, Issue 9 pp 7683—7703

Figure 4. Iron retention in lysosomes increases in senescent HSkM cells. (A) Colocalization analysis of lysosome and Fe²⁺. Proliferating and senescent cells were co-stained with Lysosome dye (Lysotracker, in green) and Fe²⁺ probes (FerroOrange, in red), and observed by a confocal laser scanning microscopy. Representative live-cell fluorescence images were presented on the left panel, and intensity profile of lysosome and Fe²⁺ signals on the line across the cells were shown on the right panel. Scale bars, 20 μm. (B) Representative images of proliferating cells and senescent cells stably expressed the tandem reporter mCherry-GFP-LC3 to visualize the autophagic flux. Nuclei were stained with DAPI. Scale bars, 20 μm. (C) Immunoblot analysis of LC3 and p62 protein expression in normal and senescent HSkM cells. And three independent biological experiment repeats have been performed. (D) Quantification of the ratio of LC3-II: LC3-I and P62 protein expression. Each protein band intensity was measured and normalized to GAPDH and compared between proliferating and senescent cells. Data represented as mean ± SD. P-values were calculated by two-tailed unpaired student’s t-test, ^*P < 0.05. (E) Dose-dependent toxicity assay of lysosomotropic agents chloroquine and LLOMe in proliferating and senescent HSkM cells. The proliferating and senescent cells were treated with a series of concentrations of CQ and LLOMe for 24 h, followed by the cell survival measurement by CCK-8 assay. Data represented as mean ± SD. And three independent biological experiment repeats have been performed. P-values were calculated by one-way ANOVA analysis with post hoc Tukey, ^***P < 0.001.