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. 2023 May 29;114(8):3176–3189. doi: 10.1111/cas.15852

FIGURE 4.

FIGURE 4

TRIM29 mediates senescence by a mechanism involving reactive oxygen species (ROS) accumulation. (A) The intracellular level of ROS was measured in CNE2 and HONE1 cells by 2′,7′‐dichlorodihydrofluorescein (DCF) fluorescence using flow cytometry. (B) Graphs show the quantification of fluorescence mean. (C) Mitochondrial superoxide of cells was measured by MitoSOX (5 μM, 30 min) and stained with Hoechst 33342. Representative immunofluorescent images are shown. (D) JC‐1 red and green fluorescence were measured by flow cytometry in HONE1 cells expressing control (shNC), shT#1, or shT#2. JC‐1 Red / JC‐1 Green ratios are plotted in the right panel. (E) HONE1 shGFP and shT#1‐Tet‐On cells were pretreated with indicated concentrations of n‐acetyl‐cysteine (NAC) (2 h), followed by cotreatment with doxycycline (1 μM) for 48 h. ROS generation was measured by DCF fluorescence using flow cytometry. (F) Senescence‐associated β‐galactosidase (β‐gal) staining was carried out in cells treated as described in (E). Photographs (left) (original magnification, ×100) and statistical graphs (right) are shown. *p < 0.05; **p < 0.01; ***p < 0.001.