Figure 3.

PLA2R induces senescence through the production of reactive oxygen species. (A) Young WI38 cells were infected with control (Ctrl) or PLA2R-expressing vectors. After 2 weeks, the cells were loaded with H₂DCFDA and the fluorescence was analysed by microscopy and flow cytometry. Pictures and relative mean fluorescence are shown (upper panel). WI38 cells approaching senescence were infected with a control vector or a shPLA2R construct, and when control cells entered senescence, the cells were loaded with H₂DCFDA and the fluorescence was analysed as above (lower panel). (B) After infection, 2.5 mM N-acetyl-cysteine (NAC) was added or not from day 1 after infection and renewed every 2 days. The cells were stained with crystal violet 2 weeks later or pictures were taken after 10 days. (C) WI38 cells were analysed for their SA-β-gal activity, and the percentage of positive cells in each condition was calculated. OE, overexpression; PLA2R, phospholipase A2 receptor; ROS, reactive oxygen species; SA-β-gal, senescence-associated β-galactosidase; sh, short hairpin.