Fig. 5: NAC treatment reduces ROS accumulation and cell death in HeLa EZR^−/− cells.

A Heatmap representing the 65 upregulated genes in HeLa treated with NSC668394 (FDR ≤ 0.05) involved in cell death and apoptotic processes. B Bubble plot representing the biological process associated to upregulated genes in HeLa treated with NSC668394. C Co-expression, obtained by GeneMANIA, highlight Ezrin and apoptotic protein binding. D HeLa WT and HeLa EZR^−/− were cultured in 6-well plates for 24 h. Subsequently, they were treated with an Annexin V/Propidium Iodide mix for 20 min and DAPI (blu). The images show an increase in cell death in HeLa EZR^−/− compared to HeLa WT, while NAC drastically reduces cell death in HeLa EZR^−/−. E HeLa WT and EZR^−/− cells were cultured in 6-well plates for 24 h. They were then treated with 1.5 mM NAC for 1 h and subsequently trypsinized. A mix of Trypan Blue and cells was prepared and counted using the LUNA cell counter. The graph shows the individual viability percentages for HeLa WT, HeLa WT + NAC, HeLa EZR^−/−, and HeLa EZR^−/− + NAC ± SEM (at least n = 3 experiments). Statistical test: unpaired t-test. F HeLa WT and EZR^−/− cells were cultured in 6-well plates for 24 h. They were then treated with 1.5 mM NAC for 1 h and incubated with the fluorescent dye H2DCFDA (green) and DAPI (blue). The images show ROS reduction when NAC is used in HeLa EZR^−/−. G HeLa WT and EZR^−/− cells were cultured in 96-well plates for 24 h. They were then treated with Presto blue reagent. The graph shows a reduction in relative absorbance levels percentage in HeLa EZR^−/− cells compared to HeLa WT, reflecting a lower cell viability in HeLa EZR^−/− cells.