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. 2021 Sep 21;9:744838. doi: 10.3389/fcell.2021.744838

FIGURE 3.

FIGURE 3

Lacking Opa1 triggers apoptosis in HEI-OC1 and Neuro 2a cells. (A,B) CCK-8 was used to assess the cell viability of HEI-OC1 (A) and Neuro 2a (B) cells cultured for 24, 48, and 72 h after transfection with scramble siRNA (Ctrl) or siOpa1. *p < 0.05, **p < 0.01, ***p < 0.001 (two-way ANOVA). (C) Representative confocal microscopic images of cleaved caspase-3 staining at 24 h after siRNA transfection of HEI-OC1 and Neuro 2a cells. Representative transmission electron microscopic images of HEI-OC1 cells at 24 h after siRNA transfection. (D) Representative confocal images of HEI-OC1 cells co-stained for mitochondria (MitoTracker) and cytochrome c at 24 h after siRNA transfection. (E) Representative flow cytometric plots generated from HEI-OC1 cells stained with Annexin V-FITC/PI to detect apoptosis at 24 h after transfection with scramble siRNA (Ctrl) or siOpa1. (F) The percentages of early and late apoptotic cells were compared between Ctrl-siRNA- and siOPA1-transfected HEI-OC1 cells. **p < 0.01 (two-way ANOVA). FITC, fluorescein isothiocyanate; PI, propidium iodide. (G) Representative flow cytometric plots generated from Neuro 2a cells stained with Annexin V-FITC/PI to detect apoptosis at 24 h after transfection with scramble siRNA (Ctrl) or siOpa1. (H) The percentages of early and late apoptotic cells were compared between Ctrl-siRNA- and siOPA1-transfected Neuro 2a cells. **p < 0.01 (two-way ANOVA). (I,J) Quantification of Bcl2 mRNA expression levels in HEI-OC1 (I) and Neuro 2a (J) cells transfected with siRNA for 24 h. *p < 0.05, **p < 0.01 (non-parametric Mann-Whitney t-test). (K) Representative Western blot images showing Bcl2 protein levels in Neuro 2a cells transfected with siRNA for 48 h.