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. 2015 Nov 26;5:17004. doi: 10.1038/srep17004

Figure 2. siRNA knockdown of key autophagy proteins sensitises cells to MPP+ induced cell death.

Figure 2

(a–c) Efficient RNAi-mediated knockdown of P62 and GABARAP in BE(2)M17 cells. Cells transfected with appropriate targeting or scrambled control siRNAs (50 nM) were harvested 72 h after transfection. Protein levels were measured by western blot and band density was normalised relative to actin. Representative images are shown (n = 3). (d–i) RNAi-mediated knockdown of P62 and GABARAP in BE(2)M17 cells exacerbates sensitivity to MPP+. Cells were transfected with appropriate targeting or scrambled control siRNAs (50 nM), treated with MPP+ (100 μM) and assayed. Combined KD indicates simultaneous P62 and GABARAP knockdown. (d,e) Cell death was assessed by counting the number of morphologically normal cells in bright field images at 48 h post-MPP+ treatment. (f,g) Cell viability was assessed by neutral red uptake at 36 h post-MPP+ treatment. (h,i) Mitochondrial membrane polarisation was measured using TMRM fluorescence 24 h post-MPP+ treatment. Bars represent mean values normalised to untreated control cells ± SEM (n = 3), data were analysed using a two way ANOVA with Sidak multiple comparison test *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.