Fig. 6: RNF166 accelerates apoptosis induced by 6-OHDA.

a–c MN9D cells transfected with blank vector or vector encoding the mouse RNF166 cDNA sequence were treated with 100 µM 6-OHDA for the indicated time periods. a Following treatment with 6-OHDA for 18 h, the rate of cell survival was determined using an MTT reduction assay. Values are expressed as a percentage of untreated control cells (100%). Bars represent the mean + SEM of five independent experiments in triplicate. ****p < 0.0001, *p < 0.05. b Soluble DNA was extracted from mock-transfected or RNF166-transfected cells in lysis buffer containing proteinase K and subjected to electrophoresis on 1.5% agarose gel. Subsequently, gels were stained with ethidium bromide and then photographed on an UV transilluminator. c Cell lysates obtained from each group at the indicated time periods after 6-OHDA treatment were subjected to immunoblotting using the indicated antibodies, including anti-cleaved caspase-3 antibody. Densitometric values of C-cas-3 are expressed as a relative fold-change after normalization against GAPDH. Bars represent the mean + SEM from three independent experiments. ****p < 0.0001, *p < 0.05. d MN9D cells were transiently transfected with RNF166 siRNA and exposed to 100 µM 6-OHDA for 12 h. Cell lysates were subjected to immunoblotting using anti-cleaved caspase-3 and anti-RNF166 antibodies. Densitometric values of C-cas-3 are expressed as fold-change relative to control siRNA–transfected cells after normalization against GAPDH (value = 1). Bars represent the mean + SEM from three independent experiments. Quantitative analysis was performed as described above. ***p < 0.001, *p < 0.05. e For rescue experiments, MN9D cells transfected with RNF166 siRNA for 48 h were further transfected with or without vector encoding V5-RNF166 for an additional 24 h. Cells were then treated with 100 µM 6-OHDA for 12 h. Cell lysates were subjected to immunoblotting using anti-cleaved caspase-3 or anti-RNF166 antibody. Densitometric values of C-cas-3 are expressed as fold-change relative to the non-transfected control cells after normalization against GAPDH (value = 1). Bars represent the mean + SEM from the independent experiments. **p < 0.01; *p < 0.05.