Figure 5

Cellular levels of RNF182 modulate the rates of cell death. N2a cells were transiently transfected with RNF182/pcDNA3.1/myc-his plasmid or mouse RNF182 on-target plus smart pool siRNAs. Cells were collected for total RNA extractions 24–48 h after transfections. Trypan Blue exclusion assay was performed 24 h after transfection or 16 h after a 7 h OGD treatment of the siRNA transfected samples. A. Over expression of RNF182 mRNA was assessed by RT-PCR. In: lane 1 – negative PCR control, lane 2 – mock transfection, lane 3-transfection with RNF182/pcDNA3.1/myc-his plasmid. B. Percentage of cell death before and after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). C. Percentage of cell death before and after transfection with transfection reagent removed 6 h after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). D. Percentage of cell death of the control and 24 h transfected samples subjected to a 7 h OGD treatment. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). E. Assessment of the siRNA silencing efficiency. RNA samples were collected 24 and 48 h after transfection with siRNAs. Down regulation of RNF182 mRNA was analyzed by RT-PCR. In: lanes 1 and 3 – 24 and 48 h after transfection with RNF182 siRNAs, lanes 2 and 4 – 24 and 48 h after transfection with non-targeting pool negative control siRNAs, respectively. F. Percentage of cell death 24 h after siRNA transfection, with or with out OGD treatment. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisks indicate a significant difference (ρ < 0.005; t-test). C24 – 24 h after transfection with non-targeting pool negative control siRNAs, siRNA24 – 24 h after transfection with mouse RNF182 on-target plus smart pool siRNAs, c24-OGD – 24 h after transfection with non-targeting pool negative control siRNAs plus OGD treatment, siRNA24-OGD – 24 h after transfection with on-target plus smart pool siRNAs plus OGD treatment.