Figure 6

Cellular levels of SDIM1 modulate the rates of cell death. N2a or ERK293 cells were transiently transfected with pSDIM1*EGFP plasmid. NT2 cells were transfected with synthetic siRNAs. Cells were collected for total RNA and protein extractions 16-24 h after transfections. Trypan Blue exclusion assay was performed 21 or 24 h after transfection or 16 h after a 6 h OGD treatment of the transfected samples. Over expression of SDIM1 mRNA was assessed by RT-PCR (A) and Western analysis (B). Lane C - negative PCR control, lane V- mock transfection with pEGFP-N1 vector, lane 16 h - transfection with pSDIM1*EGFP plasmid for 16 h, lane 18 h - transfection with pSDIM1*EGFP plasmid for 18 h, lane 21 h - transfection with pSDIM1*EGFP plasmid for 21 h, lane 24 h - transfection with pSDIM1*EGFP plasmid for 24 h. C. Percentage of cell death after pSDIM1*EGFP transfection for 21 h. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (p-value = 0.0096). D. Assessment of the siRNA silencing efficiency. RNA and protein samples were collected 24 after transfection with siRNAs. Down regulation of SDIM1 was analyzed by RT-PCR and Western blotting. E. Percentage of cell death 24 h after siRNA transfection, with or without 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 (p-value = 0.001). Ctrl24 - 24 h after transfection with negative control siRNAs, siRNA24 - 24 h after transfection with SDIM1 pool siRNAs, Ctrl24-OGD - 24 h after transfection with negative control siRNAs plus OGD treatment, siRNA24-OGD - 24 h after transfection with pool SDIM1 siRNAs plus OGD treatment.