Figure 6. Knockdown of Sepp1 transcript variants with siRNA increases apoptosis after oxidative challenge. Viability and apoptosis were assessed by flow cytometry for propidium iodide and Annexin V fluorescence following RNAi-mediated knockdown of Sepp1a, b or c transcripts in N2a murine neuronal cells. (A) Confirmation of siRNA efficacy and specificity by qPCR measurement of RNA normalized to 18S rRNA for total Sepp1 and for each variant, shown as median ± range. (B) western blot (above) showing Sepp1 in media of N2A cells following siRNA transfection, and the 67 kD albumin band (below). The (C) Quantification of the 60 kD Sepp1 band using albumin as a loading control, relative to negative control siRNA samples. (D) Quantification of 55 kD band. (E) Percent viable cells negative for both propidium iodine and annexin V. (F) Percent apoptotic cells defined as negative for propidium iodide but positive for annexin V. * indicates p < 0.05 and ** indicates p < 0.01, 2-way ANOVA with Bonferonni’s posthoc test.