Figure 6. DRP1 over-expression inhibits the apoptotic response during the onset of pluripotent stem cell differentiation.

A. ATP-b immunostaining showing mitochondrial morphology in wild-type and Drp1^OE cells during differentiation. Scale bar =10μm. B. Circularity measurement of mitochondrial particles from ATP-b immunostained images of wild-type and Drp1^OE cells at day 3 of differentiation. C. Quantitative RT-PCR showing gene expression levels of naïve and primed pluripotency markers in wild-type and Drp1^OE ESCs cultured in pluripotency (Plurip.) or differentiation (Diff.) conditions. Gene expression normalised against Gapdh. D. Fold change in basal cleaved-CASPASE 3 levels in wild-type and Drp1^OE cells at day 3 of differentiation, quantified from Supplementary Figure 4D, F and G. E. Fold change in cleaved-CASPASE 3 levels in wild-type and Drp1^OE cells untreated or treated with 1μM thapsigargin (Tg) for 5h. Quantified from Supplementary Figure 4D. F. Fold change in cleaved-CASPASE 3 levels in wild-type and Drp1^OE cells untreated or treated with 1μM sodium arsenate (NaAsO₂) for 5h. Quantified from Supplementary Figure 4F. G. Fold change in cleaved-CASPASE 3 levels in wild type and Drp1^OE cells untreated or treated with 1μM ABT-737 for 5h at day 3 of differentiation. Quantified from Supplementary Figure 4G. Protein levels in D. E. F. and G. are against α-TUBULIN and graphs show protein expression levels relative to wild-type cells. normalised Average of 3 (C, E, F) or 5 (D) independent experiments +/− SEM is shown. Students T-Test (D) or 2-way ANOVA with Šidák correction (E, F, G) ****p<0.0001. The statistical comparisons are made to untreated cells. Drp1^OE 1 and 2 represent two independent Drpl^OE lines. See also Figure S4.