Fig. 3
From: MTCH2-mediated mitochondrial fusion drives exit from naïve pluripotency in embryonic stem cells
MTCH2 loss delays exit from naïve pluripotency. a MTCH2−/− cells fail to adequately elongate mitochondria following priming. Left panels: Representative IF images (magenta, anti-TOM20 Abs; yellow, DAPI; Scale bar, 5 mM) of MTCH2F/F and MTCH2−/− cells. Right panel: Aspect ratio calculations of MTCH2F/F and MTCH2−/− cells mitochondria. Results are presented as mean ± SEM (***p<0.001; Anova statistical assay; n>60 cells were scored; n=3). Inset: Steeper elongation of MTCH2F/F mitochondria after priming. b MTCH2F/F EpiLCs preferentially increase glutamine utilization. Mass isotope tracing (Inset: schematic representation of TCA cycle) of MTCH2F/F ESCs and EpiLCs. Labeled fractions for each metabolite derived from either 13C6-glucose (13C-glc; M+2 is shown; left panel) or from 13C5-glutamine (13C-gln; M+4 or M+5 are shown; right panel) are plotted, after 6 hours incubation. Percentages were calculated using Metran software. Results are presented as mean ± SD (*p<0.05; **p<0.01, ***p<0.001; n=3). c MTCH2−/− cells fail to preferentially increase glutamine utilization following priming. Labeling and percentages were calculated as described in (B). Results are presented as mean ± SD (*p≤0.05; **p<0.01, ***p<0.001; n=3). d MTCH2−/− EpiLCs maintain high levels of acetylated histone 3. Inset: A representative Western blot is presented. Bottom panel: densitometry quantification of three separate Western blots. Results are presented as mean ± SD (*p<0.05; ***p<0.001; n=3). e MTCH2−/− ESCs and EpiLCs clustering similarities. Principal component analysis (PCA; left panel) and hierarchical clustering (right panel) of MTCH2F/F and MTCH2−/− cells based on differentially expressed genes (n=3). f MTCH2−/− cells show mild changes in gene expression following priming. Heat map showing the ratio of MTCH2F/F (left column) and of MTCH2−/− (right column) EpiLCs (AF) to ESCs (2i/L) RNA levels of stem cell maintenance genes (left panel) and stem cell differentiation genes (right panel) as obtained by RNA-seq (n=3). g MTCH2−/− EpiLCs fail to reduce the expression of naïve pluripotency genes. RNA levels of naïve pluripotency genes (top panel) and developmental genes (bottom panel) following priming. Results are presented as mean ± SD (*p<0.05; **p<0.01; ***p<0.001; n=3). h MTCH2−/− EpiLCs maintain high expression levels of Nanog. Representative IF images of MTCH2F/F and MTCH2−/− cells (n=3). (Scale bar, 10 μM)
