Journal: eLife

Article Title: A critical role for heme synthesis and succinate in the regulation of pluripotent states transitions

doi: 10.7554/eLife.78546

Figure Lengend Snippet: ( a ) Immunostaining of succinylated lysine residues (green) in mESCs in 2iL media or treated with 250 nM AA5. Representative image of n=3 independent experiments and quantified by flow cytometry. T-test. Scale bar = 20 μm ( b ) Percentage of ZSCAN4-positive cells in the whole population of naïve (2iL) mESCs or naïve treated with 250 nM AA5, with or without 1 μM diethyl butylmalonate (BM), counted from confocal micrographs with 10 images per conditions for at least 1000 cells per condition. n=3 independent biological replicates. ANOVA1 ( c ) Relative expression of the 2CLC genes in response to 250 nM AA5 or 0.5 µM retinoic acid (RA), with or without 10 µM HX531 and 100 nM AGN193109. ( d-e ) Relative expression of 2C-gene markers of mESCs assessed by RT-qPCR relative to Gapdh expression in naïve 2iL mESCs and treated with 16 mM dimethyl succinate, 40 mM sodium succinate (Na succinate), 1 μM of MCT1 and 4 inhibitor (AZD3965; AZD) and 1 μM SUCNR1 receptor antagonist (NF-56-EJ40; NF56). Data shown as mean +/-S.D. *p<0.05, **p<0.01, ***p<0.001. ANOVA-1 followed by a Tukey post-test. n=4 independent biological replicates. ( f ) Graphical representation of the succinate-induced 2CLC reprogramming. Created with https://www.biorender.com/ .

Article Snippet: For retinoic acid experiments, RA, HY-14649, HX531 (HY-108521) and AGN193109 (HY-U00449) were used at 0.5 µM, 10 µM and 100 nM, respectively (all from Medchemexpress).

Techniques: Immunostaining, Flow Cytometry, Expressing, Quantitative RT-PCR