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. 2022 Dec 2;13:7414. doi: 10.1038/s41467-022-35199-0

Fig. 3. Nuclear TCA cycle enzymes modulate somatic cell reprogramming, primed-to-naive transition and totipotency acquisition.

Fig. 3

a, b Relative somatic cell reprogramming efficiency after transducing with nuclear-targeted TCA cycle enzymes together with SKO (a) or SKOM (b). The reprogramming efficiency was determined by counting the GFP-positive colonies. c, d The relative somatic cell reprogramming efficiency after overexpression of Pdha1 or nls-Pdha1 together with SKO (c) or SKOM (d). e, f The relative somatic cell reprogramming efficiency after overexpression of nls-Pdha1 or nuclear-targeted catalytic mutants of Pdha1-S293A, S293A + S300A or S232A + S293A + S300A (Triple mutant)-together with SKO (e) or SKOM (f). g Representative images (upper panel) and flow cytometry analysis (lower panel) (gated on Oct4-GFP cells) of GFP-positive naive stem cell colonies generated from EpiSCs with nls-Pdha1, nls-Pcb, nls-Aco2, nls-Cs or nls-Idh3a overexpression. The Flag was used as control. Scale bars, 250 µm. Three independent experiments were repeated with similar results. h The relative PNT efficiency after overexpression of nls-Pdha1, nls-Pcb, nls-Aco2, nls-Cs, and nls-Idh3a. i Representative images (upper panel) and flow cytometry analysis (lower panel) (gated on Oct4-GFP cells) of GFP-positive naive stem cell colonies generated from EpiSCs with nls-Pdha1, S293A, and Triple mutant. Scale bars, 250 µm. Three independent experiments were repeated with similar results. j The relative PNT efficiency after overexpression nls-Pdha1, nls-Pdha1, S293A and Triple mutant. k Left: Schematic of OG2 ESCs with 2C:: tdTomato reporter induced into totipotency stem cells. Right: Flow cytometry (gated on 2C:: tdTomato cells) showing the relative proportion of 2C:: tdTomato-positive totipotency stem cells generated from OG2 ESCs with overexpression of nls-Pdha1 or nuclear-targeted catalytic mutants of Pdha1 (right). Data are presented as the mean ± S.D (a–f, h, j), and n = 3 independent experiments (a–f) or n = 4 independent experiments (h, j). A two-tailed unpaired Student’s t test was used (a–f, h, j).