Additional file 1 of Crotonylation of GAPDH regulates human embryonic stem cell endodermal lineage differentiation and metabolic switch
Creators: name: Zhang, Jingran
name: Shi, Guang
name: Pang, Junjie
name: Zhu, Xing
name: Feng, Qingcai
name: Na, Jie
name: Ma, Wenbin
name: Liu, Dan
name: Songyang, Zhou
Publisher: name: figshare
Year: 2023
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1. Figure S1 (A, B) H9-LTR7 GFP reporter hESCs were cultured first in LCDM medium for 15 passages to derive EPS cells that were immunostained with antibodies against NANOG (A) or OCT4 (B). Primed reporter hESCs served as controls. Scale bars, 100 μm. (C, D) Primed H9-LTR7 GFP reporter hESCs were differentiated as shown. (E, F) The differentiated endodermal cells (E) and ectodermal cells (F) were analyzed by microscopy. (G, H) RT-qPCR assays for lineage marker expression in endodermal cells (G) and ectodermal cells (H) were shown. OCT4, NANOG and SOX2 pluripotency markers. GATA4, GATA6 and SOX17, endoderm markers. SOX1, NESTIN and PAX6, ectoderm markers. Primed reporter hESCs served as controls. Scale bars, 100 μm. Error bars represent mean ± S.D. (n = 3 independent experiments). Figure S2 (A) The mouse embryonic stem cell line E14 was cultured in the presence of crotonate at the indicated concentrations for 24 h. Whole-cell lysates were probed with the indicated antibodies. (B–F) E14 cells treated for 3 days with the indicated concentrations of acetate or crotonate were examined under microscopes (scale bars of 100 μm) (B). The percentages of naïve and differentiated clones were quantitated and graphed (200 clones/group) for cells treated with acetate (C) or crotonate (D). RT-qPCR analysis of the indicated marker genes was carried out using cells treated with acetate (E) or crotonate (F). Oct4 and Nanog, pluripotency markers. Gata6 and Sox17, endoderm markers. Mixl1 and Cd34, mesoderm markersFull. Sox1 and Nestin, ectoderm markers. Error bars represent mean ± S.D. (n = 3 independent experiments). Figure S3 (A) H9-LTR7 GFP reporter hESCs and hESCs treated with crotonate for 1–6 days were immunoprecipitated (IP) with anti-GAPDH antibody. The immuoprecipitates were then western blotted as indicated. IgG served as a negative control. (B) Extracellular acidification rate (ECAR) analysis of endodermal cells and hESCs treated with 10 mM crotonate for different time points (day 0, day 1, day 2, day 3, day 6, day 12). Glucose (10 mM), oligomycin (1 mM), and 2-deoxyglucose (2-DG, 50 mM) were used. (C) Glycolytic rates and glycolytic capacity for endodermal cells and hESCs treated with 10 mM crotonate for 0 day, 1 day, 2 days, 3 days, 6 days and 12 days. Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. *p &lt; 0.05; **p &lt; 0.01. (D, E) Recombinant ENOA was incubated with crotonyl-CoA and recombinant p300 and analyzed by western blotting with the indicated antibodies (D) or assessed for ENOA activity (E). Coomassie blue staining served as loading control. Error bars represent mean ± S.D. (n = 3 independent experiments). (F) H9-LTR7 GFP hESCs were treated with 10 mM crotonate for 12 days and then harvested to assess the activity of in vivo crotonylated ENOA. Untreated cells and endodermal cells derived from the reporter cells served as controls. Error bars represent mean ± S.D. (n = 3 independent experiments). (G) Recombinant His-tagged wild-type, single and double point mutant GAPDH proteins were purified from E.coli (BL21), incubated with crotonyl-CoA and recombinant p300 and assessed for GAPDH activity. NS, not significant. **p &lt; 0.01.