Research Paper Volume 13, Issue 3 pp 4063—4078

PRPS1-mediated purine biosynthesis is critical for pluripotent stem cell survival and stemness

PRPS1 is critical for PSCs survival and stemness by controlling purine synthesis. (A) Representative images of cells at 24 h post sgRNA-PRPS1 lentivirus infection or at second passage (P2). Bars: 20 μm. (B) FACS analysis of cell apoptosis in PSCs at Day 3 after sgPRPS1 or sgPRPS2 lentivirus transduction. *** PC) WB of the expression of DNA damage and apoptosis marker proteins in the PSCs from (B). (D, E) FACS analysis of cell apoptosis (D) and cell cycle (E) in DOX-induced PRPS1 KD PSCs with or without ectopic expression of PRPS1 M115T mutant. DOX was added for 0h, 72h, or 24 h to induce PRPS1 KD. At 24 h, DOX were removed to rescue PRPS1 expression. PRPS1-M115T mutant, an enzymatically inactive PRPS1 mutant. (F) WB of the expression of DNA damage and apoptosis marker proteins in the PSCs from (D). (G) qRT-PCR analysis of the expression levels of pluripotency genes in the PSCs from (D). (H) FACS analysis of cell apoptosis in response to HX treatment based on PRPS1 KD. (I) WB of the expression of DNA damage and apoptosis marker proteins in response to HX treatment based on PRPS1 KD. (J) WB of the expression of DNA damage and apoptosis marker proteins in response to Gart inhibitor, lometrexol, treatment. (K) qRT-PCR analysis of the expression levels of pluripotency genes in the PSCs from (J). In (B, D, E, G, H and K), data are expressed as the mean ± SD. *P P t-test. Data are representative of three independent experiments with similar results.

Figure 5. PRPS1 is critical for PSCs survival and stemness by controlling purine synthesis. (A) Representative images of cells at 24 h post sgRNA-PRPS1 lentivirus infection or at second passage (P2). Bars: 20 μm. (B) FACS analysis of cell apoptosis in PSCs at Day 3 after sgPRPS1 or sgPRPS2 lentivirus transduction. *** P< 0.001. sgControl, a control sgRNA. (C) WB of the expression of DNA damage and apoptosis marker proteins in the PSCs from (B). (D, E) FACS analysis of cell apoptosis (D) and cell cycle (E) in DOX-induced PRPS1 KD PSCs with or without ectopic expression of PRPS1 M115T mutant. DOX was added for 0h, 72h, or 24 h to induce PRPS1 KD. At 24 h, DOX were removed to rescue PRPS1 expression. PRPS1-M115T mutant, an enzymatically inactive PRPS1 mutant. (F) WB of the expression of DNA damage and apoptosis marker proteins in the PSCs from (D). (G) qRT-PCR analysis of the expression levels of pluripotency genes in the PSCs from (D). (H) FACS analysis of cell apoptosis in response to HX treatment based on PRPS1 KD. (I) WB of the expression of DNA damage and apoptosis marker proteins in response to HX treatment based on PRPS1 KD. (J) WB of the expression of DNA damage and apoptosis marker proteins in response to Gart inhibitor, lometrexol, treatment. (K) qRT-PCR analysis of the expression levels of pluripotency genes in the PSCs from (J). In (B, D, E, G, H and K), data are expressed as the mean ± SD. *P <0.05, ***P <0.001, by two-tailed t-test. Data are representative of three independent experiments with similar results.