Fig 1: The effects of SOX14 ectopic expression. A: Effect of SOX14 ectopic expression on the activity of the SOX-responsive luciferase reporter gene. The plasmid 3SXluc was co-transfected into HeLa cells with either pcDNA3.1 vector or pcDNA3.1/SOX14 expression construct. Normalized luciferase activities were calculated as a fold of the 3xSX luc activity in cells co-transfected with vector pcDNA3.1+3SXluc, which was set as 100%. Data are presented as the mean ± S.E.M. of four independent transfections. Mean values of relative luciferase activities were compared with Student's t-test. The value of p≤0.01 is represented by *. B: Effects on SOX1, SOX2, SOX3 and SOX21 protein levels in HeLa cells analyzed by Western blot. Protein levels were analyzed 24 h, 48 h, and 72 h after transfection with pcDNA3.1/SOX14 expression construct. Transfection with pcDNA3.1 vector (designated as C) was used as a control for transfection. Protein extracts from NT2/D1 cells were used as positive controls for SOX1, SOX2 and SOX3 expression. Protein extract from HeLa cells transiently transfected with SOX21 expression construct was used as a positive control for SOX21 expression. α-Tubulin was used as a loading control. C: Quantification of the effects of SOX14 overexpression on SOX1 protein levels presented in B. The quantities of SOX1 protein in transfected cells were calculated as a percentage of the quantity in cells transfected with pcDNA3.1 vector, which was set as 100%. Data are presented as the means ± S.E.M. of three independent transfections experiments. Mean values were compared with Student's t-test. The value of p≤0.05 is represented by *.
Fig 2: E2 exposure up‐regulated insulin‐like growth factors (IGF)‐1 and marker genes’ expression of ectoderm layers during human embryonic stem cells (hESCs) differentiation period. (A) IGF‐1, NESTIN and SOX‐1 were measured by qPCR assay at three time‐points. E2 increased the expression of ectoderm markers of NESTIN (days 0, 3 and 7), SOX1 (days 0, 3 and 7) and IGF‐1 (days 0, 3 and 7), while ICI or JB1 singly applied repressed NESTIN (days 0, 3 and 7), SOX1 (days 3 and 7) and IGF‐1 (days 0, 3 and 7) expression partially. Inhibitors combination (ICI plus JB1) significantly reduced NESTIN (days 3 and 7), SOX1 (days 3 and 7) and IGF‐1 (day 7) expression. (B) At day 7, IGF‐1, NESTIN and SOX‐1 were measured by FACS assay. Results indicated that one inhibitor just curbed E2 effect slightly, and inhibitors combination strongly decreased the population of NESTIN+SOX‐1+ cells significantly, n = 3; Error bars indicate SD. *P < 0.05, **P < 0.01; ***P < 0.001 (compared with the DMSO group). #P < 0.05; ##P < 0.01; ###P < 0.001 (compared with the E2 group)
Fig 3: Hif-1α overexpression promoted the mesendoderm differentiation but repressed the ectoderm differentiation. A Hif-1α overexpression upregulated T protein expression under normoxia. T (red) was stained on differentiation day 4. Nuclei were stained with DPAI (blue). B Hif-1α overexpression induced by Dox supplementation repressed the expression of ectoderm markers (Pax6 and Nestin), although the difference in Nestin levels was not significant. C TOPFlash assays showed that Hif-1α knockdown significantly repressed the Wnt/β-Catenin pathway activation. D TOPFlash assays showed that Hif-1α overexpression significantly promoted the Wnt/β-Catenin pathway activation. E, F The expressions of Hif-1α in Hif-1α-iOE AB2.2 mESCs treated with 1, 10, 100, and 1000 ng/mL Dox, respectively. Normoxic and hypoxic cultures without dox treatment were used as controls. G Schematic diagram of Hif-1α-iOE AB2.2 mESC differentiation treated with 1, 10, 100, and 1000 ng/mL Dox, respectively. Normoxic and hypoxic cultures without dox treatment were used as controls. H The expression changes of mesendoderm markers (T, Eomes, Mesp1, and Gsc) on day 4 of the differentiation described in G. I The ratios of T+ and Sox1+ cells on day 4 of the differentiation assays in G were detected by flow cytometry. scramble, AB2.2/scramble cells; shHif-1α, AB2.2/shHif-1α cells; DOX, doxycycline; *, significant (P<0.05)
Fig 4: Hif-1α knockdown repressed the mesendoderm differentiation and promoted the ectoderm differentiation. A, B The expression patterns of Hif-1α in differentiating AB2.2 cells under normoxia and hypoxia. C Schematic diagram of AB2.2 mESC differentiation treated with normoxia and hypoxia on differentiation days 0–2 and 2–4, respectively. D The expression of mesendoderm markers (T, Eomes, Mesp1, and Gsc) was repressed to the same extent by hypoxia on differentiation days 0–2 and 2–4. E, F Hif-1α knockdown was verified by western blotting analysis under hypoxia. G Hif-1α knockdown inhibited T protein expression under hypoxia. T (red) was stained on differentiation day 4. Nuclei were stained with DPAI (blue). H Hif-1α knockdown promoted the expression of ectoderm markers (Pax6 and Nestin). I Schematic diagram of the parallel comparison of Hif-1α knockdown effects on AB2.2 mESC differentiation under either normoxic or hypoxic conditions. J Hif-1α knockdown inhibited the expression of mesendoderm markers (T, Eomes, Mesp1, and Gsc) on differentiation day 4 under both normoxia and hypoxia, with the effect more obvious under normoxia. The ratios of K T+ and L Sox1+ cells on day 4 of the differentiation assays in I were detected by flow cytometry. scramble, AB2.2/scramble cells; shHif-1α, AB2.2/shHif-1α cells; *, significant (P<0.05); ns, not significant
Fig 5: Hypoxia dramatically suppressed mesendoderm differentiation of mESCs. A Schematic diagram of mESC differentiation under normoxia or hypoxia. B Hypoxia significantly repressed the mRNA expression of mesendoderm markers (T, Eomes, Mesp1, and Gsc) in differentiating AB2.2 mESCs. C Hypoxia repressed T protein expression in differentiating AB2.2 mESCs. T (red) was stained on differentiation day 4. Nuclei were stained with DPAI (blue). D Hypoxia significantly upregulated the mRNA expression of ectoderm markers (Pax6 and Nestin) in differentiating AB2.2 mESCs. E Hypoxia promoted Sox1 protein expression in differentiating AB2.2 mESCs. Sox1 (green) was stained on differentiation day 4. Nuclei were stained with DPAI (blue). F The ratios of T+ and Sox1+ cells on differentiation day 4 ofAB2.2 mESCs were detected by flow cytometry. G Hypoxia significantly repressed the mRNA expression of cardiac markers (Tbx5, Mef2C, Nkx2.5, and α-MHC) in differentiating AB2.2 mESCs. H Hypoxia inhibited T protein expression in R1 mESCs. T (red) was stained on differentiation day 4. Nuclei were stained with DPAI (blue). I The ratios of T+ and Sox1+ cells on differentiation day 4 of R1 mESCs were detected by flow cytometry. *, significant (P<0.05)
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