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-1a overexpression promoted the mesendoderm differentiation but repressed the ectoderm differentiation. A Hif-1a overexpression upregulated T protein expression under normoxia. T (red) was stained on differentiation day 4. Nuclei were stained with DPAI (blue). B Hif-1a 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-1a knockdown significantly repressed the Wnt/ß-Catenin pathway activation. D TOPFlash assays showed that Hif-1a overexpression significantly promoted the Wnt/ß-Catenin pathway activation. E, F The expressions of Hif-1a in Hif-1a-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-1a-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-1a, AB2.2/shHif-1a cells; DOX, doxycycline; *, significant (P<0.05)
Fig 4: Hif-1a knockdown repressed the mesendoderm differentiation and promoted the ectoderm differentiation. A, B The expression patterns of Hif-1a 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-1a knockdown was verified by western blotting analysis under hypoxia. G Hif-1a knockdown inhibited T protein expression under hypoxia. T (red) was stained on differentiation day 4. Nuclei were stained with DPAI (blue). H Hif-1a knockdown promoted the expression of ectoderm markers (Pax6 and Nestin). I Schematic diagram of the parallel comparison of Hif-1a knockdown effects on AB2.2 mESC differentiation under either normoxic or hypoxic conditions. J Hif-1a 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-1a, AB2.2/shHif-1a 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 a-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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