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RT-qPCR was employed to examine whether the reduction in global DNA methylation (5mC and 5hmC) in response to physiological normoxia were in relation to changes in the expression of DNMTs/TETs genes. The results of SHEF1 cells showed there was no significant change in the relative expression of DNMT1, DNMT3A, TET2 and TET3 excepting DNMT3A and TET3 expression in day 5 from differentiation in 2%WKS conditions (0.35 and 0.44, respectively), and in day 20 in 2%PG and 2%WKS (0.35 and 0.43, respectively) in TET3 in comparison to those cultured in 21% ST (Figure 4.12). The level of DNMT3B was decreased significantly in undifferentiated cells following treatment with 2%PG and 2%WKS (0.37 and 0.52, respectively), and in all time course from spontaneous differentiation in 2%WKS (0.55, 0.50 and 0.51, respectively) (Figure 4.12). Similar significant reduction was observed in TET1 level in undifferentiated SHEF1 cells grown under 2%WKS conditions (0.51), and in day 5, 10 and 20 in 2%WKS from differentiation (0.54, 0.66 and 0.59, respectively). The level of DNMT3L was decreased significantly in undifferentiated cells following treatment with 2%PG and 2%WKS (0.49 and 0.65, respectively), and in day 5 and day 20 from spontaneous differentiation in 2%WKS (0.55 and 0.67, respectively), and in day 10 in 2%PG and 2% WKS (0.48 and 0.53, respectively) in comparison to those cultured in 21% ST (Figure 4.12).

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Figure 4.12 The RT-qPCR expression of the DNMTs and TETs after SHEF1 cells incubation under different oxygen level. RNA quantification of DNMTs and TETs

isolated from SHEF1 cells following incubation in air oxygen (21%ST) and normoxia physiological culturing conditions (2%PG and 2%WKS). The expression was normalized to the expression of β-actin in the SHEF1 cells. Y-axis indicates relative changes in 2^ΔΔCt _of treated cell to untreated cell. X-axis indicates time (days). Data are presented as mean ± standard deviation (SD). n=3 *P<0.05, **P<0.01 vs air oxygen (21%ST).

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Consistent results were observed for SHEF2 cells treated with physiological normoxia. Under 2%PG and 2%WKS significant reduction in DNMT3B expression was noticed in undifferentiated cells (0.44 and 0.22, respectively), and after 10 days from differentiation (0.49 and 0.64, respectively), and day 20 in 2%WKS (0.84) in comparison to those cultured in 21% ST (Figure 4.13). TET1 expression decreased in a pattern similar to that of DNMT3B expression. Furthermore, a significant decrease in the expression of DNMT3L was showed under 2%WKS conditions in undifferentiated cells (0.52), and after 20 days in 2%PG and 2%WKS (0.40 and 0.72, respectively) (Figure 4.13). No significant change was noted in the expression of DNMT1, DNMT3A, TET2 and TET3 excepting DNMT3A and TET3 expression in undifferentiation cells and in day 20 from differentiation in 2%WKS conditions (0.26 and 0.70, respectively) in comparison to those cultured in 21% ST (Figure

4.13).

Similar significant reduction was observed in DNMT3B level in undifferentiated iPS cells grown under 2%PG and 2%WKS conditions (0.50 and 0.55, respectively), and in day 5 (0.34 and 0.59, respectively) and day 20 (0.37 and 0.45, respectively) from differentiation in comparison to those cultured in 21% ST (Figure 4.14). The level of TET1 was decreased significantly in undifferentiated cells and day 10 after differentiation following treatment with 2%WKS (0.35 and 0.43, respectively), and in day 20 in 2%PG and 2%WKS conditions (0.13 and 0.4, respectively) (Figure 4.14). Significant decrease in the expression of DNMT3L was showed under 2%PG and 2%WKS conditions in undifferentiated cells (0.61 and 0.59, respectively), and after 20 days (0.62 and 0.61, respectively) (Figure 4.14). No significant change in the expression of DNMT1, DNMT3A and TET2 excepting DNMT3A and DNMT1 expression in undifferentiation cells and day 5 from differentiation in 2%WKS conditions (0.36 and 0.48, respectively) in comparison to those cultured in 21% ST (Figure

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4.14). In addition, TET3 expression increased significant only in undifferentiated cells in

both reduced oxygen 2%PG and 2%WKS conditions (0.44 and 0.46, respectively) in comparison to those cultured in 21% ST (Figure 4.14).

Figure 4.13 The RT-qPCR expression of the DNMTs and TETs after SHEF2 cells incubation under different oxygen level. RNA quantification of DNMTs and TETs

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physiological culturing conditions (2%PG and 2%WKS). The expression was normalized to the expression of β-actin in the SHEF2 cells. Y-axisindicates relative changes in 2^ΔΔCt _of treated cell to untreated cell. X-axis indicates time (days). Data are presented as mean ± standard deviation (SD). n=3 *P<0.05, **P<0.01 vs air oxygen (21%ST).

Figure 4.14 The RT-qPCR expression of the DNMTs and TETs after hiPSC line (ZK2012L) cells incubation under different oxygen level. RNA quantification of DNMTs

and TETs isolated from ZK2012L cells following incubation in air oxygen (21% ST) and h normoxia physiological culturing conditions (2%PG and 2%WKS). The expression was

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normalized to the expression of β-actin in the ZK2012L cells. Y-axis indicates relative changes in 2^ΔΔCt _{of treated cell to untreated cell. X-axis indicates time (days). Data are} presented as mean ± standard deviation (SD). n=3 *P<0.05, **P<0.01 vs air oxygen (21%ST).

The data from all three hPS cells (SHEF1, SHEF2, and hiPSCs) was combined, and the results showed there was a significant reduction in DNMT3B level in undifferentiated cells grown under 2%PG and 2%WKS conditions (0.46 and 0.44, respectively), and in day 20 (0.67 and 0.40, respectively) from differentiation in comparison to those cultured in 21% ST

(Figure 4.15).The level of TET1 was decreased significantly in undifferentiated cells

following treatment with 2%WKS (0.48), and in day 5, 20 from differentiation (0.0.55 and 0.57, respectively), and in day 10 in 2%PG and 2%WKS (0.60 and 0.37, respectively) (Figure 4.15). Significant decrease in the expression of DNMT3L was showed under 2%PG and 2%WKS conditions in undifferentiated cells (0.53 and 0.41, respectively), and after 20 days following treatment with 2%WKS (0.41). No significant change in the expression of DNMT1, DNMT3A and TET2 excepting DNMT3A expression at day 10 from differentiation in 2%WKS conditions (0.79) was observed in comparison to those cultured in 21% ST (Figure 4.15). In addition, TET3 expression increased significant only in differentiated cells in 2%PG and 2%WKS conditions at day 5 (0.69 and 0.61, respectively), and day 20 (0.62 and 0.49, respectively), and after 10 days following treatment with 2%WKS (0.80) in comparison to those cultured in 21% ST (Figure 4.15).

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Figure 4.15 The RT-qPCR expression of the DNMTs and TETs after 3 hPSC line (SHEF1, SHEF2 and ZK2012L) cells incubation under different oxygen level. RNA

quantification of DNMTs and TETs isolated from 3 hPSC line following incubation in air oxygen (21% ST) and normoxia physiological culturing conditions (2%PG and 2%WKS). The expression was normalized to the expression of β-actin in the ZK2012L cells. Y-axis indicates relative changes in 2^ΔΔCt _{of treated cell to untreated cell. X-axis indicates time}

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(days). Data are presented as mean ± standard deviation (SD). n=3 *P<0.05, **P<0.01 vs air oxygen (21%ST).

4.4.8 Immunoblotting assay demonstrated reduced DNMT3B and TET1 protein