Status of methylation at the CpG site in the maspin promoter region of TEV-1 cells upon hypoxia and DI A-D: Bisulfite sequencing revealed a methylated CpG island in the maspin promoter region of TEV-1 cells in four groups.

<p>Each square represents an individual CpG site, and the sizes of the yellow parts indicate fractional methylation. The yellow parts represent methylation-positive results, and the blue parts represent methylation-negative results. E: The extent of methylation at the CpG site was calculated using the following formula: [extent of methylation/(extent of methylation + extent of unmethylation)]. The methylation rates of the maspin promoter in the HOX group were significantly decreased compared to the NOX group at 24h and 48h. (*P < 0.05). F: DI significantly reduced the methylation rates of the maspin promoter in the NOX group, but it had no significant effect on the methylation rates of the maspin promoter in the HOX group. (*P < 0.05). G: MSP analysis of maspin promoter in TEV-1 cells of four groups. All the 4 groups show the presence of both unmethylated and methylated DNA in the placental tissues, respectively. The bands detected by the methylated primer represent methylated maspin (M), and the bands detected by the unmethylated primer represent unmethylated maspin (U).</p

Effect of recombinant maspin protein on proliferative, apoptotic, migrative and invasive ability in TEV-1 A: The number of cells in NOX, HOX, r.M and HOX+r.M groups was measured by ELISA.

<p>Recombinant maspin protein significantly weakened the proliferative ability of TEV-1 cells in NOX group at 24h, but overall it had no significant effect on the proliferative ability of TEV-1 cells in either NOX or HOX group. B: The apoptosis rate of cells in NOX, HOX, r.M and HOX+r.M groups was detected by FCM. Recombinant maspin protein had no significant effect on the apoptosis rate of TEV-1 cells in either NOX or HOX group. C: The migrated/invaded cells were counted to estimate the effect of recombinant maspin protein on normoxic EVT cells. Recombinant maspin significantly weakened the migrative ability and the invasive ability of TEV-1 cells in the normoxic EVT cells. (*P < 0.05) D: The migrated/invaded cells were counted to estimate the effect of recombinant maspin protein on hypoxic EVT cells. Recombinant maspin significantly weakened the migrative ability and the invasive ability of TEV-1 cells in the hypoxic EVT cells. (*P < 0.05).</p

Effect of DI on maspin expression, aggressive ability of TEV-1 cells.

<p>A: Representative Western blot analysis of maspin protein expression. DI increased the protein expression of maspin in TEV-1 cells in the NOX group at 24h and 48h. DI increased the protein expression of maspin in TEV-1 cells in the HOX group at 24h, but had no significant effect on the protein expression of maspin in TEV-1 cells in the HOX group at 48h. (*P < 0.05). B: We investigated the effect of decitabine on the apoptosis rate of the normoxic/hypoxic EVT cells. DI had no significant effect on the apoptosis rate of TEV-1 cells in the NOX and HOX group. C: Representative Western blot analysis of maspin protein expression in the NOX and HOX groups upon DI at two different culture time points (24h, 48h). (+: with DI;-: without DI). D: The migrated/invaded cells were counted to estimate the effect of decitabine on normoxic EVT cells. Decitabine significantly weakened the migrative ability and the invasive ability of TEV-1 cells in the normoxic EVT cells. (The control was the NOX group. *P < 0.05) E: The migrated/invaded cells were counted to estimate the effect of decitabine on hypoxic EVT cells. Decitabine significantly weakened the migrative ability and the invasive ability of TEV-1 cells in the hypoxic EVT cells. (The control was the NOX group. *P < 0.05) F: Photo of migrated/invaded cells in the NOX, DI, HOX and HOX+DI group (200X).</p

Effect of hypoxia on protein expression of maspin, apoptotic and invasive ability in TEV-1 A: The maspin protein in the normoxic and hypoxic groups expressed as a relative measure compared to GAPDH and normalized to a control sample run on each gel at two different culture time points (24h, 48h).

<p>The protein expression of maspin in the HOX group was significantly higher compared to the NOX group at both 24h and 48h, the protein expression of maspin at 48h was significantly higher compared to 24h; (*P < 0.05). B: Representative Western blot analysis of maspin protein expression in the NOX and HOX groups; C: Apoptosis rate of cells was measured by flow cytometry. The apoptosis rate in the HOX group was significantly higher compared to the NOX group at 12h, 24h and 48h. (*P < 0.05) D: The invasive ability of cells were tested by transwell method. The invaded cells in the HOX group was significantly fewer compared to the NOX group at 48h. (*P < 0.05) E: Photo of invaded cells in the NOX and HOX group (200X).</p