Fig 1: Hypoxia regulation of U2AF65 and JMJD6HUVECs cultured in hypoxia for 24 h had significantly up-regulated JMJD6, with no change in U2AF65 expression (A). Similarly, BeWo cells exposed to hypoxia also had unchanged U2AF65 but significantly elevated JMJD6 (B). Chronic hypoxia of the RUPP placenta, however, had significantly increased U2AF65 (C) and JMJD6 (D) compared with sham animals. Two-way ANOVA was performed for both cell groups. Student’s t-test was performed on the placental protein data. N = 6 in all groups. *P < 0.05; #P < 0.01 ##P < 0.001 vs. control/ sham.
Fig 2: Confirmation of knockdown/overexpression in BeWortPCR was performed to ensure adequate knockdown (A) and overexpression (B) was achieved in BeWo placental trophoblasts (N = 6). Additionally, Western blots were performed to see if the message translated to the protein expression. U2AF65 and JMJD6 knockdown Western blots and their analyses are shown in (C), while overexpression can be observed in (D) (N = 3). *P < 0.05; #P < 0.01
Fig 3: Effect of U2AF65 and JMJD6 expression on heparanaseHUVEC knockdown samples showed that there was no effect on heparanase mRNA expression (N = 4) (A) or media protein (N = 3) (B). HUVEC JMJD6 overexpression had significant increase in heparanase mRNA (N = 4) (C), but only HUVEC overexpression of U2AF65 had significant increase in media heparanase protein (N = 3) (D). BeWo knockdown of JMJD6 showed significantly decreased heparanase message (N = 6) (E), but none of the BeWo knockdown groups showed any change in heparanase media protein (N = 3) (F). BeWo overexpression of U2AF65 and JMJD6 had no effect on heparanase mRNA (N = 6) (G) or media protein (N = 3) (H). One-way ANOVE was performed for all groups. *P < 0.05 vs. control.
Fig 4: Effect of U2AF65 and JMJD6 expression on sFlt-1HUVEC knockdown (A) and overexpression (B) samples were analyzed for impact on sFlt-1 mRNA, with JMJD6 overexpression causing significantly increased sFlt-1 version 3 expression (N = 4). Media from HUVEC samples were analyzed for sFlt-1 protein. Both knockdown (C) and overexpression (D) media showed trends of sFlt-1 protein in the same direction as U2AF65 and JMJD6 expression (N = 4). BeWo knockdown (E) or overexpression (F) had no effect on sFlt-1 mRNA (N = 6). Similarly, media collected from U2AF65 or JMJD6 knockdown (G) or overexpression (H) had no difference in media sFlt-1 concentration (N = 6). Two-way ANOVA was performed on the rtPCR samples, and one-way ANOVA was performed on media sFlt-1 samples *P < 0.05 vs. control. ND = Not detectable.
Fig 5: Confirmation of knockdown/overexpression in HUVECsrtPCR was performed to ensure that adequate knockdown (A) and overexpression (B) of U2AF65 and JMJD6 was achieved in HUVECs (N = 4). Western blots were also done to see if the message translated to the protein level. Western blots for knockdown of U2AF65 and JMJD6 are shown in (C) and samples from overexpression of the proteins are seen in (D) (N = 2). *P < 0.05; #P < 0.01
Supplier Page from Abcam for Anti-U2AF65 antibody [EPR17046] - C-terminal