Figure 6.

Inhibition of intracellular ROS rescues mitochondrial respiration and EndoMT in the OCT4-deficient ECs. (A) Representative western blot showing protein levels of ABCG2 and GAPDH in OCT4^WT and OCT4^ex1_KO HUVECs. Replicates correspond to independent experiments. The relative ABCG2/GAPDH ratio based on the densitometry analysis is indicated beneath the blots. (B) Quantification of extracellular haem concentrations in OCT4^WT and OCT4^ex1_KO HUVEC cultured media. Values = mean ± S.E.M.; *P < 0.05 by unpaired t-test; n = 3 independent experiments. (C and D) Loss of OCT4 increased ROS generation within HUVEC in response to hemin chloride treatment. (C) Schematic showing the experimental design for the loss-of-function assay of ABCG2 after treatment with succinylacetone (endogenous inhibitor of haem synthesis) followed by FTC (specific ABCG2 inhibitor) to inhibit ABCG2 in OCT4^WT HUVECs and subsequent intracellular ROS estimation in OCT4^WT, OCT4^ex1_KO, and OCT4^WT+FTC HUVECs. (D) Hemin chloride-induced ROS generation was then measured by DCF intensity and showed dose–response with increasing hemin chloride concentration. OCT4^WT+FTC group recapitulated the OCT4^ex1_KO phenotype in the presence of increasing hemin chloride. Values = mean ± S.E.M; Data were analysed by linear mixed-model ANOVA followed by Tukey’s post hoc test, *P < 0.05, **P < 0.01, and ***P < 0.001; n = 3 independent experiments. (E) Seahorse mitochondrial stress test measuring the oxygen consumption rates (OCR) in OCT4^WT, OCT4^ex1_KO HUVECs, and OCT4^ex1_KO HUVECs cultured in the presence of the ROS inhibitor, N-acetyl-l-cysteine (NAC). NAC significantly improved basal mitochondrial respiration, spare respiratory capacity, and ATP production in OCT4^ex1_KO HUVECs. Error bars represent mean ± S.E.M.*P < 0.05, **P < 0.01 by linear mixed-model ANOVA followed by Tukey’s post hoc test, n = 3 independent experiments. Results for OCT4^WTand OCT4^ex1_KO HUVECs are the same as in Figure 4H. (F) Schematic showing experimental design for in vitro EndoMT experiments. (G and H) qRT–PCR analyses demonstrated that EC transfected with blocking siOct4 have lower Oct4 and Pecam1 levels (G) but higher levels of the EndoMT markers and Nfkb mRNA gene expression (H) in response to TGFβ1 treatment as compared with siNT control cells. The presence of NAC significantly improved TGFb1-induced gene expression alterations. Data were analysed by one-way ordinary ANOVA (mixed-model), followed by Tukey’s multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, and #P = 0.07). Values = mean ± S.E.M; n = 3 independent experiments.