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. 2023 Jul 13;26(8):107370. doi: 10.1016/j.isci.2023.107370

Figure 4.

Figure 4

UQCC3 was highly expressed under hypoxia and important for HIF-1α stabilization and VEGF expression

(A) Correlation analysis between representative hypoxia and UQCC3 levels in tumor tissues. Scale bar: 50 μm.

(B) Effect of hypoxia on UQCC3 levels and HIF-1α activation. Three cell lines (SMMC7721, A2780S, and A549) were cultured under hypoxia for 0, 4, or 8 h. The lysates from the harvested cells were analyzed by immunoblotting. Data are represented as means +SEM. ∗∗∗∗p < 0.0001, Student’s t test.

(C) HIF-1α expression levels were detected in Uqcc3+/+ and Uqcc3−/− mice using specific antibodies against HIF-1α, and HIF-1α intensity was analyzed (n = 5). Scale bar: 500 μm. Data are represented as means +SEM. ∗∗∗p < 0.001, Student’s t test.

(D) Western blot (WB) analysis was performed with the indicated antibodies on total cell extracts from WT or UQCC3-KO cells under normoxia (20% O2) or hypoxia (1% O2). Data are represented as means +SEM. ∗∗∗∗p < 0.0001, Student’s t test.

(E) Immunofluorescent staining of VEGF in Uqcc3+/+ and Uqcc3−/− embryos at E9.5. (Scale bars: 1 mm). VEGF-positive cells and relative VEGF intensity were analyzed (n = 3 for each group). Scale bar = 500 μm. Data are represented as means +SEM. ∗∗∗p < 0.001, Student’s t test.

(F) The secretion of VEGF in the supernatant by WT and UQCC3-KO cells was detected by ELISA. Data are represented as means +SEM. ∗∗p < 0.01, ∗∗∗p < 0.001, Student’s t test.

(G) HUVECs were cultured on matrigel-coated 48-well plates for 18 h. Tube formation networks were observed under a microscope (Fresh medium: fresh medium for HUVEC growth, Condition media: tumor cell culture medium, VEGF block: tumor cell culture medium with VEGF block, UQCC3-KO: UQCC3-knockout tumor cell culture medium, UQCC3-KO+VEGF: UQCC3-knockout tumor cell culture medium with VEGF). Scale bar: 100 μm.

(H) Analysis of the total tube formation. Each bar represents the mean ± SEM of three experiments compared to the control group. Data are represented as means +SEM. ∗p < 0.05, ∗∗p < 0.01, Student’s t test.