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. 2021 Feb 15;29(6):2134–2150. doi: 10.1016/j.ymthe.2021.02.017

Figure 6.

Figure 6

WDR5 promoted HIF-1α accumulation via suppressing the PHD2-mediated degradation pathway

(A) ChIP assay proved that the combination of shMyc (sh) and c-Myc inhibitor 10058-F4 (40 μM) almost completely suppressed c-Myc recruitment on the HIF1A promoter. (B and C) In QBC939 cells treated with shMyc and inhibitor 10058-F4, quantitative real-time PCR (B) and western blot (C) showed that WDR5 overexpression had little effect on HIF1A mRNA, but it increased HIF-1α accumulation. (D) Half-life analysis of HIF-1α protein. Cells were treated with 10 μg/mL cycloheximide (CHX) for the indicated times. The gray scale values of western blot bands were quantified with ImageJ software. (E) Effect of ubiquitination inhibitor MG-132 (10 μM) on control and WDR5-overexpressed QBC939 cells. MG-132 attenuated the HIF-1α elevation induced by WDR5 overexpression in QBC939 cells treated with shMyc and inhibitor 10058-F4. (F) In QBC939 cells treated with shMyc and inhibitor 10058-F4, WDR5 overexpression decreased hydroxylation of HIF-1α. (G and H) The mRNA levels of the PHD family and VHL in QBC939 cells transfected with scramble and WDR5 knockdown were detected by RNA-seq (G) and verified by quantitative real-time PCR (H). (I) The expression levels of the PHD family in QBC939 cells transfected with control, vector, and LV5-WDR5 were detected by western blot in the treatment with shMyc and inhibitor 10058-F4. (J) Effect of PHD1–PHD3 knockdown on the expression of total HIF-1α and hydroxy-HIF-1α in QBC939 cells treated with shMyc and inhibitor 10058-F4. (K) Survival curves of CCA were stratified with PHD1–PHD3 in TGCA database. (L) The correlations between WDR5 and PHD2 mRNA in iCCA, pCCA, and dCCA were analyzed with a Pearson correlation test. (M) Expression levels of HIF-1α in WDR5-silenced and/or PHD2-silenced QBC939 cells after treatment with shMyc and inhibitor 10058-F4. PHD2 knockdown attenuated the influence of WDR5 knockdown on HIF-1α reduction. Data were analyzed with one-way ANOVA in (A), (B), and (H). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n.s., not significant.