Figure 5.

DDX5 attenuates hypoxia-induced ferroptosis through promoting GPX4 mRNA processing

(A) Western blotting analysis of the protein levels of GPX4, ACSL4, and ptgs2 in PASMCs transfected with DDX5 plasmid (n = 6).

(B) Immunofluorescence analysis of GPX4 expression. Scale bars, 100 μm (n = 6). Green color denotes GPX4, stained with FITC, and blue color denotes nucleus, stained with DAPI.

(C–E) Detection of the GSH depletion, MDA release and ferrous ion accumulation in PASMCs overexpressed DDX5 (n = 6).

(F) Detection of the percentage of ferrous ion staining-positive cells by a specific probe of ferrous ion (red). Scale bars, 100 μm (n = 6).

(G) RIP-PCR analysis of the association of GPX4 pre-mRNA to DDX5 protein (n = 3).

(H) After transfection with DDX5 plasmid, real-time qPCR analysis of the level of GPX4 mRNA and GPX4 pre-mRNA (n = 6).

(I) After Actinomycin D treatment, real-time qPCR analysis of the impact of DDX5 overexpression on the stability of GPX4 pre-mRNA (n = 3). Data are shown as means ± SD. Statistical analysis of the graph (A, D, and H) was performed with one-way ANOVA followed by Dunn’s post-test. Statistical analysis of the graph (C) and (E) was performed with one-way ANOVA followed by Bonferroni correction. Statistical analysis of the graph (G) and (I) was performed with Student’s t test. Hyp, hypoxia; Nor, normoxia; NC, negative control, ns, not significantly different. ∗p < 0.05, ∗∗p < 0.01.