Figure 6.

Silencing Eef1a1 attenuates hypoxia-induced ferroptosis

(A) RNA pull-down and western blotting analysis were used to identify proteins associated with circMyst4 (left), and mass spectrometry of specific segments of Eef1a1 (right).

(B) RIP-PCR analysis of the association of circMyst4 to Eef1a1 protein (n = 3).

(C) Western blotting analysis of effects of silencing Eef1a1 on the protein levels of GPX4, ACSL4 and ptgs2 in hypoxic PASMCs (n = 6).

(D–F) The GSH, MDA and ferrous ion analysis of the effects of Eef1a1 on cell ferroptosis (n = 6).

(G) The percentage of ferrous ion-positive cells were detected after transfection with Eef1a1 siRNA in hypoxia-treated PASMCs. Scale bars, 100 μm. Images of fluorescence staining with a ferrous ion probe (red).

(H) Western blotting analysis of effects of circMyst4 overexpression on the protein levels of Eef1a1 in hypoxic PASMCs (n = 5).

(I) CoIP analysis of the binding of Eef1a1 with ACSL4 after circMyst4 overexpression in hypoxic PASMCs. Data are shown as means ± SD. Statistical analysis of the graph B was performed with Student’s t test. Statistical analysis of the graph (C, E, and H) was performed with one-way ANOVA followed by Dunn’s post-test. Statistical analysis of the graph (D) and (F) was performed with one-way ANOVA followed by Bonferroni correction. Hyp, hypoxia; Nor, normoxia; NC, negative control, ns, not significantly different. ∗p < 0.05, ∗∗p < 0.01.