Fig. 5

ALKBH5 stabilizes FOXM1 mRNA via the RNA binding protein HuR. HUVECs were transfected with either ALKBH5 siRNA (30 nM) or an overexpression plasmid (1 μg), and the levels of indicated mature or pre-mRNA were assessed by qRT-PCR (A–C). HUVECs were infected with lentiviruses containing ALKBH5 shRNA, ALKBH5 plasmid, or a negative control plasmid to generate ALKBH5-silenced or overexpressing stable cell lines. The relative activity of the FOXM1 promoter firefly luciferase reporter (1 μg) was measured (D). RNA from HUVECs treated with 10 μg/mL actinomycin D (Act D) for 2 to 10 h was collected and analyzed by qRT-PCR (E, F). HUVECs were transfected with exogenous FLAG-tagged ALKBH5, and the enrichment of FOXM1 mRNA or pre-mRNA with FLAG was measured by RIP followed by qRT-PCR, normalized to input (G, H). RIP analysis was used to detect the interaction of FOXM1 pre-mRNA or mature mRNA with HuR in HUVECs treated as indicated. The enrichment of RNA with HuR was quantified by qRT-PCR (I–L). Statistical analysis was conducted using one-way ANOVA with Sidak’s multiple comparisons test (A–D), two-way ANOVA with Sidak’s multiple comparisons test (E, F, I–L), and unpaired t-test (G, H). n ≥ 3, *p < 0.05, **p < 0.01