Fig. 2
From: Characterization of ACTN4 as a novel antiviral target against SARS-CoV-2
The m6A modification and its catalytic complex are essential for ACTN4 expression. a SARS-CoV-2 infection alters the expression of m6A-related proteins. WB assays of METTL3, METTL14, WTAP, ALKBH5, FTO and SARS-CoV-2 N protein levels in Huh7 cells infected with WT SARS-CoV-2 at MOI = 1 for 48 h. GAPDH was used as the loading control. b–f WTAP knockdown or overexpression affects the m6A modification level of ACTN4 mRNA. b, d WB assays of extracts from Huh7 cells treated with pFlag-WTAP (d) or shRNA (b) WTAP expression was assessed using anti-Flag/anti-WTAP Abs, and GAPDH was served as the loading control. c, e, f Modulation of ACTN4 mRNA methylation was affected by WTAP. Total RNAs were extracted from Huh7 cells with WTAP overexpression (e, f) or knockdown (c) and subjected to MeRIP-qPCR. Data are means ± SEMs (n = 3). ***P < 0.001, unpaired Student’s t-test (e, f) or one-way ANOVA (c). g, h WTAP knockdown inhibits ACTN4 expression. g WB assays of ACTN4, WTAP, N protein levels in Huh7 cells transfected with scramble shRNA (shNC) or two WTAP-specific shRNAs (shWTAP-1 and shWTAP-2). h RT-qPCR analysis of the relative ACTN4 mRNA levels in WTAP knockdown Huh7 cells. Data are means ± SEMs (n = 3). **P ≤ 0.01, one-way ANOVA. i, j WTAP enhances ACTN4 mRNA stability. Relative ACTN4 mRNA levels remaining after actinomycin D (4 µg/mL) treatment in WTAP knockdown (i) or overexpressing (j) Huh7 cells as assessed using qRT-PCR. Data are means ± SEMs (n = 3). *P ≤ 0.05, **P ≤ 0.01, unpaired Student’s t-test (j) or one-way ANOVA (i). k, l WTAP increases the ribosome loading onto ACTN4 transcripts. WTAP knockdown (k) or overexpressing (l) Huh7 cells were used to analyze input and ribosome-loaded ACTN4 RNA levels at 48 hpi, GAPDH was set as the control. Data are means ± SEMs (n = 3). ***P < 0.001, ns: not significant, unpaired Student’s t-test (l) or one-way ANOVA (k)
