Fig. 5
Effects of m6A eraser proteins on RSV gene expression. a Overexpression of m6A eraser proteins diminishes RSV gene expression. HeLa cells were transfected with plasmids encoding ALKBH5 and/or FTO, followed by rgRSV infection at an MOI of 0.5. At 18 h post-infection, cell lysates were harvested for Western blot analysis. b Overexpression of m6A eraser proteins reduces GFP expression. The GFP expression was monitored by fluorescence microscopy. Representative micrographs with 10 × magnification (scale bar of 100 μm) at 18 h post-infection were shown. c Quantification of GFP-positive cells by flow cytometry at 18 h post-infection. The P value (Student’s t-test) for ALKBH5, FTO, and ALKBH5&FTO is ****P = 1.91 × 10−6, ****P = 1.34 × 10−5, and ****P = 3.61 × 10−6, respectively. d Knockdown of m6A eraser proteins enhances RSV gene expression. HeLa cells were transfected with siRNA targeting ALKBH5 and/or FTO, followed by rgRSV infection at an MOI of 0.5. At 18 h post-infection, cell lysates were harvested for Western blot analysis. e Knockdown of m6A eraser proteins enhances GFP expression. Micrographs with 10 × magnification (scale bar of 100 μm) are shown. f Quantification of GFP-positive cells by flow cytometry. Fold of GFP signal compared to the control is shown. Western blots and GFP images shown are representatives of three independent experiments. Flow cytometry data are expressed as mean ± standard deviation. The P value (Student’s t-test) for ALKBH5, FTO, and ALKBH5&FTO is ****P = 2.06 × 10−4, ****P = 3.38 × 10−5, and ****P = 6.50 × 10−6, respectively. g Overexpression of m6A eraser protein reduces m6A level in viral RNA. A549 cells were transfected with plasmid encoding ALKBH5 or pCAGGS, followed by rgRSV infection. RSV particles were purified from supernatants. Virion RNA was subjected to m6A-IP. The P value (Student’s t-test) is ****P = 5.81 × 10−9. h Fold reduction in m6A content in viral RNA compared to controls. The P value (Student’s t-test) is ****P = 1.17 × 10−11. i Knockdown of m6A eraser protein increases m6A level in viral RNA. A549 cells were transfected with siRNA targeting ALKBH5 or control siRNA, followed by rgRSV infection. RSV particles were purified. Virion RNA was subjected to m6A-IP. The P value (Student’s t-test) is ****P = 5.09 × 10−9. j Fold increase in m6A content in viral RNA compared to controls. The P value (Student’s t-test) is ****P = 2.57 × 10−6. Results are from three or four independent experiments
