Fig 9. Transfection of m6A-altered HIV-1 RNA into primary MDM modulates IFN-I expression.
(A) m6A levels of HIV-1 RNA (100 ng) of virions generated from vector control HEK293T cells, FTO-overexpressed HEK293T cells (FTO-OE), control-KO HEK293T cells (Ctrl-KO), or FTO-KO HEK293T cells were measured by dot-blotting. Methylene blue (MB) staining was used as an RNA loading control. (B-E) MDM (5×105) were mock-transfected (no RNA) or transfected with 125 ng isolated HIV-1 RNA or poly (I:C) as indicated. At 24 hr post-transfection, (B and D) IFN-I mRNA levels in MDM were measured by qRT-PCR. (C and E) IFN-I protein levels in the supernatants of transfected MDM were detected by ELISA. These experiments were repeated three times with MDM from three different donors. Data shown were from one representative experiment with means ± S.D. of three biological repeats. Un-paired t-test was used for statistical analysis. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 compared to the indicated controls (B-C, compared to the vector control; D-E, compared to the Ctrl-KO). (B-E) IFN-I levels in mock samples were undetectable and used as a background control. (F) Flow cytometry detection of GFP expression in MDM mock-transfected (light grey peak) or transfected with gfp mRNA (dark grey peaks). Percentage of GFP-positive cells (88%) and mean fluorescence intensity (MFI) are shown.