Fig. 5
MA-induced m6A doesn’t affect the protein expression of target genes or NK cytotoxicity in NK cells. A Western blot analysis of protein expression of target genes in NK cells following MA treatment. ERK was used as a loading control. (Different lanes from the same gel are shown, with cropped sections in the middle. Full-length blots are provided in the Supplementary file). B Quantification of protein expression using relative density by ImageJ. Data is presented as mean ± SD from 3 different donors (n = 3). C Flow cytometry (FACS) analysis of protein expression in NK cells treated with 12.5 µM MA compared to DMSO-treated cells. Histogram overlays were generated using FlowJo. The red peak represents an unstained control, the blue peaks represent NKG2D, PRF1 and TRAIL protein expression, respectively, in a DMSO-treated sample, and the orange peak represents the same proteins in an MA-treated sample, all from the same donor. Similar results were obtained from 5 different subjects. Controls included a negative unstained control, a positive control for each primary antibody, and compensation controls for each dye (Zombie NIR, NKG2D-PE, PE anti-human CD253, FITC-anti-Perforin). D 51Cr release cytotoxicity assay showing NK cell cytotoxicity after treatment with 12.5 µM MA compared to DMSO at different E: T ratios. Data is representative of 5 donors and expressed as mean ± SD. Controls: Maximum release (target cells in 1% Triton X-100) as the positive control and spontaneous release (target cells without NK effectors) as the negative control. Percent-specific lysis was calculated based on these controls. E Percentage of CD107a expression in 1.ry expanded NK cells from 7 different subjects following DMSO or MA treatment. Controls included a solvent-treated control (DMSO), and NK-only controls (unstained/untreated for auto-fluorescence exclusion and stained/untreated to account for background degranulation). Compensation controls were included for each dye. F Mean percentage of CD107a expression in NK cells after treatment with DMSO or MA, expressed as mean ± SD (n = 7)