Fig. 7

Demethylase knockdown doesn’t affect the protein expression of target genes or NK cytotoxicity. A Western blot analysis of protein expression in NK cells after demethylase knockout in one representative subject. ERK was used as a loading control. (Different lanes from the same gel. Full-length blots are provided in the Supplementary file). B Quantification of protein expression of target genes using ImageJ. Data is from two different knockouts and is presented as mean ± SD. C Flow cytometry (FACS) analysis of protein expression in NK cells for NKG2D, PRF1, and TRAIL in transfected NK cells compared to untransfected controls using FlowJo. The purple peak represents an unstained control, the orange peak represents protein expression in an untransfected sample, the red peak represents protein expression in an ALKBH5 knockout sample, and the blue peak represents protein expression in an FTO knockout sample. Data is from a representative subject with similar results obtained in 2 independent KOs. 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 Cytotoxicity Assay of demethylases-knock out NK cells against K562 cells, showing mean percentage-specific lysis from 3 donors. Data is presented as mean ± SD (n = 3). 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 Mean percentage of CD107a expression in transfected NK cells from 3 different subjects. Data expressed as mean ± SD (n = 3). Controls included an untransfected control (electroporated cells without synthetic guide RNAs) and NK-only controls (unstained/untransfected for auto-fluorescence exclusion and stained/untransfected to account for background degranulation). Compensation controls were included for each dye