Extended Data Fig. 6: Site-specific m6A editing validates functional regulation of CHD9.
From: METTL3-based epitranscriptomic editing screening identifies functional m6A sites in cancers
Unless otherwise indicated, data are presented as mean ± SD from independent experiments (n as specified in each panel). a) Writing efficiency evaluated by m6A meRIP-qPCR in 22Rv1 cells. n = 3; one-way ANOVA with Dunnett’s test. b) CHD9 abundance after dCasRx-M3-based m6A writing in 22Rv1 cells detected by western blot. Representative of two independent experiments with similar results. c) DeepSRAMP prediction of candidate m6A sites within CHD9 mRNA. The x-axis represents nucleotide position, and the y-axis indicates predicted m6A probability scores (0-1). Five sites within the targeted peak are highlighted in red. d) SELECT-based quantification of m6A abundance at predicted sites in 22Rv1 cells after dCasRx-M3 editing. n = 3; two-way ANOVA with Dunnett’s test. e) Relative m6A abundance at the CHD9 A8466 site measured by SELECT assay in 22Rv1 cells transfected with non-targeting control (sgNT) or five CHD9-targeting guide RNAs (g1-g5). n = 3; one-way ANOVA with Dunnett’s test. f, g) Cell proliferation (f) and relative m6A abundance at the CHD9 A8466 site (g) in 22Rv1 cells transfected with the dCasRx-M3 guided by CHD9-targeting guides (sg6, sg7) or sgNT. n = 3; two-way (f) or one-way (g) ANOVA with Dunnett’s test. h-i) Representative images of migration and invasion assay (h) and the quantification (i) after writing CHD9 in 22Rv1 cells. n = 3; two-way ANOVA with Dunnett’s test. Scale bar, 200 μm. j) Tumor weight (left) and tumor size (right) of xenografts derived from 22Rv1-M3 cells after writing CHD9 by CHD9-targeting sgRNAs. n = ten mice; one-way ANOVA with Dunnett’s test. k-l) Western blot showing CHD9 protein abundance (k; representative of two independent experiments with similar results) and cell proliferation analysis (l) in 22Rv1 cells after dCasRx-ALKBH5-mediated m6A erasing at CHD9. n = 3; two-way ANOVA with Dunnett’s test. m, n) Representative images (m) and quantification (n) of migration and invasion assays after CHD9 m6A erasing by dCasRx-ALKBH5 in 22Rv1 cells. n = 3; two-way ANOVA with Dunnett’s test. Scale bar, 200 μm.
