Fig. 4

ALKBH5 negatively regulates CD58 via m⁶A RNA demethylation. A Heat map showed the expression of ALKBH5 was negatively correlated with CD58 in TCGA GC database. B Scatter plot showed the correlation between the expression of ALKBH5 and CD58 in patients with GC in TCGA (left) and GSE13861 (GEO, right). C CD58 was evaluated in GC tumor tissues and normal gastric tissues from GSE54129 cohort. D The protein level of ALKBH5 was analyzed in GC cell lines and pooled non-tumor tissues (10 cases of non-tumor tissues). β-Tubulin was used as a loading control. E The protein level of ALKBH5 was determined in ALKBH5 knockdown AGS and MKN45 cells. β-Tubulin was used as a loading control. F, G The expression of CD58 in ALKBH5 knockdown cells (AGS and MKN45) (F) and HGC27-HSPA4 cells (G) was analyzed by flow cytometry. Representative pictures and mean fluorescence intensity (MFI) of CD58 was summarized. H Immunofluorescence images of ALKBH5 (green) and CD58 (red) in xenografts formed by MKN45-HSPA4 and control cells in nude mice (Figure 2F) (scale bar=20 µm). I The primers were designed according to the possible m⁶A methylation sites on CD58 mRNA (upper). MeRIP-qPCR was performed in ALKBH5-KD AGS cells and control cells and m⁶A+ CD58 fragments enrichments were assayed. (*, P<0.05; **, P<0.01; ****, P<0.0001)