Fig. 5
ALKBH5 deficiency increases m6A modification of the CSF3R mRNA to promote its decay. A qRT‒PCR analysis of Csf3r mRNA level in bone marrow neutrophils isolated from Alkbh5-deficient mice and their WT littermates 12 h after CLP (n = 10). B qRT‒PCR analysis of CSF3R mRNA level in ALKBH5-deficient and WT dHL-60 cells infected with E. coli (left, n = 6) or stimulated with LPS (right, n = 5). mRNA level data were normalized to Gapdh (A) or GAPDH (B) expression. C m6A abundance on the CSF3R mRNA in dHL-60 cells infected with E. coli as indicated by our m6A-seq data (GSE201060, NM_000760.4). Two independent biological replicates (Rep 1 and Rep 2). Red, m6A-IP; gray, input; blue box, m6A peaks. The y-axis shows the normalized m6A signal along the gene. D m6A-RIP-qPCR analysis of m6A enrichment of the CSF3R mRNA in ALKBH5-deficient and WT dHL-60 cells infected with E. coli for 2 h (n = 4). Different regions of human EEF1A1 mRNA were used as positive and negative controls (ctrl). The data were presented relative to those obtained with IgG. E RNA decay assay of CSF3R mRNA degradation in ALKBH5-deficient and WT dHL-60 cells treated with actinomycin D for the indicated times (n = 5). The data were normalized to the 18S rRNA level, and residual mRNA abundances were normalized to the abundance at t = 0 h. All data are the mean ± SEM of biologically independent samples. Two-tailed unpaired Student’s t-test (A, B, D and E). *P < 0.05; **P < 0.01; ****P < 0.0001; ns not significant
