Fig 2: The expression of CSF3R in the family measured by: (A) direct PCR using cDNA transcription of total RNA extracted from subject's neutrophils; (B) rt-PCR showing fold change of relative CSF3R expression (normalized to GAPDH) compared to healthy controls. (C) Western blot (n = 3) and (D) the relative levels of CSF3R protein in neutrophils adjusted by GAPDH (Quantification of band intensities was performed using ImageJ). Results are presented as mean ± SD, *p < 0.05, **p < 0.01. HC, healthy control; M, marker; MW, molecular weight.

Fig 3: ALKBH5 imprints production- and mobilization-promoting gene signatures in both mouse and human neutrophils. A Volcano plot of gene expression profiles in bone marrow neutrophils from Alkbh5-deficient mice and their WT littermates 12 h after CLP. The significantly differentially expressed genes (DEGs) upon Alkbh5 deletion are colored red to indicate upregulation or blue to indicate downregulation. The x-axis is limited to genes with |log2FC| ≤ 4. Two independent biological replicates. B Visualization of the top 10 biological processes (top) and top 10 KEGG pathways (bottom) identified by GO and KEGG enrichment analyses of the significant DEGs upon Alkbh5 deficiency based on RNA-seq as in (A). C Heatmap showing the variations in the expression of the DEGs related to neutrophil production and mobilization based on RNA-seq as in (A). Two independent biological replicates (R1 and R2). D Volcano plot of gene expression profiles in ALKBH5-deficient and WT dHL-60 human neutrophils infected with E. coli. The significant DEGs upon ALKBH5 deletion are colored red to indicate upregulation or blue to indicate downregulation. Two independent biological replicates. E Visualization of the top 10 biological processes (top) and top 10 KEGG pathways (bottom) identified by GO and KEGG enrichment analyses of the significant DEGs upon ALKBH5 deficiency based on RNA-seq as in (D). F Heatmap showing the variations in the expression of the DEGs related to neutrophil production and mobilization based on RNA-seq as in (D). Two independent biological replicates (R1 and R2). G Integrative analysis of overlapping genes from the indicated KEGG pathways as in (B) and (E). H FACS analyses of the protein level of G-CSFR on the surface of bone marrow neutrophils from Alkbh5-deficient mice and their WT littermates 12 h after CLP (n = 5). I FACS analyses of the G-CSFR protein level on the surface of ALKBH5-deficient and WT dHL-60 cells 4 h after E. coli infection (n = 6). All data are the mean ± SEM of biologically independent samples. The data are representative of 5–6 independent experiments with similar results (H, I). Two-tailed unpaired Student’s t-test (H, I). ***P < 0.001; ****P < 0.0001

Fig 4: Reduced binding of ALKBH5 to the CSF3R mRNA leads to decreased G-CSFR expression in bacteria-infected neutrophils. A, B FACS analyses of the protein levels of G-CSFR on the surface of WT dHL-60 cells stimulated with E. coli (A) or LPS (B) for the indicated times (n = 6). C, D FACS analyses of total intracellular G-CSFR protein in WT dHL-60 cells stimulated with E. coli (C) or LPS (D) for the indicated times (n = 6). E, F qRT‒PCR analysis of CSF3R mRNA level in WT dHL-60 cells stimulated with E. coli (E) or LPS (F) for the indicated times (n = 5). G RIP-qPCR analysis of ALKBH5 binding to the CSF3R mRNA in WT dHL-60 cells without or with E. coli infection for 4 h (n = 4). The data were presented relative to those obtained with IgG. All data are the mean ± SEM of biologically independent samples. The data are representative of 6 independent experiments with similar results (A–D). Two-tailed unpaired Student’s t-test (A–G). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig 5: Loss of ALKBH5 impairs G-CSFR signaling in neutrophils during bacterial infection. A FACS analyses of total intracellular levels of G-CSFR protein in ALKBH5-deficient and WT dHL-60 cells infected with E. coli for the indicated times (n = 6). B, C FACS analyses of cell surface (B) and total intracellular (C) levels of G-CSFR protein in ALKBH5-deficient and WT dHL-60 cells stimulated with LPS for the indicated times (n = 6). D Analyses of STAT3 signaling in bone marrow neutrophils from Alkbh5-deficient mice and their WT littermates 12 h after CLP by immunoblotting (left) and integrated density measurement (right, n = 4). E Analyses of STAT3 signaling in ALKBH5-deficient and WT dHL-60 cells infected with E. coli and then treated with human G-CSF (100 ng/ml) for the indicated times by immunoblotting (left) and integrated density measurement (right, n = 5). The integrated density was determined using the ImageJ program (D, E). All data are the mean ± SEM of biologically independent samples. The data are representative of 4 or 5 independent experiments with similar results (D, E). Two-tailed unpaired Student’s t-test (A–E). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001