Machine learning-based prediction reveals kinase MAP4K4 regulates neutrophil differentiation through phosphorylating apoptosis-related proteins
Fig 6
Knocking out MAP4K4 reduces phosphorylation levels in the apoptosis pathway, and overexpression STAT5A rescued MAP4K4 deficiency-caused neutrophil apoptosis.
(A) Schematic of mass spectrometry analysis, created using Biorender. (B) The number of proteins with significant differences in protein and phosphorylation levels after MAP4K4 knockout. (C) Comparison heatmap of log 2 (fold-change) in protein and phosphorylation levels after MAP4K4 knockout. (D) Differential phosphorylation level between control and MAP4K4 KO parasites is involved in the positive regulation of the apoptotic process, as determined by Gene Set Enrichment Analysis (GSEA). RES represents the running enrichment score. RLM represents the ranked list metric. (E) Volcano plot of the apoptosis-related proteins with differentially expressed phosphorylation levels. The x-axis shows the phosphorylation difference of log2 (fold-change) between MAP4K4 KO and Ctrl. The y-axis shows –log10 (adjusted p value) of the t-test between these two groups. Red indicates proteins with up-regulated phosphorylation after MAP4K4 KO, and blue, proteins with down-regulated phosphorylation after MAP4K4 KO. The top 20 proteins with the most significant differences in phosphorylation among the apoptosis-related proteins are visualized. (F) Western blot (WB) for MAP4K4, p-STAT5A, STAT5A, p-STAT5B, STAT5B, and GAPDH in sgAASV1 and sgMAP4K4 cells. (G) Representative FACS analysis of HL-60 cells were first infected with overexpression of STAT5A or STAT5B or control plasmids and then treated with 10 µM MAP4K4 inhibitor (PF-06260933, PF) for 48 hours, followed by a subsequent 96-hour treatment with both PF and all-trans-retinoic acid (ATRA) for staining of anti–annexin V and SYTOX was shown (left). Percentages of annexin V+ and SYTOX− cells (right) (n=4; mean ± SD). Mann-Whitney U test. Fig 6A was created with Biorender.