Fig 2: (A) Immunoblotting of indicated proteins after siE6 or siE6+siSet8. (B) Principal component analysis of the RNA seq results which show that the RNA seq from the knockdown conditions are reproducible and cluster as expected. (C,D) Volcano plots with Log2 fold change (X-axis) vs. −Log10 p-value (Y-axis) show that many genes are significantly up or down-regulated when E6 is knocked down (C) or when Set8 is co-knocked down with E6, relative to E6 alone (D). Red dots: genes with Log2FC > |0.5| and −Log10 adjusted p-value> 1.3. Genes with a −Log10 p-value > 15 were plotted at 15.

Fig 3: SET8 is downregulated in acute myeloid leukemia (AML) cells after LukS-PV treatment. (A) Downregulated histone epigenetic regulators after LukS-PV treatment. (B) HL-60 and (C) NB4 cells were treated with LukS-PV at different concentrations, and SET8 mRNA and protein expression levels were determined by quantitative real-time PCR and Western blotting. (D) HL-60 and (E) NB4 cells were treated with 3.0 μM of LukS-PV at different timepoints, and SET8 mRNA and protein expression levels were determined by quantitative real-time PCR and Western blotting. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 4: PIK3CB is the target gene for LukS-PV-SET8/H4K20me1. (A) Gene Ontology (GO) analysis of downregulated peak related gene binding by LukS-PV-mediated H4K20me1 through ChIP-seq. (B) Functional groups in downregulated peak-related genes binding by LukS-PV-mediated H4K20me1. (C) Heatmap of different expression genes upon LukS-PV or phosphate-buffered saline (PBS) treatment. (D) The binding of LukS-PV and PBS on target gene PIK3CB. (E) The binding of H4K20me1 at the PIK3CB promoter was significantly reduced upon LukS-PV treatment via quantitative chromatin immunoprecipitation (ChIP)–PCR analysis. Data are presented as fold-change relative to the control with PBS as a negative control. (F, G) HL-60 and NB4 cells were treated with LukS-PV at different concentrations for 24 h, and PIK3CB gene and protein expression levels were assessed via quantitative real-time PCR and Western blotting. (H, I) Western blotting showed that LukS-PV downregulated PIK3CB via SET8/H4K20me1 in HL-60 (H) and NB4 (I) cells. **p < 0.01; ***p < 0.001.

Fig 5: LukS-PV induces apoptosis via the PIK3CB signal pathway by targeting SET8. (A) Levels of PIK3CB, FOXO1, AKT, apoptosis‐associated (BAK/BCL2) proteins, and AKT phosphorylation in HL-60 and NB4 cells were assessed through Western blotting. (B) Expression levels of SET8, H4K20me1, PIK3CB, FOXO1, AKT, apoptosis‐associated (BAK/BCL2), proteins, and AKT phosphorylation in primary acute myeloid leukemia (AML) blasts were assessed through Western blotting. (C) Proposed mechanism of action of LukS-PV in acute myeloid leukemia cells. SET8-OE, SET8 overexpression; SET8-NC, SET8 negative control; SET8-SiRNA, SET8 small interfering RNA.