Figure 5.
Pharmacological mediated phosphorylation of PHF8 orchestrates cell-intrinsic immune response in human AML. (A) Barcode plot showing GSEA of ATRA gene signature. (B) Bar charts illustrating the impact of ATRA treatment on human AML cells. It displays normalized colony numbers of THP1 cells transduced with empty vector (EV) (control) or PHF8 wt (PHF8) and treated with ATRA (12 hours) at the indicated concentration (10–8 M). Nontreated cells were taken as the reference for each group. Error bars are representative of 4 independent experiments. Tukey multiple comparison test (2-way ANOVA, ∗P < .01; ∗∗∗∗P < .0001). (C) Bar charts display normalized percentage of Annexin-V–positive cells after flow cytometry analysis. Nontreated cells were taken as the reference for each group. Error bars indicate standard deviation of 4 independent experiments. Tukey multiple comparison test (2-way ANOVA, ∗P < .03; ∗∗∗P < .0005; ∗∗∗∗P < .0001). (D) Scatterplot showing enriched GO terms of significantly regulated phosphoproteins in THP1 PHF8 cells upon 12 hours of ATRA treatment (10–8 M). Changes of phosphoproteins were compared with the unchanged phosphoproteome in the data set based on GO, KEGG, and keyword terms; y-axis shows the negative log of P value obtained from the Fisher exact test. (E) Dot plot showing changes in phosphosites of displayed proteins (12 hours of ATRA) in THP1 cells transduced with EV or PHF8 (PHF8). Red color and asterisk highlight specific IFIT5 targets. (ANOVA test permutation-based FDR <0.05). Sizes and colors of the dots are proportional to the averaged phosphosite intensity, z score (log2 intensity; n = 4 biological replicates per group). (F) Representative western blot analyses of time-course experiments (n = 3) after treatment with indicated ATRA concentration. Actin was used as a loading control for total extract and Lamin B1 for nuclear extract of THP1 cells expressing PHF8. (G) Western blot analysis of NF-κB (p65) protein levels (12 hours of ATRA) in nuclear (Lamin B1 loading control) and cytosolic (tubulin loading control) extracts of THP1 cells (n = 3). Cells were transduced with EV (control) or PHF8 wt (PHF8). (H) Phosphoproteome map of early IFN-I response and apoptosis in PHF8 AML cells after ATRA stimulation. Significantly regulated phosphoresidues are depicted on proteins participating in the process (n = 4 biological replicates per group; 1-way ANOVA, permutation-based FDR <0.05). Arrows indicate protein-protein interactions and phosphorylation events curated from experimentally defined databases. GSEA, gene set enrichment analysis; RELA, REL-associated; S, serine; T, threonine; Y, tyrosine.

Pharmacological mediated phosphorylation of PHF8 orchestrates cell-intrinsic immune response in human AML. (A) Barcode plot showing GSEA of ATRA gene signature. (B) Bar charts illustrating the impact of ATRA treatment on human AML cells. It displays normalized colony numbers of THP1 cells transduced with empty vector (EV) (control) or PHF8 wt (PHF8) and treated with ATRA (12 hours) at the indicated concentration (10–8 M). Nontreated cells were taken as the reference for each group. Error bars are representative of 4 independent experiments. Tukey multiple comparison test (2-way ANOVA, ∗P < .01; ∗∗∗∗P < .0001). (C) Bar charts display normalized percentage of Annexin-V–positive cells after flow cytometry analysis. Nontreated cells were taken as the reference for each group. Error bars indicate standard deviation of 4 independent experiments. Tukey multiple comparison test (2-way ANOVA, ∗P < .03; ∗∗∗P < .0005; ∗∗∗∗P < .0001). (D) Scatterplot showing enriched GO terms of significantly regulated phosphoproteins in THP1 PHF8 cells upon 12 hours of ATRA treatment (10–8 M). Changes of phosphoproteins were compared with the unchanged phosphoproteome in the data set based on GO, KEGG, and keyword terms; y-axis shows the negative log of P value obtained from the Fisher exact test. (E) Dot plot showing changes in phosphosites of displayed proteins (12 hours of ATRA) in THP1 cells transduced with EV or PHF8 (PHF8). Red color and asterisk highlight specific IFIT5 targets. (ANOVA test permutation-based FDR <0.05). Sizes and colors of the dots are proportional to the averaged phosphosite intensity, z score (log2 intensity; n = 4 biological replicates per group). (F) Representative western blot analyses of time-course experiments (n = 3) after treatment with indicated ATRA concentration. Actin was used as a loading control for total extract and Lamin B1 for nuclear extract of THP1 cells expressing PHF8. (G) Western blot analysis of NF-κB (p65) protein levels (12 hours of ATRA) in nuclear (Lamin B1 loading control) and cytosolic (tubulin loading control) extracts of THP1 cells (n = 3). Cells were transduced with EV (control) or PHF8 wt (PHF8). (H) Phosphoproteome map of early IFN-I response and apoptosis in PHF8 AML cells after ATRA stimulation. Significantly regulated phosphoresidues are depicted on proteins participating in the process (n = 4 biological replicates per group; 1-way ANOVA, permutation-based FDR <0.05). Arrows indicate protein-protein interactions and phosphorylation events curated from experimentally defined databases. GSEA, gene set enrichment analysis; RELA, REL-associated; S, serine; T, threonine; Y, tyrosine.

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