Global proteome and phosphoproteome alterations in AML cells upon ASF1 and TLK depletion. Global proteomics and phosphoproteomics analysis of AML cell line MOLM-14 was performed upon knockdown of ASF1s or TLKs as described in supplemental Figure 6A and “Methods.” (A) STRING-DB Gene Ontology analysis for the biological process for proteins that were significantly downregulated for protein levels or their phosphorylation upon ASF1A/B knockdown (left) compared with nontargeting controls (shNT). Analysis of variance (ANOVA) t test, false discovery rate (FDR) <0.05. Additionally, the top 50 downregulated phosphorylation sites for the proteins associated with cell cycle (middle, GO:0007049) and DNA repair (right, GO:0006281) Gene Ontology terms are shown. Scale bar represents normalized TMT intensity using medium centering approach. If multiple phosphorylation sites of the same protein were observed, only the most downregulated phosphorylation sites were shown in the heat maps. (B) The x-axis corresponds to the normalized TMT intensity with medium centering approach of global or phosphorylated protein of all proteins that were significantly altered in shASF1A/B cells (ANOVA t test, FDR < 0.05), whereas the y-axis shows their normalized TMT intensity in shASF1A, shASF1B, or shNT cells. The correlation coefficients (r) are shown for shASF1A/B cells with shASF1A (red), shASF1B (blue), and shNT (gray) cells within cell cycle and DNA repair Gene Ontology terms. (C) Venn diagram showing the overlap of proteins with reduced phosphorylation in MOLM-14 cells with ASF1A/B or TLK1/2 knockdown. (D) STRING-DB Gene Ontology analysis for the biological process for the 446 proteins with reduced phosphorylation in MOLM-14 cells with both ASF1A/B or TLK1/2 knockdown. (E) Representative phosphorylated DNA damage proteins upon ASF1 and TLK knockdown. (F) Immunoblot of DNA damage and cell cycle–related proteins upon ASF1 and TLK knockdown. TMT, tandem mass tags.