FIGURE 3 from Targeting CHAF1B Enhances IFN Activity against Myeloproliferative Neoplasm Cells

Gene-targeted inhibition of CHAF1B increases IFNα-inducible mRNA expression of ISGs in JAK2V617F-positive leukemic cells. qRT-PCR analyses of the indicated genes in control (Ctrl) siRNA or CHAF1B siRNA-transfected JAK2V617F-positive HEL (A) and SET-2 (B) cells, either left untreated or treated with IFNα for 6 hours. GAPDH mRNA expression was used for normalization. Data are expressed as fold change over control siRNA-transfected untreated cells (control) and represent means ± SEM of three independent experiments for each cell line. Statistical analyses were performed using one-way ANOVA followed by Tukey multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. C, ChIP assay was performed in untreated and IFNα-treated (for 6 hours at 5,000 IU/mL) HEL cells at the ISG15 promoter and the IFIT1 promoter for CHAF1B binding using an anti-CHAF1B antibody. IgG antibody was used for each promoter region as negative control. Scatter dot plots show data as percent enrichment relative to input ± SEM for three independent experiments. Statistical analyses were performed using one-way ANOVA followed by Tukey multiple comparisons test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. D, Immunoblot analysis of ULK1 expression in control siRNA (Ctrlsi) and ULK1 siRNA (ULK1si) transfected HEL cells either left untreated or treated with IFNα (for 6 hours at 5,000 IU/mL), as indicated. GAPDH levels were used as loading control. Blots are representative of three independent experiments used to perform ChIP assays shown in E. E, ChIP assay was performed in untreated (UT) and IFNα-treated control (Ctrl) siRNA- and ULK1 siRNA-transfected HEL cells at the ISG15 promoter and the IFIT1 promoter for CHAF1B binding using an anti-CHAF1B antibody. F, HEL cells were pretreated for 1 hour with either DMSO (Ctrl and IFNα groups) or SBI-0206965 (SBI; 10 μmol/L; SBI and SBI+IFNα groups) followed by 6 hours of treatment with either DMSO (Ctrl), SBI (10 μmol/L), IFNα (5,000 IU/mL) or SBI+IFNα, as indicated. ChIP assay was performed in HEL cells at the ISG15 promoter and the IFIT1 promoter for CHAF1B binding using an anti-CHAF1B antibody. E and F, IgG antibody was used for each promoter region as negative control. Scatter dot plots show data as percent enrichment relative to input ± SEM for three independent experiments. Statistical analyses were performed using two-way ANOVA followed by Tukey multiple comparisons test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Relevant statistical differences are shown for the binding of CHAF1B to ISG15 and IFIT1 promoter regions between experimental conditions.