FIGURE 3 from Targeting CHAF1B Enhances IFN Activity against Myeloproliferative Neoplasm Cells
Authors: Saleiro Diana , Kosciuczuk Ewa M. , Fischietti Mariafausta , Perez Ricardo E. , Yang G. Sohae , Eckerdt Frank , Beauchamp Elspeth M. , Hou Ye , Wang Qixuan , Weinberg Rona Singer , Fish Eleanor N. , Yue Feng , Hoffman Ronald , Platanias Leonidas C. 
Published: May 2023

<p>Gene-targeted inhibition of <i>CHAF1B</i> increases IFNα-inducible mRNA expression of ISGs in <i>JAK2</i><sup>V617F</sup>-positive leukemic cells. qRT-PCR analyses of the indicated genes in control (Ctrl) siRNA or <i>CHAF1B</i> siRNA-transfected <i>JAK2</i><sup>V617F</sup>-positive HEL (<b>A</b>) and SET-2 (<b>B</b>) cells, either left untreated or treated with IFNα for 6 hours. <i>GAPDH</i> 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. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001. <b>C,</b> ChIP assay was performed in untreated and IFNα-treated (for 6 hours at 5,000 IU/mL) HEL cells at the <i>ISG15</i> promoter and the <i>IFIT1</i> 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; *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001. <b>D,</b> Immunoblot analysis of ULK1 expression in control siRNA (Ctrlsi) and <i>ULK1</i> siRNA (<i>ULK1</i>si) 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 <b>E</b>. <b>E,</b> ChIP assay was performed in untreated (UT) and IFNα-treated control (Ctrl) siRNA- and <i>ULK1</i> siRNA-transfected HEL cells at the <i>ISG15</i> promoter and the <i>IFIT1</i> promoter for CHAF1B binding using an anti-CHAF1B antibody. <b>F,</b> 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 <i>ISG15</i> promoter and the <i>IFIT1</i> promoter for CHAF1B binding using an anti-CHAF1B antibody. <b>E</b> and <b>F,</b> 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: *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; ****, <i>P</i> < 0.0001. Relevant statistical differences are shown for the binding of CHAF1B to <i>ISG15</i> and <i>IFIT1</i> promoter regions between experimental conditions.</p>