FIGURE 2 from Targeting CHAF1B Enhances IFN Activity against Myeloproliferative Neoplasm Cells

CHAF1B interacts with ULK1 in the nuclear compartment of JAK2^V617F-positive cells and is overexpressed in patients with MPN. A and B, Left, ULK1–protein complexes were co-IP using an anti-ULK1 antibody from cytosolic and nuclear protein fractions isolated from untreated or IFNα-treated (10 or 240 minutes) HEL (A) or SET-2 (B) cells and then resolved by SDS-PAGE. As control, cytosolic and nuclear lysates isolated from cells treated with IFNα for 240 minutes were incubated with normal rabbit IgG (RIgG) antibody. Interaction between ULK1 and CHAF1B was assessed by immunoblotting with anti-ULK1 and anti-CHAF1B antibodies. Note: ǂ, unspecific band. A and B, Right, Equal amounts of cytosolic and nuclear protein lysates isolated from untreated and IFNα-treated HEL (A) and SET-2 (B) cells used for co-IPs were resolved by SDS-PAGE, transferred to PVDF membranes and then immunoblotted with antibodies against ULK1, CHAF1B, α-tubulin (cytosolic marker), and lamin A/C (nuclear marker), as indicated. A and B, Blots are representative of three independent experiments. C, Scatter dot plot of log₂CHAF1B mRNA expression in neutrophils from healthy individuals (normal, n = 11) and patients with ET (n = 47), PMF (n = 18), and PV (n = 28). Data were extracted from NCBI GEO: GSE54646 study (21) and analyzed using GraphPad Prim 8. Shown are means ± SEM. Statistical analysis was performed using one-way ANOVA followed by Dunnett multiple comparisons test to assess P values between patients with MPN and healthy individuals. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Scatter dot plots of log₂CHAF1B mRNA expression in neutrophils from patients with ET (green triangles), PMF (purple diamonds), and PV (red squares) carrying wild-type (WT) or mutant (MUT) JAK2 (D), CALR (E), and TET2 (F) genes (JAK2 WT: ET n = 20, PMF n = 10, PV n = 5; JAK2 MUT: ET n = 26, PMF n = 8, PV n = 23; CALR WT: ET n = 27, PMF n = 11, PV n = 26; CALR MUT: ET n = 15, PMF n = 5, PV n = 1; TET2 WT: ET n = 45, PMF n = 17, PV n = 24; TET2 MUT: ET n = 2, PMF n = 1, PV n = 4). Data were extracted from NCBI GEO: GSE54646 study (21) and analyzed using GraphPad Prim 8. Patients for which no information was available for the mutational status for the JAK2, CALR, or TET2 genes were excluded from the analysis. Shown are means ± SEM. Statistical analyses were performed using two-sample two-tailed t test: *, P < 0.05.