Myeloid Cells Transduced With the JAK2V617F Allele Exhibit Enhanced Proinflammatory Properties
(A) Immunoblot analysis reveals modest overexpression of exogenous human JAK2WT and JAK2V617F in THP-1 cells using the lentivirus system (left). Green fluorescent protein (GFP) was expressed in control cells. Signal transducer and activator of transcription (STAT) activities in each experimental group of cells were evaluated by immunoblot with antibodies that detect the level of activating phosphorylation. (B) THP-1 cells were treated with 1 μM of ruxolitinib or vehicle. STAT1 phosphorylation was evaluated by immunoblot analysis. (C) THP-1 cells harboring JAK2V617F were transduced with a lentivirus encoding clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, red fluorescent protein, and single guide RNA targeting human interferon gamma receptor 1. STAT1 phosphorylation was evaluated by immunoblot analysis that detected decreased levels of phosphorylation in IFNGR1 knockout Jak2V617F THP-1 cells. (D) Gene expression analysis of THP-1 cells transduced with lentivirus encoding GFP, JAK2WT, or JAK2V617F at 8 h after stimulation with 10 ng/ml lipopolysaccharide (LPS). n = 3 in each group. Data are shown as mean ± SEM. Statistical analysis was performed using 2-way analysis of variance with Tukey’s multiple comparison tests. Significance stars are from Tukey’s tests. *p < 0.05, **p < 0.01, ***p < 0.001. AIM2 = AIM2, interferon-inducible protein A2; CCL2 = C-C motif chemokine ligand 2; IL = interleukin; p-STAT = phosphorylated; signal transducer and activator of transcription; Rux = ruxolitinib; TNF = tumor necrosis factor; other abbreviations as in Figure 1.