Fig 2: In vitro transformation potential of the NPM1–TYK2 fusion gene.A Detection of overexpressed NPM1–TYK2 mRNA transcript levels in transformed Ba/F3 cells by qRT-PCR. Myla and SU-DHL-1 cell lines were used as positive and negative controls, respectively. Columns represent the mean of three independent experiments; bars represent the SEM. B Subcellular localization of NPM1–TYK2. Ba/F3-Vector and transformed Ba/F3 (NPM1–TYK2) cells were cytospun onto glass slides, fixed, permeabilized, and stained for FLAG antibody and DAPI. Images were acquired with a fluorescent microscope using a 60× oil immersion lens. C Transformation of Ba/F3 cells to IL-3 independent growth. Ba/F3-Vector and transformed Ba/F3 (NPM1–TYK2) cells were grown in RPMI medium in the absence of IL-3. Cell viability from each condition was determined daily by trypan blue exclusion assay. Points represent the mean of three independent experiments; bars represent SEM. ∗∗∗P < 0.0005 was considered as statistically extremely significant. D The clonogenic potential of transformed Ba/F3 (NPM1–TYK2) cells. Ba/F3-Vector and transformed Ba/F3 NPM1–TYK2 cells were mixed with MethoCult medium and plated. After 7 days of incubation, the number of colonies was counted. Columns represent the mean of three independent experiments; bars represent the SEM.

Fig 3: Visual overview.NPM1-TYK2 fusion mediated oncogenic signaling.

Fig 4: JAK and vIRF-1 regulation of pSTAT3 in HHV-8-infected cells.(A) HHV-8 (BAC16) infected iSLK cells were depleted of either TYK2 or JAK1 via lentiviral vector-mediated shRNA expression, using two different shRNAs for each target; non-silencing (ns) shRNA was used as a control. The cultures were left untreated (latent) or treated with Dox/NaB to induce lytic replication, and cells were harvested after six days for immunoblot analysis of pSTAT3, total-STAT3, and lytically expressed vIRF-1 and vIL-6. Relative levels of pSTAT3, normalized to total-STAT3, are shown below the pSTAT3 blots. (B) Analysis of pSTAT3 and pTYK2 levels as a function of vIRF-1 expression in iSLK cells, infected with native (WT) and vIRF-1-knockout (v1ttg) BAC16 viruses. Cultures were ether untreated (latent) or treated with Dox/NaB (lytic) for six days. Immunoblotting of whole-cell lysates was carried out to detect levels of pSTAT3 and pTYK2 relative to total STAT3 and TYK2 and lytic markers vIRF-1 and vIL-6. Charts show quantified data from triplicate lytic cultures, with WT values set at 1. Student’s t-test P values (two-tailed) are shown.

Fig 5: Mapping and functional analysis of vIRF-1 interaction with TYK2.(A) Verification of screening-identified vIRF-1Δ9 (deleted of residues 198–222) as defective for TYK2 interaction. This variant, along with STAT3-binding-defective Δ2 and Δ3 (Fig 3A), were compared for TYK2 and STAT3 interactions by immunoprecipitation (IP) assay employing vIRF-1-Flag as “bait” and immunoblotting for vector-expressed S-tagged TYK2 or endogenous STAT3 in the precipitates. (B) Reciprocal precipitation for vIRF-1-TYK2 interaction, using affinity precipitation (AP) of TYK2-CBD and detection of coexpressed vIRF-1 or vIRF-1Δ9. (C) Testing of vIRF-1Δ9 for suppression of vIL-6-induced STAT3 signaling by transfection-based 4xM67-Luc reporter assay. The variant was inactive in this assay but retained inhibitory effect on the Smad-responsive 4xSBE-Luc reporter, used in parallel transfections. Immunoblotting for Flag-tagged vIRF-1, Smad3, and vIL-6 confirmed appropriate expression of the respective proteins in transfected-cell lysates (combined triplicates). (D) Diminished functional interaction of vIRF-1Δ9 with TYK2 was confirmed by analyzing cell lysates for pTYK2 and pSTAT3 levels in lentiviral vector-transduced, TYK2-overexpressing 292T cells (293T-TYK2) transfected with vIRF-1, vIRF-1Δ9, or RFP (negative control) expression plasmids. Levels of pTYK2 and pSTAT3, normalized to TYK2 and STAT3, respectively, and expressed relative to levels (set at 1) in the RFP vector-transfected culture are shown below the respective blots.