Ruxolitinib

Universal Type I IFN Alpha (PBL) was used at 5–500 U/mL. Lipofectamine 3000 (Thermo Fisher Scientific) was used to deliver cGAMP (InvivoGen). Z-AAD-CMK (Enzo Life Sciences) was dissolved in water at 10 mM and diluted in medium at a final concentration of 100 μM. Ruxolitinib (LC Laboratories) was dissolved in DMSO at 5 mM and then diluted in medium at a final concentration of 2.5 μM.

Ruxolitinib (R-6688, LC Laboratories) was formulated into Nutra-gel diet (F5769-Kit, Bio-Serv) at a concentration of 1 g/kg. Mice were acclimated to untreated Nutra-gel diet (replaced daily) alongside normal rodent chow for 7 days. Mice were provided with Ruxolitinib diet or Nutra-gel diet ad libitum (replaced daily) for 2 days prior to infection and continued throughout infection study period. In experiments where mice were treated with Amphotericin B during infection, Ruxolitinib diet was added on day 18 post-infection and continued throughout the study period.

Selumetinib (AZD6244, MEK inhibitor), trametinib (GSK1120212, MEK inhibitor), KX2-391 (Src inhibitor) and tofacitinib (CP-690550, JAK3 inhibitor) were purchased from Selleck Chemicals (Houston, TX, USA). PD98059 (MEK inhibitor) and ruxolitinib (INCB081424, JAK1/2 inhibitor) were purchased from LC Laboratories (Woburn, MA, USA). LY5 (STAT3 inhibitor [67 (link)]) was synthesized by Chenglong Li's Lab (College of Pharmacy, The Ohio State University). The drug compounds were dissolved in sterile dimethyl sulfoxide (DMSO) to make a 20 mM stock solution stored at −20°C.

Tumor cells from lymph nodes of moribund Pax5^Jak2/+ and Pax5^Jak2‐Luc/+ mice as well as B220^low and B220⁺ B cells from the bone marrow of Pax5^Jak2/+ mice were sorted by flow cytometry and transferred by intravenous injection (10⁵ cells per mouse) into sublethally irradiated C57BL/6 mice (4.5 Gy). Ruxolitinib (R‐6688; LC Laboratories) was administered twice daily by oral gavage of 45 mg/kg in 0.5% methylcellulose (M0512; Sigma). For bioluminescence imaging, mice were intraperitoneally injected with D‐luciferin (150 mg/kg, Goldbio) and imaged with an IVIS Spectrum Xenogen machine (Caliper Life Sciences).

Isolation, sorting and culture of muscle CD45⁻Ter119⁻CD31⁻CD34⁺Sca1⁻ satellite cells and CD45⁻Ter119⁻CD31⁻CD34⁺Sca1⁺ interstitial cells was carried out as previously described (15 (link)). For pSTAT3 phos-flow analysis, cultured muscle satellite cells, interstitial cells, and the mouse mesenchymal progenitor cell line Kusa4b10 cells (29 (link), 30 (link)) were detached by incubating cell monolayers in PBS plus 4 mM EDTA for 5 min at 37°C. Once in suspension, cells were washed in Dulbecco modified essential medium (DMEM, Gibco, Life Technologies), centrifuged and resuspended in DMEM. Cell aliquots (1 × 10⁶) were then preincubated with or without 1 μM ruxolitinib (LC Laboratories), for 30 min at 37°C and subsequently stimulated by addition of 25 ng/mL recombinant mouse OSM (R&D Systems) for 10 min. Cells were then immediately washed in 10 mL ice cold Tris-buffered saline pH 7.4 containing 1 mM Na₃VO₄, centrifuged, and cell pellets resuspended for fixation and permeabilization (BD Cytofix, Perm buffer IV, BD Biosciences) for 10 and 30 min respectively, cells were then stained with AlexaFluor647-conjugated mouse anti-pSTAT3 (pY705) monoclonal antibody (BD Biosciences, catalog # 557815) for 30 min on ice, cells were then washed and subsequently run on a LSR Fortessa x20 flow cytometer (BD Biosciences). Files were subsequently analyzed with Flow Jo software version 10.4.

Bryostatin-1, prostratin, ingenol-3,20-dibenzoate and ingenol-3-hexanoate, also known as ingenol B, were obtained from the Martin Delaney Collaboratory of AIDS Researchers for Eradication (CARE) Pharmacology Core, University of North Carolina, Chapel Hill, NC. The kinase inhibitor library was obtained from the University of Utah Drug Discovery Core Facility. CD3/CD28 antibody-coated magnetic beads (Dynabeads^® Human T-Activator CD3/CD28) were purchased from Life Technologies (ThermoFisher Scientific). Ruxolitinib was purchased from LC Laboratories, Woburn MA.