Casin

CASIN (catalog no. 5050, Tocris Biosciences) powder was resuspended in dimethyl sulfoxide (DMSO) at 100 mM, stored at −20 °C, thawed and diluted in PBS before injection. Ruxolitinib (Jakavi, Novartis) was resuspended in polyethylene glycol (catalog no. 202371-500g, Sigma-Aldrich) for administration to mice. Mice were treated with CASIN (5 mg kg⁻¹, intraperitoneally), Ruxolitinib (70 mg kg⁻¹, once daily) or vehicle every other day for 5 weeks. For intravital imaging, 8–12-week-old Nes-GFP recipient mice were irradiated and transplanted with 1,000–5,000 Lin⁻CD150⁺CD48⁻ HSCs isolated from β-actin-DsRed donors (B6.FVB-Tg(Acta2-DsRed)1Rkl/J mice intercrossed with Vav-Cre;JAK2^V617F or Mx1-Cre;JAK2^V617F mice). Recipient mice were treated with CASIN (10 mg kg⁻¹, intraperitoneally), Ruxolitinib (70 mg kg⁻¹, once daily) or vehicle 24, 48 and 72 h after transplantation. The last treatment was performed 2 h before the beginning of the surgery for intravital imaging.

F12‐based cultures performed as described previously (Wilkinson et al, 2020 (link)). Briefly, single or bulk HSCs were cultured on BioCoat fibronectin 96 well plates (Corning) in 200 μl of Ham's F12 nutrient mix (Thermo) supplemented with 1% Insulin‐Transferrin‐Selenium‐Ethanolamine (ITSX, Gibco), 10 mM 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES, Gibco), 1% Penicillin/Streptomycin/L‐Glutamate (P/S/G, Gibco), 100 ng/ml mouse TPO (Preprotech), 10 ng/ml mouse SCF (Peprotech), and 0.1% PVA (Sigma) or HSA (Albumin Bioscience) at 37°C with 5% CO₂. Where indicated, 20 ng/ml of human IL‐11 (SCT or Bio‐Techne) were also used. Complete medium changes were made every 2–3 days after the first 5–6 days. Where indicated, 10% of the cultures were taken out for flow cytometric analysis detailed below. For RhoGTPase inhibitor and activator cultures, indicated concentrations of CASIN (Tocris), NSC23766 (Tocris), Rhosin (Tocris), and ML‐099 (Merck) were used for the entirety of the culture, with medium changes performed as normal.