Gsk1016790a

Human CD34⁺ cells were isolated, separated and cultured, as described previously.^{27 (link),28 (link)} All human samples were collected in accordance with the ethical committee of the IRCCS Policlinico San Matteo Foundation, Pavia, Italy, and the principles of the Declaration of Helsinki. For collagen receptor inhibition, MKs at day 13 of culture were incubated with 10 μg/mL anti-β1 integrin blocking antibody (Millipore, clone P5D2), 10 μg/mL anti-GPVI blocking antibody (a kind gift of Prof. Jandrot Perrus) or with 200 nM (125 ng/mL) Discoidin Domain Receptor 1 (DDR1)-IN-1 (Tocris), a selective DDR1 tyrosine kinase inhibitor, for one hour prior to being plated on type I or type IV collagen for three hours (h). For Akt inhibition experiments, MKs at day 13 of culture were treated with 10 μM Akt1/2 inhibitor (Sigma Aldrich) for 30 minutes (min) and then plated on collagens for 16 h for PPT evaluation. For treatment with the TRPV4 inhibitors (RN-1734, HC067047, Sigma Aldrich), MKs at day 13 of culture were incubated with vehicle or 10 μM of the indicated TRPV4 inhibitor for 30 min prior to being plated on collagens for 3 or 16 h. For treatment with the TRPV4 agonist (GSK1016790A, Sigma Aldrich), MKs at day 13 of culture were incubated or not with 10 μM GSK1016790A for 10 min prior to being plated on collagens for 3 h.

Pipette solution was prepared and calculated for free 2.5 μM Ca²⁺ concentration (based on equilibrium constants for EGTA chelation of Ca²⁺ and Mg²⁺; Winmaxc32 software, C. Patton, Stanford University, USA), according to the TMEM16B EC₅₀ reported [48 (link)]. Intracellular solution contained (in mM): 20.2 TEA-Cl, 9.9 CaCl2, 50 HEPES, 85 sucrose and 25 HEDTA; adjusted to pH = 7.3 with TEA-OH. Extracellular solution contained (in mM): 139 TEA-Cl, 0.5 CaCl₂, 20 HEPES, 110 sucrose; adjusted to pH = 7.4 with TEA-OH. Niflumic acid, GSK1016790A and GSK2193874 compounds (from Sigma-Aldrich, St. Louis, MO) were prepared in DMSO, kept as stock solution 1000 times concentrated for each different concentration used, and stored at −20^oC. Bath solution was used for dilution to obtain the Niflumic acid, GSK1016790A or GSK2193874 required concentrations.