Xl 90

HEK 293T cells were used for lentiviral production, followed by concentration by ultra-centrifugation. Briefly, a second generation packaging system composed of three plasmids (transfer vector with expression construct LV-GFP, the packaging plasmid psPAX2, and the envelope protein expression plasmid pMD2.G – pMD2.G, a gift from Didier Trono (Addgene plasmid # 12259¹; RRID: Addgene_12259), was mixed in a ratio of 2:1:1 in DMEM (Thermofisher Scientific) and Fugene6 transfection reagent (Roche) and 293T cells were transfected during 4 h to overnight. The supernatant from the transfected plate was collected every 24 h in serum-free conditions and concentrated by ultracentrifugation (Beckman XL-90) at 90.000 rpm for 2 h at 4°C. The concentrated viral solution was passed through a 0.45 μm low-protein binding filter and aliquots were used to transfect the hiPSCs lines. The cell lines F002.1A.13, Gibco^® iPSC6.2 and EMC24i/R2 (C6) were successfully transfected, by first incubating the cells in a 24-well plate during 30 min at 37°C with E8 medium, 10 μM ROCKi and 5 μg/mL of Polybreme (Sigma). Then, LV-GFP previously dissolved in E8 was added dropwise and the cells were incubated during 1 h30 min. Culture medium was then changed daily. After cell growth and expansion, FACS sorting of GFP⁺ cells was performed using BD FACSAria^TM III (BD Biosciences-US) in order to obtain pure populations.

Samples were homogenized in hypotonic homogenization buffer (20 mM Tris-HCl, pH 7.8, 3 mM MgCl₂, 10 mM NaCl, 0.0005 mg/ml, 2 mM sodium vanadate, 20 mM sodium fluoride, 0.5 mM DTT, and 1 mM PMSF) on ice and centrifuged at 15,000 g for 30 min at 4°C to separate cytosolic proteins from intracellular and plasma membranes. The pellet was resuspended in 0.5 M Na₂CO₃, transferred to a 5%/35%/45% sucrose (in Na₂CO₃) flotation gradient and spun at 36,000 rpm for 18 h using a preparative ultracentrifuge model XL-90 (NVT90 rotor; Beckman Coulter Life Sciences). Fractions obtained from the sucrose gradient were diluted in hypotonic buffer and spun at 15,000 g for 30 min at 4°C. Pellets (25 μl) were resuspended in RIPA buffer and 2× Laemmli buffer. About 30 μl of each sample was loaded in 10% SDS-PAGE and transferred to PVDF membranes for 1 h at 220 mA. Nonspecific binding was blocked with 5% nonfat milk and 2% BSA. The samples were incubated overnight at 4°C with TRPV4 (1:500; Alomone Labs), flotillin (1:200; Santa Cruz Biotechnology), Cav-1 (1:1,000; Cell Signaling), and α-SMA (1:500; Sigma-Aldrich) antibodies, followed by anti-mouse (1:5,000; BioRad) or anti-rabbit (1:5,000; Cell Signaling) HRP-conjugated secondary antibodies. The blotted proteins were developed with an enhanced chemiluminescence kit (Thermo Fisher Scientific).