Knockdown of TRPV4 in HUVECs

To knockdown TRPV4, HUVECs at greater than 80% confluence in 60-mm dishes were used for transfection [46 (link)]. In brief, lipofectamine 2000 or lipofectamine 3000 (6 μl; Invitrogen) was mixed with Opti-MEM (250 μl; Invitrogen), and then siGenome siRNA human TRPV4 (25 nM, D-004195-03, GE Dharmacon, Lafayette, CO) or non-target control siRNA (25 nM) diluted in 250 μl Opti-MEM was added to the solution, mixed gently, and incubated at room temperature for 20 min. A total of 0.5 ml of this mixture was added to HUVECs in 4 ml Opti-MEM and incubated for 4 h. Then medium was changed back to M200 complete medium and cells were treated after 48 h after transfection.