Fig 2: Ca2+ influx activates SPAK and OSR1. (A) HDMECs were incubated with ionomycin (2 µM) for the indicated time points and subjected to immunoblotting with the specified antibodies. Graphs show the relative levels of phosphorylated SPAK or OSR1. Data are presented as mean ± SEM. Statistical significance is indicated as follows: n, not significant; a, P < 0.05; b, P < 0.01; c, P < 0.001; d, P < 0.0001, compared with 0 min, determined by one-way ANOVA with Tukey’s multiple comparison test (n = 3). (B–D) HDMECs were loaded with Fluo-8 AM (2 µM) for 40 min at 37 °C. After 30 s baseline was recorded, DMSO, ionomycin (2 μM), or WNK463 (1 μM) were added, as indicated by arrows. (B) Changes in intracellular Ca2+ levels were measured at the specified time points. Data are presented as mean ± SD (8 replicates, 2 independent experiments). (C) showed the relative intensity of Fluo-8 AM measured 5 min after treatment with DMSO and ionomycin (2 μM). Data are presented as mean ± SD, ****P < 0.0001 (unpaired two-tailed t test, n = 3). (D) Representative Fluo-8 AM Ca2+ imaging (green) of HDMECs at 5 min. (Scale bar, 10 μm.) (E) Immunoblot analysis of HDMECs treated with DMSO, Yoda1 (5 μM), or ionomycin (2 μM) in the presence or absence of WNK463 (1 μM) for 5 min. Data are presented as mean ± SD from three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001 (unpaired two-tailed t test, nontreated vs. WNK463-treated). (F) Immunoblot analysis of HDMECs treated with siCTRL or siWNK1, followed by treatment with DMSO, Yoda1 (5 μM), or ionomycin (2 μM) for 5 min. Data are presented as mean ± SD, **P < 0.01, ***P < 0.001 (unpaired two-tailed t test, n = 3, nontreated vs. WNK463-treated). (G) Immunoblot analysis was performed on HDMECs treated with siCTRL or siPiezo1, followed by 5 min’ treatments with DMSO, Yoda1 (5 μM), or Ionomycin (2 μM), using the indicated antibodies. Statistical significance was determined by the unpaired two-tailed t test (mean ± SD), *P < 0.05, **P < 0.01, ***P < 0.001 (n = 3, siCTRL vs. siPiezo1).

Fig 3: Hypoosmotic stress activates OSR1 and SPAK through Piezo1. (A) Schematic of a cell responding to osmotic stress, either hyperosmotic or hypoosmotic, resulting in changes in membrane tension and Ca2+ influx. (B) Immunoblot analysis of HDMECs treated with osmotic stress in the presence of DMSO or WNK463 (1 μM) for 5 min. The bar graph is presented as mean ± SD from three independent experiments. **P < 0.01, ***P < 0.001 (unpaired two-tailed t test, nontreated vs. WNK463-treated); #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 (one-way ANOVA with Tukey’s multiple comparison test within the DMSO group). (C) siCTRL- and siPiezo1-treated HDMECs were exposed to osmotic stress for 5 min and analyzed by immunoblotting using the indicated antibodies. The results are expressed as mean ± SD. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001 (n = 3), unpaired two-tailed t test (siCTRL vs. siPiezo1). (D and E) Intracellular Ca2+ levels were measured in Fluo-8 AM (2 µM)-labeled HDMECs exposed to isotonic, hypertonic (500 mM NaCl), or hypotonic [3:2 (v/v) deionized H2O/culture medium mixture] solutions, as indicated by the arrows. Data are expressed as mean ± SD (8 replicates, 2 independent experiments) (D). The graph represents the relative Fluo-8 AM intensity measured 3 min after osmotic stress exposure (E). Data are shown as mean ± SD. ***P < 0.001 (unpaired two-tailed t test, 8 replicates). (F) Ca2+-starved HDMECs, maintained in Ca2+/Mg2+-free HBSS (−Ca2+) containing 0.1% FBS for 1 h, were exposed to osmotic stress in the Ca2+-free HBSS for 5 min. Cell lysates were analyzed by SDS-PAGE and immunoblotting. Data are shown as mean ± SD. **P < 0.01; ns, not significant (n = 3). (G) Immunoblot analysis of HDMECs preincubated with DMSO or BAPTA-AM (10 μM) for 10 min, followed by exposure to osmotic stress for 5 min. The results are presented as mean ± SD. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001 (n = 3), unpaired two-tailed t test (DMSO-treated vs. BAPTA-AM-treated).

Fig 4: Piezo1 agonist Yoda1 activates WNK1-OSR1/SPAK signaling. (A) Immunoblot analysis of HDMECs treated with siCTRL (negative control) or siPiezo1 for 72 h. Equal amounts of cell lysates were subjected to SDS-PAGE and immunoblotted with indicated antibodies. Data are representative for three independent experiments (mean ± SD), ***P < 0.001, ****P < 0.0001, unpaired two-tailed t test. (B) Immunoblot analysis of HDMECs treated with DMSO or WNK463 (1 μM) for 5 min. Data are presented as mean ± SD. ***P < 0.001, ****P < 0.0001 (unpaired two-tailed t test, n = 3). (C) HDMECs were treated with Yoda1 (5 µM) for the indicated time points and analyzed by immunoblotting using the specified antibodies. Graphs depict the relative levels of phosphorylated SPAK or OSR1. Data are presented as mean ± SEM. Statistical significance is indicated as follows: n, not significant; b, P < 0.01; c, P < 0.001; d, P < 0.0001, compared with 0 min, determined by one-way ANOVA with Tukey’s multiple comparison test (n = 3). (D–F) HDMECs were loaded with Fluo-8 AM (2 µM) for 40 min at 37 °C. After recording 30 s baseline, cells were treated with DMSO or Yoda1 (5 μM) indicated by arrows. (D) Changes in intracellular Ca2+ levels were measured at the specified time points. Data are presented as mean ± SD (8 replicates, 2 independent experiments). (E) The relative intensity of Fluo-8 AM measured 5 min after treatment. Data are presented as mean ± SD. ****P < 0.0001, unpaired two-tailed t test (n = 3). (F) Representative Fluo-8 AM Ca2+ imaging (green) of HDMECs at 5 min. (Scale bar, 10 μm.) (G) Immunoblot analysis was performed on HDMECs treated with siCTRL or siPiezo1, followed by 5 min’ treatments with DMSO or Yoda1 (5 μM), using the indicated antibodies. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparison test (mean ± SD), **P < 0.01, ****P < 0.0001 (n = 3, siCTRL vs. siPiezo1). (H) Intracellular Ca2+ levels were measured in Fluo-8 AM (2 µM)-labeled siCTRL- or siPiezo1-HDMECs treated with Yoda1 (5 μM). Data are representative of three independent experiments (mean ± SD) ***P < 0.001, ****P < 0.0001, unpaired two-tailed t test (4 replicates).