Thapsigargin

[Ca²⁺]i was measured by a Fluo-4 NW Calcium Assay Kit (Life Technologies). Because the calcium ion concentration of the NHEK culture medium is low, we used low calcium contained-balanced salt solution (10 mM HEPES, pH7.4, 120 mM NaCl, 4 mM KCl, 1 mM KH₂PO₄, 1 mM MgCl₂, 5 mM glucose, and 0.05 mM CaCl₂) (Karvonen et al., 2000 (link)) instead of the Kit-included Assay buffer. To study calcium ion influx, cells were incubated with dye loading solution (balanced salt solution with Fluo-4 NW dye mix and 2.5 mM probenecid) at 37°C for 30 minutes, and then at room temperature for an additional 30 minutes. TIP39 peptide (5–25 μM), ionomycin (1 μM), thapsigargin (1 μM) (Enzo Life Science), bafilomycin A (0.5 μM) (Sigma-Aldrich), xestospongin C (5 μM), U73122 (5 μM), and W-7 (50 μM) (TOCRIS Bioscience, Bristol, UK) were added as indicated in dye loading solution, and then [Ca²⁺]i was monitored at an emission of 516 nm with excitation of 494 nm using a SpectraMax Gemini EM microplate Reader (Molecular Devices, Sunnyvale, CA) (Jiang et al., 2011 (link)). Fluo-4 signal changes (%) were defined as (F − F0)/F0 × 100. F was the target fluorescence signal intensity and F0 was the baseline calculated by averaging five time points just before the application of the stimulus.

We obtained the compounds for this study from the following sources: DAPT (N-[N-(3,5-difluorophenacetyl)-1-alanyl]-(S)-phenylglycine) (Selleckchem, Houston, TX USA, #S2215), thapsigargin (Enzo Biochem, Inc., USA #BML-PE180–0005).

Intracellular calcium measurement was performed as previously described (Sato et al., 2016). NHEK were seeded at 4×10⁴/well (or 1×10⁴/well for siRNA experiment) one day before experiments. For testing calcium reaction in the endoplasmic reticulum, we used calcium free balanced salt solution with 150μM EGTA. 10 or 25μM TIP39 peptide (Bachem), 5μM thapsigargin, 5 or 10μM ionomycin (Enzo Life Science) and 2 or 2.5 mM CaCl₂ were added as indicated in dye loading solution then Intracellular calcium was monitored at an emission of 516 nm with excitation of 494 nm using SpectraMax Gemini EM microplate Reader (Molecular Devices, Sunnyvale, CA). Fluo-4 signal changes (%) were defined as (F-F0) / F0 × 100. F was the target fluorescence signal intensity and F0 was the baseline calculated by averaging five time points just prior to the application of the stimulus. Intracellular calcium images were captured using a BX41 microscope (Olympus).