Figure 2

Ca²⁺ influx and Ca²⁺ stores in SLC10A7 overexpressing and SLC10A7 knockout cells. Calcium imaging was performed in HAP1 (control), HAP1-KOP7 (SLC10A7 knockout), HEKP7-tet (control, cells without tetracycline treatment), and HEKP7+tet (SLC10A7 overexpression after tetracycline treatment) cells pre-loaded with 2 µM Fluo-4 AM. Cells were treated with 2 µM ionomycin (a,b), 1 µM TG (c,d), or 100 µM ATP+Crb (e,f) in the absence of extracellular calcium to allow ER depletion. Then, 2 mM Ca²⁺ were added to allow store-operated Ca²⁺ entry. Fluorescence recording was performed every 10 s, and cell-based fluorescence was determined at defined regions of interest for each cell line (n = 6–10 for the HAP1 cells and n = 9–20 for the HEK293 cells), with a total number of about 84–260 cells. The bar graphs indicate the maximum peak data after ionomycin/TG/ATP+Crb (first peak) and calcium (second peak) treatment, respectively. (g) Cells were incubated in growth medium supplemented with 4 mM Ca²⁺ for 20 min. Total cellular Ca²⁺ was detected using a colorimetric calcium assay and was calculated after measuring the absorbance at 575 nm. All data were related to the total protein content of the cells. Data represent means ± SD of triplicate determinations of a representative experiment. (h) The Ca²⁺ content of intracellular stores was calculated by measuring the peak of ionomycin-mediated Ca²⁺ release in the presence of 3 mM extracellular EGTA (inset). Cell-based fluorescence was analyzed in each cell line at 10 defined regions of interest with a total number of about 180 to 260 cells. All data represent means ± SD of a representative experiment. *Significantly different with p  < 0.05 (Student’s t-test).