hGAAP increases calpain activity by enhancing SOCE. Changes in [Ca²⁺]_i or rates of Mn²⁺ entry were measured in fura-2–loaded U2-OS cells expressing the indicated hGAAPs or neo control or treated with siRNA. (A) Typical traces show changes in [Ca²⁺]_i after restoration of extracellular Ca²⁺ (2 mM) to populations of cells bathed in nominally Ca²⁺-free HBS. (B) Summary results (means ± SEM, n = 3) show the peak increases in [Ca²⁺]_i. (C) Addition of 1 mM Mn²⁺ to populations of cells in normal HBS quenches cytosolic fura-2 fluorescence as Mn²⁺ enters cells via channels in the plasma membrane. Typical traces show fluorescence normalized to the initial fluorescence intensity. (D) Summary results (means ± SEM, n = 3) show rates of fluorescence quenching, in arbitrary fluorescence units per second (AFU/s), determined from the gradients of lines fitted by linear regression to intervals of 100–200 s after Mn²⁺ addition. (E) Effects of 50 µM 2-APB or 1 µM Gd³⁺ on [Ca²⁺]_i in cells overexpressing hGAAP after restoration of extracellular Ca²⁺ (2 mM) to populations of cells bathed in nominally Ca²⁺-free HBS. (F) Summary results (means ± SEM, n = 3) show the normalized peak Ca²⁺ signals. (G) Effects of 50 µM 2-APB or 1 µM Gd³⁺ on the quenching of fura-2 fluorescence after addition of 1 mM Mn²⁺ to populations of cells overexpressing hGAAP. (H) Summary results (means ± SEM, n = 3) show normalized rates of Mn²⁺-evoked fluorescence quenching. (I) Typical traces show changes in [Ca²⁺]_i after addition of 1 µM thapsigargin (Tg) in nominally Ca²⁺-free HBS to populations of cells overexpressing hGAAP followed by restoration of extracellular Ca²⁺ (2 mM). The initial thapsigargin-evoked increases in [Ca²⁺]_i were smaller in cells overexpressing hGAAP (139 ± 4 nM, n = 5) than in neo cells (154 ± 4 nM, n = 5; P < 0.05) or cells expressing hGAAP Ctmut (176 ± 8 nM, n = 5). (J) Summary results (means ± SEM, n = 3–4) show normalized peak changes in [Ca²⁺]_i after restoration of extracellular Ca²⁺ (2 mM) to thapsigargin-treated cells. Effects of 50 µM 2-APB or 1 µM Gd³⁺ are indicated. Responses are normalized to parallel measurements from cells overexpressing hGAAP. (K) Single-cell analysis shows changes in [Ca²⁺]_i after treatment with 1 µM thapsigargin and restoration of extracellular Ca²⁺ (2 mM) to U2-OS cells overexpressing hGAAP and transfected with either DN-Orai1 or control pcDNA3.1 plasmid. Transfected cells were identified using cotransfected pmCherry-C1. Typical traces show average responses from 11 transfected cells in each condition. (L) Summary results show inhibition of thapsigargin-evoked SOCE by DN-Orai1 in hGAAP and neo cells, calculated from the differences between parallel measurements of SOCE in cells expressing DN-Orai1 or pcDNA3.1 (means ± SEM, from three independent transfections, with responses from 6–18 transfected cells measured in each). (M) Effect of hGAAP siRNA on rates of Mn²⁺-evoked quenching of fura-2 fluorescence (means ± SEM, n = 3). (N) U2-OS cells transfected with the calpain FRET biosensor were plated on fibronectin, treated with 2 µM Gd³⁺ or 50 µM 2-APB, and imaged for 20 min. Typical images, taken from videos, show the IRM and FRET signals recorded 5 min after addition of the inhibitors. Bar, 5 µm. (O) Summary results (means ± SEM, from ≥10 cells) show the percentage of low FRET pixels (<1.5 ratio, i.e., high calpain activity) within 5 µm of the plasma membrane in the image collected at t = 0. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired Student’s t test).