Fig 2: Orai1 regulates Ca2+/CaM/MLCK mediated phosphorylation of MLC. A) Quantification of entotic cells cultured for 4 h in DMSO and MLCK inhibitors, ML-9 (10 µm), and P18 (10 µm). Data represent mean ± SEM of triplicate experiments (n > 300 in each experimental group). Significance was determined using unpaired two-tailed t-test. ***p < 0.001. B,C) Immuno-fluorescence images of suspended cells after 2.5 h of culturing show endogenous pMLC (red) and MLC (blue) in GFP-Orai1-expressing MCF7 cells. pMLC2 is enriched at ID (B) and PJ (C) compared with total MLC. Line graphs show pMLC, MLC, and GFP intensities for the indicated line scans. X axis: 5 µm, Y axis: 5000. D) Immuno-fluorescent images of suspended cells taken after 2.5 h of culturing show endogenous pMLC (green) and CellBrite (red, PM marker). Line graphs show relative pMLC (green) and PM marker (red) intensities for the indicated line scans. X axis: 5 µm, Y axis: 1 (A.U.). E,F) Immunofluorescence images of endogenous pMLC (green) and F-actin (phalloidin, red) cultured with DMSO, P18, or SKF96365 at ID (E) and PJ (F). Line graphs show pMLC and F-actin intensities for the indicated line scans. X axis: 5 µm, Y axis: 20 000. G,H) Quantification of pMLC2 intensity at ID (G, n = 27 in each group) and PJ (H, n = 11 in each group). Significance was determined using unpaired two-tailed t-test. ***p < 0.001; **p < 0.01. I) Quantification of entotic cells cultured for 4 h in MLCK inhibitors, P18 (10 µm) and SOC channel inhibitor, SKF-96365 (10 µm). Data represent mean ± SEM of triplicate experiments (n > 300 in each experimental group). Statistical analysis was performed using one-way ANOVA followed by Dunnett's test. ***p < 0.001. ns: not significant. J) Quantification of entotic cells in Orai1 KO MCF7 cell with MLCK inhibitors, ML-9 (10 µm) and P18 (10 µm). Significance was determined using unpaired two-tailed t-test. ns: not significant. Data represent mean ± SEM of triplicate experiments (n > 300 in each experimental group). K) Immuno-fluorescence images of suspended cells after 2.5 h of culturing show endogenous pMLC (red) and MLC (green) in MCF7 cells. L) Quantification of pMLC/MLC intensity ratio at ID (n = 28, 20, 19). Statistical analysis was performed using one-way ANOVA followed by Dunnett's test. ***p < 0.001. ns: not significant. Scale bar = 5 µm. ED: engulfing cell distal region; CC: cell–cell contact site; ID: invading cell distal region; PJ: peripheral junction.