Journal: bioRxiv

Article Title: Orai1 is required for Ca 2+ -dependent plasma membrane repair and mechanoadaptation

doi: 10.64898/2026.05.13.724989

Figure Lengend Snippet: A. Western blot confirmation of loss of Orai1 expression upon CRISPR/Cas9-mediated KO in MDA-MB-231 and Hs578T cells; shown are pools of three clones maintained individually. B-C. Impairment of SOCE upon Orai1 KO in TNBC cells. SOCE was measured using thapsigargin-induced ER Ca 2+ depletion. Typical profiles are shown on left and quantification of fold-change in peak fluorescence intensity from 3 independent experiments is shown on right. Welch’s t test, ***p<0.001. D-E. Orai1-KO impairs the repair of mechanically induced plasma membrane injury. WT vs. Orai1-KO cells were subjected to mechanical injury to plasma membrane and repair assay was performed as in . Representative confocal images are shown in D and quantified data are shown in E. Scale bar, 200 µm. Data represents mean +/- SEM of three experiments, two-way ANOVA, **,p<0.01. F-G. Orai1-KO impairs the repair of SLO-induced membrane injury. Plasma membrane damage using SLO and the repair assay on MDA-MB-231 ( F ) and Hs578T ( G ) cell lines were as in . Representative confocal images are shown on left. Quantification of PI-stained cells from 3 independent experiments is shown on right. Welch’s t test, ***p<0.001. H-J. Orai1 inhibitors impair plasma membrane repair. Panel H shows Thapsigargin (TG)-induced SOCE measurements (initial peak, Ca 2+ store release in the absence of extracellular Ca 2+ ; second peak, SOCE in the presence of extracellular Ca 2+ ) performed on the indicated cell lines cultured with or without CM4620 (10 μM, 4 hours pretreatment). Note significant SOCE inhibition by CM4620 in WT cells but not in Orai1-KO cells, supporting Orai1-selelctive effect of CM4620. Data represents mean +/- SEM of three experiments, Welch’s t test, ***p<0.001. The I panel shows the impairment of mechanically induced plasma membrane repair by CM4620 in MDA-MB-231. Cells were cultured without or with pretreatment with CM4620 (10 μM) and analyzed for repair as in . Scale bar, 200 µm. Data represents mean +/- SEM of three experiments, two-way ANOVA, **,p<0.01. The J panel shows the impairment of SLO-induced membrane damage in MDA-MB-231. Left panel shows representative FACS analysis of membrane repair in cells without or with CM4620 treatment. Right panel shows quantification of cells that failed to repair (PI staining) from 3 independent experiments. Data represents mean +/-SEM of three experiments. Welch’s t test, ***, p<0.001.

Article Snippet: All-in-One sgRNA CRISPR/Cas9 Lentivectors from Applied Biological Materials (Richmond, BC, Canada) were used to derive Orai1 (pLenti-U6-sgRNA-SFFV-Cas9-2A-Puro, #35720125) KO cell lines.

Techniques: Western Blot, Expressing, CRISPR, Clone Assay, Fluorescence, Clinical Proteomics, Membrane, Staining, Cell Culture, Inhibition