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. 2021 Mar 10;220(5):e202006035. doi: 10.1083/jcb.202006035

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© 2021 Chandra et al.

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Figure 3. — ANO5 is an ER-resident ion channel that regulates [Ca²⁺]_c during PMR. (A and B) Pseudocolored images (A) and quantification (B) of the kinetics of change in fluorescence of Cl⁻-sensitive YFP and Cl⁻-insensitive RFP fluorescence targeted to ER in healthy and ANO5-deficient patient myoblasts. n = 18 (healthy control) and 15 (patient) cells. (C and D) Pseudocolored images (C) and plot (D) showing quantification of T1ER FRET ratios before and 60 s after injury of healthy and patient cells. n = 20 (healthy) and 19 (patient) cells. (E and F) Images (E) and plots (F) showing FM1–43 dye entry in healthy and patient cells treated or untreated with the SERCA inhibitor TG. n = 16 (healthy), 31 (healthy, TG), 33 (patient), and 45 (patient, TG) cells. (G) Percentage of myoblasts in E and F that repaired following PM injury after indicated treatments. (H) Images before and 60 s after injury. (I and J) Plots showing average kinetics of annexin A1–GFP (AnxA1-GFP; I) and (J) annexin A2–RFP (AnxA2-RFP) accumulation at the repair site (marked by boxes) of healthy and patient myoblasts coexpressing annexin A1–GFP and annexin A2–RFP. n = 22 (healthy) and 18 (patient) cells. (K) Pseudocolored images of healthy and patient myoblasts expressing the mitochondrial calcium sensor CAR-GECO1 imaged during the course of PMR. (L and M) Plots showing quantification of normalized CAR-GECO1 fluorescence in healthy and patient myoblasts during PMR. n = 26 (healthy) and 18 cells (patient). A.U, arbitrary units. (N and O) Healthy and patient myoblasts were colabeled using Tom20-YFP (green) and the mitochondrial potential–sensitive dye TMRE (red). (N) Images of cells before and 20 s after injury. (O) Schematic and plot for quantification of the TMRE/YFP ratio in mitochondria in the injury-distal region of the cell (indicated by dotted line). n = 7 (healthy) and 15 (patient). Scale bars, 10 µm. Plots show mean ± SD; @, P < 0.05; $, P < 0.001; #, P < 0.0001 by Mann–Whitney test or unpaired t test with Welch's correction. Arrowheads indicate site of injury.