Plasma membrane repair is defective in ANO5-KO mouse muscle fibers. (A) Workflow for laser injury assay. FDB myofibers were isolated and damaged with 405-nm light directed at the plasma membrane on the lateral edge of the fiber. (B) Representative images of cytosolic Ca²⁺ detected with Cal-520 following membrane damage in WT or ANO5-KO fibers. t = 0 is before injury (WT and ANO5-KO data points superimposed). Scale bars = 10 µm. (C and D) Cal-520 fluorescence time course after injury, normalized to initial fluorescence (C) and AUC values (D) for individual WT or KO fibers. ANO5-KO n = 12 fibers from three mice, WT n = 18 fibers from two mice. (E) Representative images of FM1-43 infiltration following membrane damage in WT or ANO5-KO fibers. Scale bars = 10 µm. (F and G) Time course of FM1-43 fluorescence normalized to average maximal fluorescence in ANO5-KO fibers (F_maxKO; G) and AUC values for individual WT or KO fibers (G). ANO5-KO n = 17 fibers from three mice, WT n = 22 fibers from three mice. (H) Maximum intensity projections of FM1-43 fluorescence generated from z-stacks of WT or ANO5-KO fibers acquired ∼10 min after injury. (I and J) Quantification of FM1-43–labeled blebs in the repair patch following injury (I) and patch volumes (J). Blebbing of the patch in ANO5-KO fibers contributed to a significant increase in overall patch volume. ANO5-KO n = 17 fibers from three mice, WT n = 14 fibers from two mice. Data are mean ± SEM. *, P < 0.05. Scale bar = 10 μm.