Lysosomal K⁺ homeostasis and cytosolic Ca²⁺ increase are both required for lysosomal Ca²⁺ store refilling. (A) Lysosome-targeted genetically encoded Ca²⁺ indicator GCaMP3-ML1. (B) In HEK293 cells stably expressing GCaMP3-ML1 (HEK-GCaMP3-ML1 cells), lysosomal Ca²⁺ release, as indicated by elevated GCaMP3 fluorescence (F₄₇₀), was induced by bath application of ML-SA1 (25 µM), a membrane-permeable ML1-specific agonist, in a 0 Ca²⁺ (<10 nM) external solution. Averaged responses of 15–30 cells in one coverslip are shown. (C) Short-term (5-min) application of valinomycin (20 µM) in the refilling phase abolished the second ML-SA1 response. (D) Effect of valinomycin on lysosome Ca²⁺ refilling. (E and F) Effect of BAPTA-AM treatment on recovery of lysosomal Ca²⁺ content upon ML-SA1 treatment in ML1-expressing HEK293 cells that were loaded with OG-BAPTA dextran dyes. Statistical comparisons were made with variance analysis (Student’s t test). **, P < 0.01. Error bars indicate SEM.