Figure 4: Lysosomal Ca²⁺ release via TRPML1 is reduced in NPC cells.

(a,b) ML-SA1-induced lysosomal Ca²⁺ release in GCaMP3-ML1-transfected WT (a) and NPC (b) CHO cells. (c) Colocalization of GCaMP3-ML1 fluorescence with Lamp-1 in NPC CHO cells doubly transfected with GCaMP3-ML1 and Lamp-1-mCherry. Scale bar=5 μm. (d) Comparable expression levels of GCaMP3-ML1 in WT and NPC CHO cells. Expression level of GCaMP3-ML1 was estimated by the maximal GCaMP3 fluorescence signal induced by ionomycin (1 μM). (e) Comparable GPN-induced GCaMP3 responses in WT, NPC or U18666A-treated GCaMP3-ML1-transfected CHO cells. (f) NH₄Cl (10 mM) and GPN (200 μM) induced comparable levels of Ca²⁺ increases (measured with Fura-2 ratios) in WT, NPC and U18666A-treated CHO cells. (g) NH₄Cl (10 mM) and GPN (200 μM) induced comparable levels of Fura-2 Ca²⁺ responses in WT and NPC human fibroblasts. (h) TRPML1-mediated, but not GPN-induced, lysosomal Ca²⁺ release was reduced in GCaMP3-ML1-transfected NPC human fibroblasts compared with WT cells. (i) WT CHO cells were treated with U18666A (2 μg ml⁻¹) for 16 h, and then subjected to filipin staining. Scale bar=10 μm. (j) SMs were stained with Lysenin (0.1 μg ml⁻¹) in WT CHO cells, WT CHO cells treated with U18666A (2 μg ml⁻¹) for 16 h and NPC CHO cells. Scale bar=10 μm. (k) Ca²⁺ responses in GCaMP3-ML1-transfected WT CHO cells treated with U18666A (2 μg ml⁻¹) for 16–20 h. (l) ML-SA1-induced peak GCaMP3 responses were reduced in GCaMP3-ML1-transfected NPC or U18666A-treated WT CHO cells. (m,n) Endogenous TRPML1-mediated lysosomal Ca²⁺ release (measured with Fura-2 ratios) in WT (m) and NPC (n) CHO cells. (o) Average endogenous ML-SA1-induced lysosomal Ca²⁺ responses in WT and NPC CHO cells, and WT, NPC1^−/− and ML1^−/− mouse macrophages. For panels d, e, f, g, h, k, l and o, the results were averaged for 40–100 cells from at least three independent experiments; data are presented as the mean±s.e.m. Statistical comparisons were made using analysis of variance: *P<0.05.