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. Author manuscript; available in PMC: 2012 Sep 13.
Published in final edited form as: Nat Commun. 2012 Mar 13;3:731. doi: 10.1038/ncomms1735

Figure 4. TRPML1-mediated lysosomal Ca2+ release, but not lysosome Ca2+ store, is reduced in NPC cells.

Figure 4

(a, b) ML-SA1-induced lysosomal Ca2+ release in GCaMP3-ML1-transfected WT (a) and NPC (b) CHO cells. (c) Co-localization 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 to WT cells. (i) WT CHO cells were treated with U18666A (2 μg/ml) for 16 h, and then subjected to filipin staining. Scale bar = 10 μm. (j) Sphingomyelins were stained with Lysenin (0.1 μg/ml) in WT CHO cells, WT CHO cells treated with U18666A (2 μg /ml) 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) 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 3 independent experiments; data are presented as the mean ± SEM. Statistical comparisons were made using analysis of variance: * P <0.05.