Figure 1.

NAADP Signaling Maintains Late Endosome and Lysosomal Morphology

(A–C) Representative confocal images of LAMP1 staining (white) in fibroblasts treated overnight with either DMSO (0.1%) (A) or the NAADP antagonists, Ned-19 (100 μM) (B) and Ned-K (100 μM) (C). Nuclei were stained using DAPI (blue). Zoomed images are displayed in the right panels. Scale bars, 10 μm.

(D) Representative electron micrographs of the endo-lysosomal system in fibroblasts treated overnight with either DMSO (0.1%) (left) or Ned-19 (100 μM) (right). Regions of interest corresponding to endo-lysosome clusters, the nucleus, and the cell entirety are highlighted. Scale bars, 5 μm.

(E–G) LAMP1 staining in fibroblasts treated overnight with the TPC blocker, tetrandrine (Tet, 10 μM) (E) or for 2 hr with acetoxymethyl (AM) esters of the Ca²⁺ chelators BAPTA (50 μM) (F) or EGTA (50 μM) (G).

(H–J) Summary data quantifying LAMP1 intensity in cells treated with NAADP antagonists (H) (95–299 cells from 3–11 independent treatments), clustering of endo-lysosomes in cells treated with Ned-19 expressed as a percentage of the area occupied relative to non-nuclear cytoplasm (I) (ten cells under each condition), and LAMP1 intensity in cells treated with tetrandrine/Ca²⁺ chelators (J) (73–116 cells from three to five independent treatments). Data are presented as a percentage of DMSO controls (±SEM).

See also Figure S1 and Table S1.