Fig 2: I1061T NPC1 human fibroblasts are more sensitive to lysosomal damage.(A) Primary human fibroblasts homozygous for WT NPC1 (Ctrl) or I1061T NPC1 (I1061T) were treated with Veh or indicated doses of LLOMe for 1 hour and stained for Gal3 to detect damaged lysosomes. Gal3 puncta per cell quantified below. (B) Ctrl and I1061T human fibroblasts were treated with Veh or 2 mM LLOMe for 1 hour and stained for Gal3 and LAMP-1. Colocalization was performed on 4 fields each from 3 independent experiments, with 100–200 cells per experiment. Data are shown as mean ± SEM from 3 independent experiments. *P = 0.05, ***P = 0.001, ****P = 0.0001 by (A) 1-way ANOVA with Tukey’s multiple comparisons (B) t test [(A) F = 24.04, (B) F = 12.78]. Scale bar: 25 µm. NPC, Niemann-Pick disease type C; LLOMe, L-leucyl-L-leucine methyl ester; Ctrl, control; Gal3, galectin-3; Veh, vehicle.

Fig 3: Intracellular galectin 3 (GAL3) accumulation upon treatment with LLOMe. Human ARPE-19 retinal pigment epithelial cells were stained for GAL3 using a specific antibody (green). Untreated cells show no GAL3 puncta, whereas after 30 minutes of treatment with LLOMe (a potent inducer of lysosome membrane permeabilization), the number of GAL3 spots increase, indicating accumulation. Washout reverts the GAL3 accumulation. The graphs show the number of GAL3 spots per cell. Scale bar, 20 µm

Fig 4: Thermoneutrality-driven macrophage accumulation in brown fat is reversed upon acclimation to cold. (A) Schematic representation of experimental groups. (B–E) Gene expression levels of tissue-resident macrophage marker gene (B), general macrophage marker genes (C), M1 macrophage marker genes (D) and M2 macrophage marker genes (E) in IBAT of long-term thermoneutral mice (n = 6) and in IBAT of cold-acclimated, previously long-term thermoneutral mice (n = 5). Values are means ± S.E. ***P < 0.001, significant difference between temperatures by Student’s unpaired t-test. (F–I) Representative confocal images of IBAT from long-term thermoneutral mice (F, G) and cold-acclimated, previously long-term thermoneutral mice (H, I), stained for MAC-2 (green), perilipin (red) and nuclei (blue) using immunohistochemistry. Scale bar 50 µm (applies also to H and I). (J) Representative Western blots of MAC-2 and perilipin in IBAT of the mice as in (B–I). (K–M) MAC-2 protein levels (K), MAC-2 protein content (L) and MAC-2 protein density (M) in IBAT of the mice as in (B–I). The values in IBAT of long-term thermoneutral mice were set to 1.0, and the levels in IBAT of cold-acclimated, previously long-term thermoneutral mice were expressed relative to this value. Values are means ± S.E. *P < 0.05, significant difference between temperatures by Student’s unpaired t-test.

Fig 5: The effect of age on macrophage accumulation in brown fat. (A) Schematic representation of experimental groups. (B–I) Representative confocal images of IBAT from long-term thermoneutral mice (B–E), ~ 11 month old mice housed at room temperature (F, G) and standard mice (H–I) stained for MAC-2 (green), perilipin (red) and nuclei (blue) using immunohistochemistry. Scale bar 50 µm (also applies to D–I). (J–N) Gene expression levels of tissue-resident macrophage marker gene (J), general macrophage marker genes (K), M1 macrophage marker genes (L), M2 macrophage marker genes (M) and thermogenesis-related genes (N) in IBAT of long-term thermoneutral mice (~ 11 month old) (n = 6), in IBAT of ~ 11-month-old mice housed at room temperature (n = 6) and in IBAT of ~ 2-month-old mice housed at room temperature (n = 5). The tissues were analyzed in parallel. Values are means ± S.E. Statistical significance by one-way ANOVA with Tukey post-test (different letters denote groups that are significantly different from each other, with p < 0.05).