Fig 1: miR-25 directly targets NPC1(A) Predicted binding sites of miR-25 and NPC1 3'UTR.(B) Luciferase activity of HEK293T cells transfected with either PmirGLO-NPC1-WT-UTR or PmirGLO-NPC1-MUT-UTR plus control mimic and miR-25 mimic.(C) THP-1 cells pretreated with control mimic, miR-25 mimic, control inhibitor or miR-25 inhibitor were infected with Bacillus Calmette–Guérin for a specified period of time, and qRT-PCR detection of NPC1 levels was performed,(D and E) western blotting of the lysate was performed to detect the amount of NPC1. (F and G) THP-1 cells pre-transfected with control siRNA or NPC1 siRNA were infected with BCG or H37Rv for a specified period of time, and then CFU detection was performed, indirect immunofluorescence (IF) analysis of the co-localization of autophagosomes (LC3, green) and lysosomes (LAMP-1, red) bar, 10 µM (H).(I) Pearson correlation coefficients (PCCs) of images of internalized Alexa Fluor 488-LC3 and Alexa Fluor 594-LAMP1 in THP-1 cells. Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ns, not significant.
Fig 2: Inhibition of cathepsin B/L and not cathepsin D causes lysosomal dysfunction.(A) EGFR degradation assay. Western blot analysis of EGFR in SH-SY5Y cells treated with different inhibitors at different time points. α-Tubulin was used as a loading control. (B) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Student t-test was used for statistical analysis. Error bars present the mean ± standard deviation (* p < 0.05, ** p < 0.01). (C) Confocal microscopy of CHOwt cells treated with different inhibitors and CHO NPC1-null cells. LysoTracker (red) and Hoechst (blue). (D) Western blot of CHOwt cells treated with different inhibitors and CHO NPC1-null cells using LC3 antibody. β-Actin was used as a loading control. (E) Quantification of Western blot results of the 3 independent experiments was performed by ImageJ. Student t-test was used for statistical analysis. Error bars present the mean ± standard deviation (* p < 0.05, ** p < 0.01).
Fig 3: Correlation between model genes and clinical characteristics. NPC1 showed differential expression levels in (a) different stages (stage III–IV was higher, P < 0.001), (b) grades (grade 3–4 was higher, P < 0.001), and (c) TNM (T3–4 was higher, P < 0.001). ABCC1 showed higher expressions in high (d) grades, (e) stages, and (f) T (P = 0.014, P = 0.008, and P = 0.016, respectively). Patients with higher risk scores had advanced pathological staging, including the (g) stage, (h) grade, and (i) T (P = 0.026, P < 0.001, and P = 0.027, respectively).
Fig 4: Subcellular distribution of free cholesterol and colocalization with lysosomes and NPC1.The cells were grown in the presence of 10% FCS or set to serum-free medium for 16 h before measurements (16 h SF). (A) For staining of free cholesterol, the cells were fixed with paraformaldehyde and stained filipin III (cyan). Bars, 20 µm. (B) For concomitant staining of lysosomes, the cells were incubated with LysoTracker Red DND-99 (orange) before fixing with paraformaldehyde and staining with filipin. Bars, 20 µm. (C) The cells were transfected with NPC1-YFP before fixing with paraformaldehyde and staining with filipin. Bars, 10 µm.
Fig 5: Pharmacological Inhibition of NPC1 Increases Neuronal Excitability(A) Schematic of the hypothesis: lysosomal cholesterol efflux regulates neuron excitability.(B) Inverted confocal micrographs of healthy (left) and NPC1I1061T (right) fibroblasts fixed and stained with filipin. Insets show the accumulation of cholesterol within the lysosome lumen.(C) Comparison of normalized cumulative frequencies of vesicle size between healthy (black, 16 cells, 777 vesicles) and NPC1I1061T (red, 14 cells, 545 vesicles) fibroblasts. *p < 0.0005, Mann-Whitney-Wilcoxon test.(D) Inverted confocal micrographs of control (left) and U18-treated (right; 1 µM, 18 h) sympathetic neurons fixed and stained with filipin.(E) Comparison of normalized cumulative frequencies of vesicle size between neurons cultured without (black, 17 cells, 2,186 vesicles) and with (red, 20 cells,1,373 vesicles, *p < 0.0005, Mann-Whitney-Wilcoxon test) U18.(F) Top: schematic of current injection protocol. Membrane potential was held at -65 mV, and current steps were given from -40 to 100 pA in 10-pA steps. Center: voltage traces from control neuron. Bottom: voltage traces from U18-treated neuron. Note that the U18-treated neuron fires several APs with 40 pA current injection (bottom, black trace, 9 APs) compared to a neuron cultured without U18 (middle, black trace, 1 AP). Dotted lines: -65 mV.(G) Summary of Vrest measured from neurons cultured without (empty circles, n = 8) and with (red circles, n = 8) U18.(H) Quantification of the amount of current required to fire at least 1 AP (rheobase) in control (empty circles, n = 8) and U18-treated (red circles, n = 8) neurons.(I) Comparison of the number of APs induced with increasing current injections in control (empty circles, n = 8) and U18-treated (red circles, n = 8) neurons. In all figures, average data are presented as mean ± SEM. See also Figure S1.
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