Cathepsin D genetic depletion in MEF cells does not cause cholesterol nor lysosomal proteins accumulation.

<p>(A) Confocal microscopy of CtsD KO MEFs. Cholesterol (filipin staining, white) and NPC1 (green). (B) Western blot analysis of CtsD KO MEFs using LC3, LAMP1 and NPC1 antibody. α-Tubulin was used as a loading control. (C) 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 < 0.01).</p

Cathepsin B/L inhibition causes NPC disease-like cholesterol accumulation in CHO cells.

<p>(A) Confocal microscopy of CHOwt cells treated with different inhibitors. Cholesterol (filipin staining, white) and NPC1 (green). (B) Western blot of CHOwt cells treated with different inhibitors using ABCA1, NPC1 and NPC2 antibody. β-Actin was used as a loading control. (C) 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 < 0.01, *** p < 0.001).</p

Cathepsin B/L genetic depletion in MEF cells causes accumulation of cholesterol and lysosomal proteins.

<p>(A) Confocal microscopy of CtsB KO, CtsL KO and CtsB/L double KO MEFs. Cholesterol (filipin staining, white) and NPC1 (green). (B) Western blot analysis of CtsB KO, CtsL KO and CtsB/L double KO MEFs using LC3, LAMP1 and NPC1 antibody. α-Tubulin was used as a loading control. (C) 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 < 0.01, *** p < 0.001).</p

Cathepsin B/L inhibition causes NPC disease-like cholesterol accumulation in SH-SY5Y cells.

<p>(A) Confocal microscopy of SH-SY5Y control and PADK treated cells. Cholesterol (filipin staining, white) and NPC1 (green). (B) Western blot of SH-SY5Y control and PADK treated cells using LC3, ABCA1 and NPC1 antibody. α-Tubulin was used as a loading control. (C) 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 < 0.001). (D) RT-PCR Expression of the cholesterol egress genes in the control, PADK, U18666A- and Leu/NH₄Cl-treated SH-SY5Y cells, normalized to β-actin and quantified by 2−ΔΔCt method using control sample as calibrator. ATP-Binding Cassette sub-family A member 1 (ABCA1) and Niemann-Pick C1 protein (NPC1) mRNA levels presented as a fold change. The error bars present the mean ± SEM (* p < 0.05).</p

Inhibition of cathepsin B/L and not cathepsin D causes lysosomal dysfunction.

<p>(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 <i>NPC1</i>-null cells. LysoTracker (red) and Hoechst (blue). (D) Western blot of CHOwt cells treated with different inhibitors and CHO <i>NPC1</i>-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).</p