Journal: EMBO Reports

Article Title: Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol

doi: 10.1038/s44319-024-00328-x

Figure Lengend Snippet: ( A ) Graph representing the percentage of peroxisome-lysosome colocalization in HeLa cells after infection with WT STM with MOI = 10. Interaction of PEX14 and LAMP1 was monitored at 3, 6, and 12-h post-infection. Data for three independent experiments containing more than 200 cells are shown. ( B ) Cholesterol levels on isolated peroxisomes from HeLa cells infected with indicated bacterial strains (MOI = 10) for 6 h. Data represent the mean ± SEM of three independent experiments. ( C ) Representative confocal micrographs of HeLa and PEX5 knockout (KO) HeLa cells infected with either GFP-tagged WT STM or GFP-tagged Δ sseI STM (MOI = 50) for 6 h. Cells were immunostained with anti-LAMP1 antibody to label SCVs (red); cholesterol was stained using filipin (blue) and STM (green). Scale bars: 10 µm (main panel), 2 µm (inset). ( D ) Graph representing the change in fluorescence intensity of filipin, indicating cholesterol on SCVs. Data represent the mean ± SEM from three independent experiments with more than 180 cells analyzed. ( E ) Graph representing the changes in fold proliferation of STM (MOI = 10) after silencing NPC1 in HeLa cells. Data represent the mean ± SEM of three independent experiments. ( F ) Graph representing the changes in fold proliferation of STM (MOI = 10) after silencing ABCD1 in HeLa cells. Data represent the mean ± SEM of three independent experiments. ( G ) Change in fold proliferation of WT STM with LDL/U18666A in the media containing 1% FBS in HeLa cells. Data represent the mean ± SEM of three independent experiments. ( H ) Graph representing the changes in fold proliferation of STM (MOI = 10) after treatment with Atorvastatin (1 µM), the media containing 1% FBS in HeLa cells. Data represent the mean ± SEM of three independent experiments. ( I ) Representative confocal micrographs of HeLa cells infected with either GFP-tagged WT STM with Syt-7 Flag overexpressing plasmid. In Zoom, the yellow arrowhead represents the colocalization between Syt-7 (red), LAMP1 (green), and STM (blue). Scale bars: 10 µm (main panel), 2 µm (inset). ( J ) Representative confocal micrographs of uninfected HeLa cells with Syt-7 flag overexpressing plasmid. In Zoom, the yellow arrowheads represent the colocalization between Syt-7 (red) and PEX14 (green). Scale bars: 10 µm (main panel), 2 µm (inset). ( K ) Representative confocal micrographs of Syt-7 flag overexpressing HeLa cells infected with GFP-tagged WT STM (blue). In Zoom, the yellow arrowheads represent the colocalization between Syt-7 (red), PEX14 (green), and STM (blue). Scale bars: 10 µm (main panel), 2 µm (inset). ( L ) Graph representing the percentage colocalization of peroxisome and Syt-7 in HeLa cells after infection with WT STM and uninfected cells. Data of three independent experiments containing more than 80 cells are shown. ( M , N ) Change in fold proliferation of WT STM (MOI = 10) after silencing Syt-7 ( M ) or E-Syt1 ( N ) in HeLa cells. Data represent the mean ± SEM of three independent experiments. ( O ) Cholesterol levels on isolated peroxisomes from HeLa cells silenced for NPC1 , SYT7 , ARF1 , and PIP4K2A and infected with WT STM (MOI = 10) for 6 h. Data represent the mean ± SEM of three independent experiments. Data analysis: Data were analyzed using one-way ANOVA (Sidak’s multiple comparisons test), ( A ) (* p = 0.0452), ( B ) (*** p = 0.0007, ** p = 0.0023) ( D ) (** p = 0.0018), ( G ) (** p = 0.0019), and ( O ) (* p = 0.0144). Data were analyzed using student’s t -test, ( E ) (*** p = 0.0004), ( F ) (*** p = 0.0006), ( H ) (** p = 0.0039), ( L ) (* p = 0.0313), ( M ) (** p = 0.0041). ‘ns’ denotes non-significant difference. .

Article Snippet: NPC1 , Santa Cruz , SC-41588.

Techniques: Infection, Isolation, Knock-Out, Staining, Fluorescence, Plasmid Preparation

Journal: EMBO Reports

Article Title: Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol

doi: 10.1038/s44319-024-00328-x

Figure Lengend Snippet: ( A ) Fold change in mRNA levels of NPC1 after its silencing in HeLa cells. Graph represent the ± SEM of three biological replicates. ( B ) Fold change in mRNA levels of ABCD1 after its silencing in HeLa cells. Graph represent the ± SEM of three biological replicates. ( C ) Graph representing the changes in fold proliferation of STM (MOI = 10) after silencing LDLR in HeLa cells. Three independent experiments were performed. Each dot represents the mean of an independent experiment. ( D ) Fold change in mRNA levels of LDLR after its silencing in HeLa cells. Graph represent the ± SEM of three biological replicates. ( E ) Fold proliferation of STM (MOI = 10) in HeLa cells under LDL-free FBS conditions following Atorvastatin (1 µM). HeLa cells were treated with Atorvastatin and DMSO as Vehicle control. Three independent experiments were performed. Each dot represents the mean of an independent experiment. ( F ) Fold proliferation of STM (MOI = 10) in HeLa cells under 1% FBS conditions following HMGCR silencing. HeLa cells were transfected with siRNA targeting HMGCR or scrambled siRNA (Scr) control. Three independent experiments were performed. Each dot represents the mean of an independent experiment. ( G ) Graph representing the changes in fold proliferation of STM (MOI = 10) after silencing DHCR24 in HeLa cells at 1% FBS condition. Three independent experiments were performed. Each dot represents the mean of an independent experiment. ( H ) Fold change in mRNA levels of HMGCR after its silencing in HeLa cells. Graph represent the ± SEM of three biological replicates. ( I ) Fold change in mRNA levels of DHCR24 after its silencing in HeLa cells. Graph represent the ± SEM of three biological replicates. ( J ) Graph representing the changes in intracellular fold proliferation of STM (MOI = 10) in HeLa cells at 10% and 1% FBS condition. Three independent experiments were performed. Each dot represents the mean of an independent experiment. ( K ) Fold change in mRNA levels of SYT7 after its silencing in HeLa cells. Graph represent the ± SEM of three biological replicates. ( L ) Fold change in mRNA levels of ESYT1 after its silencing in HeLa cells. Graph represent the ± SEM of three biological replicates. ( M ) Representative microscopy images of HeLa cells infected with GFP-tagged STM (blue) (MOI = 50) and immunostained for endogenous Syt-7/E-syt1 (green) and LAMP1 (red) antibody. The intensity profile indicating the overlap of signals of Syt-7-LAMP1 and E-syt1-LAMP1 is shown on the right. Scale bars: 10 µm, 2 µm. ( N ) Immunoblot Analysis of Syt-7 Localization on Peroxisomes in HeLa cells. Cells were either infected with STM or left uninfected. Whole-cell lysates (WCL) and isolated peroxisome protein samples (both washed and unwashed) were subjected to immunoblot analysis using antibodies against Syt-7, the peroxisomal marker PEX14, and GAPDH as a loading control. Data information: ( A – F ) Data were analyzed using student’s t-test; ( A ) (**** p = 0.0001), ( B ) (** p = 0.0043), ( C ) (* p = 0.0297), ( D ) (* p = 0.0156), ( E ) (** p = 0.0053), ( F ) (* p = 0.0160), ( G , H ) (** p = 0.0043), ( I ) (*** p = 0.0002), ( J ) (* p = 0.0253), and ( K ) (**** p = 0.0001), ( L ) (* p = 0.0182), ‘ns’ denotes non-significant.

Article Snippet: NPC1 , Santa Cruz , SC-41588.

Techniques: Control, Transfection, Microscopy, Infection, Western Blot, Isolation, Marker

Journal: EMBO Reports

Article Title: Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol

doi: 10.1038/s44319-024-00328-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: NPC1 , Santa Cruz , SC-41588.

Techniques: Derivative Assay, Recombinant, Plasmid Preparation, Synthesized, Control, Sequencing, Cloning, Protease Inhibitor, Software

Journal: The Journal of biological chemistry

Article Title: NPC1 plays a role in the trafficking of specific cargo to melanosomes.

doi: 10.1016/j.jbc.2023.105024

Figure Lengend Snippet: Figure 1. Generation of the NPC1-KO cell line. A, immunoblot analysis of NPC1 protein in MNT-WT, MNT-bulk, and NPC1-KO (four clones). Calnexin (CNX) was used as loading control. B, relative lysosomal volume of NPC1-KO cells (four clones) to MNT-WT cells measured by LysoTracker staining and FACS analysis. Data analysis was based on mean fluorescence of three independent experiments and represented as mean ± SD (one-way ANOVA analysis; ****p < 0.0001). C, immunofluorescence staining with anti-LAMP-2; the scale bar represents 10 μm. D, graphic representation of mean fluorescence intensity of LAMP-2 immunostaining, n = 52 cells (two-tailed student’s t test; ****p < 0.0001). E and F, immunoblot analysis and quantification of LAMP-2 (E) and cathepsin D (F) in MNT-WT versus NPC1-KO (clone 2). Band densitometry plot of LAMP-2 (n = 5 biological replicates) and cathepsin D (n = 3 biological replicates) was represented as mean ± SD. Two-tailed Student’s t test; ****p < 0.0001,*p < 0.05. FACS, fluorescence-activated cell sorting; LAMP, lysosome- associated membrane protein; NPC1, Niemann–Pick type C1.

Article Snippet: NPC1 RNAi silencing and NPC1 transfection 2 × 106 MNT-1 cells were seeded in 6-well plate and were transfected with 50 nM NPC1 siRNA or control siRNA-A (sc37007, Santa Cruz Biotechnology) using lipofectamine 2000.

Techniques: Western Blot, Clone Assay, Control, Staining, Immunostaining, Two Tailed Test, FACS, Membrane

Journal: The Journal of biological chemistry

Article Title: NPC1 plays a role in the trafficking of specific cargo to melanosomes.

doi: 10.1016/j.jbc.2023.105024

Figure Lengend Snippet: Figure 2. Characterization of the lipid storage phenotypes in the NPC1-KO cell line. A, cholesterol measurement by Amplex Red assay, data are presented as the mean ± SD, MNT-WT versus NPC1-KO, n = 3 biological replicates. B, D, and E, assay for total GSLs (B), for different GSL species (D) and for GM2, the main storage species (E) by HPLC, n = 2 biological replicates. C, representative GSLs trace from MNT-WT cells. F, the mean ± SD of GlcCer in MNT- WT and NPC1-KO, n = 3 biological replicates. NPC1, Niemann–Pick type C1; GlcCer, glucosylceramidase; GSL, glycosphingolipid.

Article Snippet: NPC1 RNAi silencing and NPC1 transfection 2 × 106 MNT-1 cells were seeded in 6-well plate and were transfected with 50 nM NPC1 siRNA or control siRNA-A (sc37007, Santa Cruz Biotechnology) using lipofectamine 2000.

Techniques: Amplex Red Assay

Journal: The Journal of biological chemistry

Article Title: NPC1 plays a role in the trafficking of specific cargo to melanosomes.

doi: 10.1016/j.jbc.2023.105024

Figure Lengend Snippet: Figure 3. NPC1-KO cells present pigmentation defects. A, visual comparison of MNT-WT and NPC1-KO cell pellets. B, C, and D, immunoblot analysis and quantification of TYR in NPC1-KO (clone 3 and 2) versus MNT-WT cells (B), in MNT-WT cells transfected with siControl (CTRL) or with siNPC1 (C) and in NPC1- KO cells transfected with empty vector—pcDNA3.1+ or with hNPC1pcDNA3.1+ (hNPC1) (D). Intensities of TYR protein bands were quantified and repre- sented as mean ± SD (two-tailed Student’s t test; **p < 0.01; *p < 0.05, n = 3 biological replicates). E, co-immunoprecipitation of NPC1 protein using rabbit anti-NPC1 antibody in MNT-WT and NPC1-KO cell lysates was assessed. The resulted immunocomplexes were eluted with LB, and NPC1 and TYR were visualized by immunoblotting (n = 3 biological replicates). F, in-gel analysis of TYR activity. G, TYR relative gene expression level determined by semi- quantitative qRT-PCR, n = 3 biological replicates. H–J, immunoblot analysis and quantification of MITF (H), TYRP-1 (I), and DCT (J) protein levels normalized to CNX or actin, divided by WT average and represented as mean ± SD, n = 3 biological replicates. CNX, calnexin; DCT, dopachrome-tautomerase; MITF, microphthalmia-associated transcription factor; NPC1, Niemann–Pick type C1; qRT-PCR, real time quantitative reverse transcription PCR; TYR, tyrosinase; TYRP, tyrosinase-related protein.

Article Snippet: NPC1 RNAi silencing and NPC1 transfection 2 × 106 MNT-1 cells were seeded in 6-well plate and were transfected with 50 nM NPC1 siRNA or control siRNA-A (sc37007, Santa Cruz Biotechnology) using lipofectamine 2000.

Techniques: Comparison, Western Blot, Transfection, Plasmid Preparation, Two Tailed Test, Immunoprecipitation, Activity Assay, Gene Expression, Quantitative RT-PCR, Reverse Transcription

Journal: The Journal of biological chemistry

Article Title: NPC1 plays a role in the trafficking of specific cargo to melanosomes.

doi: 10.1016/j.jbc.2023.105024

Figure Lengend Snippet: Figure 4. Proteome analysis of MNT-WT versus NPC1-KO cells by mass spectrometry. A, principal component analysis (PCA) plot showing clustering of replicates according to their characteristics. B, volcano plot indicating protein expression differences of MNT-WT versus NPC1-KO according to two sample t test (p < 0.05 and absolute log2FC ≥1), n = 3 biological replicates. C, heatmap of log2 transformed LFQ intensity values showing the statistically significant upregulated and downregulated proteins in NPC1-KO cell line. D, box plot of log2 transformed LFQ intensity values of upregulated proteins involved in lipid metabolism. E, pie chart presenting protein classes of significantly downregulated protein in NPC1-KO cell line based on the Panther database. F, dot plot of log2 transformed LFQ intensity of selected proteins belonging to melanogenesis pathway. LFQ, label-free quantitation; NPC1, Niemann–Pick type C1.

Article Snippet: NPC1 RNAi silencing and NPC1 transfection 2 × 106 MNT-1 cells were seeded in 6-well plate and were transfected with 50 nM NPC1 siRNA or control siRNA-A (sc37007, Santa Cruz Biotechnology) using lipofectamine 2000.

Techniques: Mass Spectrometry, Expressing, Transformation Assay, Quantitation Assay

Journal: The Journal of biological chemistry

Article Title: NPC1 plays a role in the trafficking of specific cargo to melanosomes.

doi: 10.1016/j.jbc.2023.105024

Figure Lengend Snippet: Figure 5. NPC1 impairs TYR processing and degradation. A, cells were untreated or treated with bafilomycin A (100 nM, 5 h) and analyzed by Western blotting for TYR N-glycan processing after Endo H and PNGase F glycosidase treatment of cell lysates. Relative expression levels of PNGase F-sensitive TYR and the ratio of Endo H-resistant to Endo H-sensitive TYR were plotted. Quantitative representation as mean ± SD of relative protein expressions and the ratio of Endo H resistant to Endo H sensitive bands (one-way ANOVA analysis; *p < 0.05, **p < 0.01, n = 3 biological replicates). B, visual comparison of cell pellets of MNT-WT and NPC1-KO cells untreated or treated with bafilomycin A1 (100 nM, 5 h). C, immunoblot analysis for TYR, LAMP-2, and CNX in cell lysates and in the enrichment of secreted extracellular vesicles from culture supernatant. D, effect of MG132 (50 μM, 5 h) or inhibitor mix (INH = 14.5 uM leupeptin and 106 uM pepstatin, 5 h) cell treatment on TYR expression, analyzed by Western blotting and densitometry, represented as fold change relative to cells treated with DMSO. Quantitative representation as mean ± SD of relative protein expression, n = 3 biological replicates (one-way ANOVA analysis; *p < 0.05). E, CHX treated MNT-WT and NPC1-KO cells analyzed by immunoblotting for TYR normalized to tubulin and graphic representation of TYR degradation rate as a percentage of control (CHX, 0 h) (n = 2 biological replicates). CHX, cycloheximide; CNX, calnexin; DMSO, dimethyl sulfoxide; Endo H, endoglycosidase H; LAMP, lysosome-associated membrane protein; NPC1, Niemann–Pick type C1; PNGase F, peptide-N glycosidase F; TYR, tyrosinase.

Article Snippet: NPC1 RNAi silencing and NPC1 transfection 2 × 106 MNT-1 cells were seeded in 6-well plate and were transfected with 50 nM NPC1 siRNA or control siRNA-A (sc37007, Santa Cruz Biotechnology) using lipofectamine 2000.

Techniques: Western Blot, Glycoproteomics, Expressing, Comparison, Control, Membrane

Journal: The Journal of biological chemistry

Article Title: NPC1 plays a role in the trafficking of specific cargo to melanosomes.

doi: 10.1016/j.jbc.2023.105024

Figure Lengend Snippet: Figure 7. Investigation of the PMEL17 pathway in the absence of NPC1. A, immunoblot analysis of PMEL17 protein recognized by PEP-13 (immature PMEL17-P1 and M-beta) and HMB-45 (M-alpha/P2, M-alpha-C and RPT) antibodies. The insoluble fraction was extracted with 1% SDS and 50 mM DTT and boiled at 95 C. The KO HMB-45 positive fraction was normalized to loading control, and divided by WT average and the relative fold change was rep- resented as mean ± SD (Two-tailed Student’s t test; ***p <0.001, n = 3 biological replicates). B, the surface expression of PMEL17 analyzed by flow cytometry represented in a bar plot as the median of fluorescence related to MNT-WT. C, immunoblot analysis of ApoE protein and graphical representation as mean ± SD of relative protein expressions(Two-tailed Student’s t test; *p <0.05, n = 3 biological replicates). D, subcellular fractionation of MNT-WT and NPC1-KO cell homogenates on a sucrose gradient. Twelve fractions were collected and analyzed by immunoblotting with antibodies directed against NPC1, EEA1, LAMP- 2, TYR, TYRP-1, and HMB-45. Graphic representation of quantified proteins expression level as mean of two independent experiments. For TYR, three independent replicates were quantified and plotted as mean ± SD. ApoE, apolipoprotein E; HMB, human melanoma black; LAMP, lysosome-associated membrane protein; NPC1, Niemann–Pick type C1; TYR, tyrosinase; TYRP, tyrosinase-related protein.

Article Snippet: NPC1 RNAi silencing and NPC1 transfection 2 × 106 MNT-1 cells were seeded in 6-well plate and were transfected with 50 nM NPC1 siRNA or control siRNA-A (sc37007, Santa Cruz Biotechnology) using lipofectamine 2000.

Techniques: Western Blot, Control, Two Tailed Test, Expressing, Cytometry, Fractionation, Membrane

Journal: The Journal of biological chemistry

Article Title: NPC1 plays a role in the trafficking of specific cargo to melanosomes.

doi: 10.1016/j.jbc.2023.105024

Figure Lengend Snippet: Figure 8. Schematic representation of intracellular TYR trafficking during melanosome maturation. In this model, we propose a model of the mechanism of melanosome biogenesis by highlighting PMEL17 and the TYR pathways in NPC1-KO cells compared to MNT-WT cells. In MNT-WT, as in NPC1- KO cells the PMEL17 protein is trafficked from the TGN to the plasma membrane (PM) and is then endocytosed, while TYR is targeted to early endosomes directly or via PM. The early endosomes progressively mature into MVB, from which late endosomes, premelanosome or multiple vesicle exosomes (MVE) are developed. As illustrated, exosome secretion is not affected in NPC1 deficient cells mutant cells, while the pathways which lead to melanosome maturation are impaired in NPC1 KO cells. PMEL17 is sorted to intraluminal vesicles in premelanosome and forms a sheet-like matrix of amyloid fibrils, where melanin can be deposited. In the absence of the NPC1 protein, PMEL17 fibrils accumulates and the maturation of the immature melanosomes is perturbed (dashed arrows). In MNT-WT cells, TYR is sorted from the MVB to immature melanosomes, where it initiates the melanin biosynthesis process. In contrast, in NPC1-deficient cells, TYR delivery to immature melanosomes is reduced, due to its target misrouting for lysosomal degradation. The key to the proteins indicated in the figure are presented in the right hand panel. The modified pathways in NPC1-deficient cells are indicated by dashed red arrows. MVB, multivesicular bodies; NPC1, Niemann–Pick type C1; TGN, trans-Golgi network; TYR, tyrosinase.

Article Snippet: NPC1 RNAi silencing and NPC1 transfection 2 × 106 MNT-1 cells were seeded in 6-well plate and were transfected with 50 nM NPC1 siRNA or control siRNA-A (sc37007, Santa Cruz Biotechnology) using lipofectamine 2000.

Techniques: Clinical Proteomics, Membrane, Mutagenesis

Journal: The Journal of biological chemistry

Article Title: NPC1 plays a role in the trafficking of specific cargo to melanosomes.

doi: 10.1016/j.jbc.2023.105024

Figure Lengend Snippet: Figure 1. Generation of the NPC1-KO cell line. A, immunoblot analysis of NPC1 protein in MNT-WT, MNT-bulk, and NPC1-KO (four clones). Calnexin (CNX) was used as loading control. B, relative lysosomal volume of NPC1-KO cells (four clones) to MNT-WT cells measured by LysoTracker staining and FACS analysis. Data analysis was based on mean fluorescence of three independent experiments and represented as mean ± SD (one-way ANOVA analysis; ****p < 0.0001). C, immunofluorescence staining with anti-LAMP-2; the scale bar represents 10 μm. D, graphic representation of mean fluorescence intensity of LAMP-2 immunostaining, n = 52 cells (two-tailed student’s t test; ****p < 0.0001). E and F, immunoblot analysis and quantification of LAMP-2 (E) and cathepsin D (F) in MNT-WT versus NPC1-KO (clone 2). Band densitometry plot of LAMP-2 (n = 5 biological replicates) and cathepsin D (n = 3 biological replicates) was represented as mean ± SD. Two-tailed Student’s t test; ****p < 0.0001,*p < 0.05. FACS, fluorescence-activated cell sorting; LAMP, lysosome- associated membrane protein; NPC1, Niemann–Pick type C1.

Article Snippet: NPC1 RNAi silencing and NPC1 transfection 2 × 106 MNT-1 cells were seeded in 6-well plate and were transfected with 50 nM NPC1 siRNA or control siRNA-A (sc37007, Santa Cruz Biotechnology) using lipofectamine 2000.

Techniques: Western Blot, Clone Assay, Control, Staining, Immunostaining, Two Tailed Test, FACS, Membrane

Journal: The Journal of biological chemistry

Article Title: NPC1 plays a role in the trafficking of specific cargo to melanosomes.

doi: 10.1016/j.jbc.2023.105024

Figure Lengend Snippet: Figure 2. Characterization of the lipid storage phenotypes in the NPC1-KO cell line. A, cholesterol measurement by Amplex Red assay, data are presented as the mean ± SD, MNT-WT versus NPC1-KO, n = 3 biological replicates. B, D, and E, assay for total GSLs (B), for different GSL species (D) and for GM2, the main storage species (E) by HPLC, n = 2 biological replicates. C, representative GSLs trace from MNT-WT cells. F, the mean ± SD of GlcCer in MNT- WT and NPC1-KO, n = 3 biological replicates. NPC1, Niemann–Pick type C1; GlcCer, glucosylceramidase; GSL, glycosphingolipid.

Article Snippet: NPC1 RNAi silencing and NPC1 transfection 2 × 106 MNT-1 cells were seeded in 6-well plate and were transfected with 50 nM NPC1 siRNA or control siRNA-A (sc37007, Santa Cruz Biotechnology) using lipofectamine 2000.

Techniques: Amplex Red Assay

Journal: The Journal of biological chemistry

Article Title: NPC1 plays a role in the trafficking of specific cargo to melanosomes.

doi: 10.1016/j.jbc.2023.105024

Figure Lengend Snippet: Figure 3. NPC1-KO cells present pigmentation defects. A, visual comparison of MNT-WT and NPC1-KO cell pellets. B, C, and D, immunoblot analysis and quantification of TYR in NPC1-KO (clone 3 and 2) versus MNT-WT cells (B), in MNT-WT cells transfected with siControl (CTRL) or with siNPC1 (C) and in NPC1- KO cells transfected with empty vector—pcDNA3.1+ or with hNPC1pcDNA3.1+ (hNPC1) (D). Intensities of TYR protein bands were quantified and repre- sented as mean ± SD (two-tailed Student’s t test; **p < 0.01; *p < 0.05, n = 3 biological replicates). E, co-immunoprecipitation of NPC1 protein using rabbit anti-NPC1 antibody in MNT-WT and NPC1-KO cell lysates was assessed. The resulted immunocomplexes were eluted with LB, and NPC1 and TYR were visualized by immunoblotting (n = 3 biological replicates). F, in-gel analysis of TYR activity. G, TYR relative gene expression level determined by semi- quantitative qRT-PCR, n = 3 biological replicates. H–J, immunoblot analysis and quantification of MITF (H), TYRP-1 (I), and DCT (J) protein levels normalized to CNX or actin, divided by WT average and represented as mean ± SD, n = 3 biological replicates. CNX, calnexin; DCT, dopachrome-tautomerase; MITF, microphthalmia-associated transcription factor; NPC1, Niemann–Pick type C1; qRT-PCR, real time quantitative reverse transcription PCR; TYR, tyrosinase; TYRP, tyrosinase-related protein.

Article Snippet: NPC1 RNAi silencing and NPC1 transfection 2 × 106 MNT-1 cells were seeded in 6-well plate and were transfected with 50 nM NPC1 siRNA or control siRNA-A (sc37007, Santa Cruz Biotechnology) using lipofectamine 2000.

Techniques: Comparison, Western Blot, Transfection, Plasmid Preparation, Two Tailed Test, Immunoprecipitation, Activity Assay, Gene Expression, Quantitative RT-PCR, Reverse Transcription

Journal: The Journal of biological chemistry

Article Title: NPC1 plays a role in the trafficking of specific cargo to melanosomes.

doi: 10.1016/j.jbc.2023.105024

Figure Lengend Snippet: Figure 4. Proteome analysis of MNT-WT versus NPC1-KO cells by mass spectrometry. A, principal component analysis (PCA) plot showing clustering of replicates according to their characteristics. B, volcano plot indicating protein expression differences of MNT-WT versus NPC1-KO according to two sample t test (p < 0.05 and absolute log2FC ≥1), n = 3 biological replicates. C, heatmap of log2 transformed LFQ intensity values showing the statistically significant upregulated and downregulated proteins in NPC1-KO cell line. D, box plot of log2 transformed LFQ intensity values of upregulated proteins involved in lipid metabolism. E, pie chart presenting protein classes of significantly downregulated protein in NPC1-KO cell line based on the Panther database. F, dot plot of log2 transformed LFQ intensity of selected proteins belonging to melanogenesis pathway. LFQ, label-free quantitation; NPC1, Niemann–Pick type C1.

Article Snippet: NPC1 RNAi silencing and NPC1 transfection 2 × 106 MNT-1 cells were seeded in 6-well plate and were transfected with 50 nM NPC1 siRNA or control siRNA-A (sc37007, Santa Cruz Biotechnology) using lipofectamine 2000.

Techniques: Mass Spectrometry, Expressing, Transformation Assay, Quantitation Assay

Journal: The Journal of biological chemistry

Article Title: NPC1 plays a role in the trafficking of specific cargo to melanosomes.

doi: 10.1016/j.jbc.2023.105024

Figure Lengend Snippet: Figure 5. NPC1 impairs TYR processing and degradation. A, cells were untreated or treated with bafilomycin A (100 nM, 5 h) and analyzed by Western blotting for TYR N-glycan processing after Endo H and PNGase F glycosidase treatment of cell lysates. Relative expression levels of PNGase F-sensitive TYR and the ratio of Endo H-resistant to Endo H-sensitive TYR were plotted. Quantitative representation as mean ± SD of relative protein expressions and the ratio of Endo H resistant to Endo H sensitive bands (one-way ANOVA analysis; *p < 0.05, **p < 0.01, n = 3 biological replicates). B, visual comparison of cell pellets of MNT-WT and NPC1-KO cells untreated or treated with bafilomycin A1 (100 nM, 5 h). C, immunoblot analysis for TYR, LAMP-2, and CNX in cell lysates and in the enrichment of secreted extracellular vesicles from culture supernatant. D, effect of MG132 (50 μM, 5 h) or inhibitor mix (INH = 14.5 uM leupeptin and 106 uM pepstatin, 5 h) cell treatment on TYR expression, analyzed by Western blotting and densitometry, represented as fold change relative to cells treated with DMSO. Quantitative representation as mean ± SD of relative protein expression, n = 3 biological replicates (one-way ANOVA analysis; *p < 0.05). E, CHX treated MNT-WT and NPC1-KO cells analyzed by immunoblotting for TYR normalized to tubulin and graphic representation of TYR degradation rate as a percentage of control (CHX, 0 h) (n = 2 biological replicates). CHX, cycloheximide; CNX, calnexin; DMSO, dimethyl sulfoxide; Endo H, endoglycosidase H; LAMP, lysosome-associated membrane protein; NPC1, Niemann–Pick type C1; PNGase F, peptide-N glycosidase F; TYR, tyrosinase.

Article Snippet: NPC1 RNAi silencing and NPC1 transfection 2 × 106 MNT-1 cells were seeded in 6-well plate and were transfected with 50 nM NPC1 siRNA or control siRNA-A (sc37007, Santa Cruz Biotechnology) using lipofectamine 2000.

Techniques: Western Blot, Glycoproteomics, Expressing, Comparison, Control, Membrane

Journal: The Journal of biological chemistry

Article Title: NPC1 plays a role in the trafficking of specific cargo to melanosomes.

doi: 10.1016/j.jbc.2023.105024

Figure Lengend Snippet: Figure 7. Investigation of the PMEL17 pathway in the absence of NPC1. A, immunoblot analysis of PMEL17 protein recognized by PEP-13 (immature PMEL17-P1 and M-beta) and HMB-45 (M-alpha/P2, M-alpha-C and RPT) antibodies. The insoluble fraction was extracted with 1% SDS and 50 mM DTT and boiled at 95 C. The KO HMB-45 positive fraction was normalized to loading control, and divided by WT average and the relative fold change was rep- resented as mean ± SD (Two-tailed Student’s t test; ***p <0.001, n = 3 biological replicates). B, the surface expression of PMEL17 analyzed by flow cytometry represented in a bar plot as the median of fluorescence related to MNT-WT. C, immunoblot analysis of ApoE protein and graphical representation as mean ± SD of relative protein expressions(Two-tailed Student’s t test; *p <0.05, n = 3 biological replicates). D, subcellular fractionation of MNT-WT and NPC1-KO cell homogenates on a sucrose gradient. Twelve fractions were collected and analyzed by immunoblotting with antibodies directed against NPC1, EEA1, LAMP- 2, TYR, TYRP-1, and HMB-45. Graphic representation of quantified proteins expression level as mean of two independent experiments. For TYR, three independent replicates were quantified and plotted as mean ± SD. ApoE, apolipoprotein E; HMB, human melanoma black; LAMP, lysosome-associated membrane protein; NPC1, Niemann–Pick type C1; TYR, tyrosinase; TYRP, tyrosinase-related protein.

Article Snippet: NPC1 RNAi silencing and NPC1 transfection 2 × 106 MNT-1 cells were seeded in 6-well plate and were transfected with 50 nM NPC1 siRNA or control siRNA-A (sc37007, Santa Cruz Biotechnology) using lipofectamine 2000.

Techniques: Western Blot, Control, Two Tailed Test, Expressing, Cytometry, Fractionation, Membrane

Journal: The Journal of biological chemistry

Article Title: NPC1 plays a role in the trafficking of specific cargo to melanosomes.

doi: 10.1016/j.jbc.2023.105024

Figure Lengend Snippet: Figure 8. Schematic representation of intracellular TYR trafficking during melanosome maturation. In this model, we propose a model of the mechanism of melanosome biogenesis by highlighting PMEL17 and the TYR pathways in NPC1-KO cells compared to MNT-WT cells. In MNT-WT, as in NPC1- KO cells the PMEL17 protein is trafficked from the TGN to the plasma membrane (PM) and is then endocytosed, while TYR is targeted to early endosomes directly or via PM. The early endosomes progressively mature into MVB, from which late endosomes, premelanosome or multiple vesicle exosomes (MVE) are developed. As illustrated, exosome secretion is not affected in NPC1 deficient cells mutant cells, while the pathways which lead to melanosome maturation are impaired in NPC1 KO cells. PMEL17 is sorted to intraluminal vesicles in premelanosome and forms a sheet-like matrix of amyloid fibrils, where melanin can be deposited. In the absence of the NPC1 protein, PMEL17 fibrils accumulates and the maturation of the immature melanosomes is perturbed (dashed arrows). In MNT-WT cells, TYR is sorted from the MVB to immature melanosomes, where it initiates the melanin biosynthesis process. In contrast, in NPC1-deficient cells, TYR delivery to immature melanosomes is reduced, due to its target misrouting for lysosomal degradation. The key to the proteins indicated in the figure are presented in the right hand panel. The modified pathways in NPC1-deficient cells are indicated by dashed red arrows. MVB, multivesicular bodies; NPC1, Niemann–Pick type C1; TGN, trans-Golgi network; TYR, tyrosinase.

Article Snippet: NPC1 RNAi silencing and NPC1 transfection 2 × 106 MNT-1 cells were seeded in 6-well plate and were transfected with 50 nM NPC1 siRNA or control siRNA-A (sc37007, Santa Cruz Biotechnology) using lipofectamine 2000.

Techniques: Clinical Proteomics, Membrane, Mutagenesis

Journal: Redox biology

Article Title: Lipophagy-mediated cholesterol synthesis inhibition is required for the survival of hepatocellular carcinoma under glutamine deprivation.

doi: 10.1016/j.redox.2023.102732

Figure Lengend Snippet: Fig. 3. Glutamine deprivation increases ER cholesterol via NPC1. (A) The mRNA and protein levels of SREBF2 were detected in HepG2 and Huh7 cells after cultured with Com and Gln-for 24 h by RT-qPCR (left panel) and western blot (right panel). (B and C) ER were isolated from HepG2 and Huh7 cells after cultured with Com and Gln-for 24 h. (B) The purity of isolated ER were determined by examining Calnexin expression. (C) ER cholesterol were measured by using the Amplex™ Red Cholesterol Assay Kit. (D–G) HepG2 and Huh7 were treated with U18666A (0.5 μM) under glutamine-deprived and complete conditions for 24 h. (D and E) ER were isolated from these cells to measure cholesterol. Mature SREBF2 protein (F) and HMGCR and SQLE mRNA (G) were detected, respectively. Data present as mean ± SD of at least three independent biological experiments. *p < 0.05, **p < 0.01, and ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Scrambled siRNAs were synthesized in Sangon Biotech. siRNAs that target on SREBF2 (sc-36559), Insig 1 (sc-44432), NPC1 (sc-41588), ATG7 (sc-41447), TSC2 (sc-36762) and NPRL2 (sc-62699) are the products of Santa Cruz.

Techniques: Cell Culture, Quantitative RT-PCR, Western Blot, Isolation, Expressing, Amplex Red Cholesterol Assay

Journal: bioRxiv

Article Title: Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol for better intracellular growth

doi: 10.1101/2023.02.27.530266

Figure Lengend Snippet: LDL-derived cholesterol routed via lysosome-peroxisome is essential for Salmonella growth A. Graph representing percentage of peroxisome-lysosome co-localization in the HeLa cells after infection with the WT STM (MOI=10) for different time points. B. WT HeLa cells were infected with WT STM or STM ΔsseI for 6 h with MOI= 50. pex5 -/- HeLa cell were infected with WT STM for 6 h with MOI= 50. Cells were fixed and immunostained with LAMP1(red), cholesterol using filipin(blue) and STM (green). Zoom part represented SCV and Lysosome cholesterol (blue) level in the cells. Graph representing change in the filipin fluorescent intensity is shown. (n=50 cells). Scale bar 10 uM. C. PEROXO- tagged stable cell was generated using transfection with lentivirus overexpressing 3X myc-EGFP-Pex26. Stable PEROXO tag HeLa cells were next infected with the WT and ΔsseI STM with MOI=10 for 6 hr. Isolated peroxisomal cholesterol amount was measured using cholesterol assay kit. Graph representing the fold proliferation of STM silencing D. NPC1 E. ABCD1 in HeLa cells. Changes in fold proliferation of WT STM after 2 and 16 hr post-infection is plotted in these cells. F. Cells were kept untreated or treated with lovastatin (10uM). Changes in fold proliferation of WT STM after 2 and 16 hr post-infection is plotted in these cells. G. Cells were kept untreated or treated with LDL/U18666A in the media containing 1% FBS in HeLa cells. Changes in fold proliferation of WT STM after 2 and 16 hr post-infection is plotted in these cells. Individual data points represent mean± SD. Result is representative of 3 independent experiments. ‘MOI’ denotes multiplicity of infection. ‘src’ denoted scrambled siRNA. ‘UI’ denotes uninfected, ‘UT’ denotes untreated. ‘ns’ denotes non- significant ****p<0.0001, ***p<0.001, *p<0.05, **p<0.01, unpaired two-tailed Student T-test.

Article Snippet: All siRNAs SMART pool were obtained from Santa Cruz and Dharmacon: siRNA constructs (SC-105086(ARF1); Synaptotagmin VII siRNA (h): sc-41320; E-Syt1 siRNA (h): sc-95714; NPC1 siRNA (h): sc-41588); Pex14 (h): L-012676-00-0005, ON-Target plus TM Control Pool, D-001810-10-20 (Dharmacon DM ); ABCD (h) sc-41143; non-specific non-targeting pool using Lipofectamine RNAiMAX reagent according to the manufacturer’s instructions.

Techniques: Derivative Assay, Infection, Stable Transfection, Generated, Transfection, Isolation, Cholesterol Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol for better intracellular growth

doi: 10.1101/2023.02.27.530266

Figure Lengend Snippet: Knockdown efficiency and cell viability after knockdown. A-B. Graph representing the silencing efficiency of ABCD1 and NPC1 in HeLa cells. C-F. Cell viability was checked by MTT assay in HeLa cells transfected/treated with si-NPC1, U18666A, lovastatin and si-ABCD1. Individual data points represent mean± SD. Result is representative of 3 independent experiments. ‘MOI’ denotes multiplicity of infection. ‘ns’ denotes non-significant, **p<0.01 unpaired two-tailed Student T-test.

Article Snippet: All siRNAs SMART pool were obtained from Santa Cruz and Dharmacon: siRNA constructs (SC-105086(ARF1); Synaptotagmin VII siRNA (h): sc-41320; E-Syt1 siRNA (h): sc-95714; NPC1 siRNA (h): sc-41588); Pex14 (h): L-012676-00-0005, ON-Target plus TM Control Pool, D-001810-10-20 (Dharmacon DM ); ABCD (h) sc-41143; non-specific non-targeting pool using Lipofectamine RNAiMAX reagent according to the manufacturer’s instructions.

Techniques: MTT Assay, Transfection, Infection, Two Tailed Test

Journal: bioRxiv

Article Title: Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol for better intracellular growth

doi: 10.1101/2023.02.27.530266

Figure Lengend Snippet: SYT-7 present on SCV is essential for interaction with peroxisome. A. PEROXO-tagged stable cell was generated using transfection with lentivirus overexpressing 3X myc-EGFP-Pex26. Stable PEROXO tag HeLa cells were next transfected with non-targeting si RNA (scr) or si RNA against NPC1, SYT7 and ARF1 and incubated for 48 hours. Then the transfected cells were infected with WT STM with MOI=10 for 6 hr. Peroxisomal cholesterol is shown as measured by using cholesterol assay kit. B. Representative microscopy images showing syt7(green) co-localization with lysosome (LAMP1) and SCV (red) with STM of MOI=50, scale bar 25 uM. C. HeLa cells were transfected with non-targeting si RNA (scr) or si RNA against syt7 for 48 hr. Silencing efficiency of syt7 was measured by q-PCR. D. Changes in fold proliferation of WT STM after 2 and 16 hr post-infection is plotted in these cells. Individual data points represent mean ± SD. Result is representative of 3 independent experiments. ‘MOI’ denotes multiplicity of infection. ‘ns’ denotes non- significant ****p<0.0001, *p<0.05, unpaired two-tailed Student T-test.

Article Snippet: All siRNAs SMART pool were obtained from Santa Cruz and Dharmacon: siRNA constructs (SC-105086(ARF1); Synaptotagmin VII siRNA (h): sc-41320; E-Syt1 siRNA (h): sc-95714; NPC1 siRNA (h): sc-41588); Pex14 (h): L-012676-00-0005, ON-Target plus TM Control Pool, D-001810-10-20 (Dharmacon DM ); ABCD (h) sc-41143; non-specific non-targeting pool using Lipofectamine RNAiMAX reagent according to the manufacturer’s instructions.

Techniques: Stable Transfection, Generated, Transfection, Incubation, Infection, Cholesterol Assay, Microscopy, Two Tailed Test