Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: Terminal organ weights (mg wet tissue) and body weights (grams) in female and male NPC1 −/− mice treated with either vehicle or THLs.

Article Snippet: A human NPC1 expression plasmid (#SC120010), under the influence of the CMV promoter, is designated pCMV-NPC1, and was purchased from Origene (Rockville, MD).

Techniques:

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: H&E stains of cerebral cortex ( A – C ), spleen ( D – F ), and liver ( G – I ) from NPC1 −/− mice or control mice at euthanasia. Panels ( A , D , G ) are NPC1 −/− mice treated with vehicle; panels ( B , E , H ) are NPC −/− mice treated with TfRMAb/pPDGFB-NPC1 THLs. Panels ( C , F , I ) are control mice. Arrows in panels ( A , D , G ) point to cholesterol-laden cells in brain, spleen, and liver, respectively. Brain sections are through cortical layers I–IV. Magnification is the same for all panels and the panel A magnification bar = 100 microns.

Article Snippet: A human NPC1 expression plasmid (#SC120010), under the influence of the CMV promoter, is designated pCMV-NPC1, and was purchased from Origene (Rockville, MD).

Techniques: Control

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: Electron microscopy of cerebral cortex of vehicle ( A , B ) or THL ( C , D ) treated NPC1 −/− mouse at euthanasia. Multi-lamellated vacuoles of varying size are present in the cytoplasm of brain cells. Magnification in panels ( A ) and ( C ) is the same and magnification bar in panel ( A ) is 1,000 nm; magnification in panels ( B ) and D is the same and magnification bar in panel ( B ) is 1,000 nm.

Article Snippet: A human NPC1 expression plasmid (#SC120010), under the influence of the CMV promoter, is designated pCMV-NPC1, and was purchased from Origene (Rockville, MD).

Techniques: Electron Microscopy

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: GFAP immunocytochemistry of thalamus of NPC1 −/− mouse treated with vehicle ( A ) or the TfRMAb/pPDGFB-NPC1 THLs ( B ). The GFAP immunocytochemistry of thalamus from a control mouse is shown in ( C ). Sections are counter-stained with hematoxylin. Magnification of all 3 panels is the same and the panel ( A ) magnification bar = 100 microns.

Article Snippet: A human NPC1 expression plasmid (#SC120010), under the influence of the CMV promoter, is designated pCMV-NPC1, and was purchased from Origene (Rockville, MD).

Techniques: Immunocytochemistry, Control, Staining

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: Organ concentrations (pg plasmid DNA per mg wet tissue) in female and male NPC1 −/− mice at 4 days following the last IV injection of either vehicle or THLs.

Article Snippet: A human NPC1 expression plasmid (#SC120010), under the influence of the CMV promoter, is designated pCMV-NPC1, and was purchased from Origene (Rockville, MD).

Techniques: Plasmid Preparation, IV Injection

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: Organ enrichment of NPC1 mRNA, relative to GAPDH mRNA, in THL treated NPC1 −/− mice as compared to vehicle treated NPC1 −/− mice.

Article Snippet: A human NPC1 expression plasmid (#SC120010), under the influence of the CMV promoter, is designated pCMV-NPC1, and was purchased from Origene (Rockville, MD).

Techniques:

Journal: Scientific Reports

Article Title: Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

doi: 10.1038/s41598-020-70290-w

Figure Lengend Snippet: NPC1 Western blot of the spleen of 3 vehicle treated NPC −/− mice and 3 THL treated NPC −/− mice. The spleens of the THL treated mice express the 200 kDa immunoreactive NPC1 protein.

Article Snippet: A human NPC1 expression plasmid (#SC120010), under the influence of the CMV promoter, is designated pCMV-NPC1, and was purchased from Origene (Rockville, MD).

Techniques: Western Blot

Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: (A) Representative confocal microscopy images of 14 and 24 DIV rat primary cortical neurons treated with 1 µM BODIPY-cholesterol (green) for 24 h and stained with antibodies against an early endosomal marker (EEA1, red), which stains endosomal compartments where internalized material ends up first, or a late endosomal/lysosomal marker (LIMP-I, red), which stains a mature state of endosomal compartments. 24 DIV neurons accumulate BODIPY-cholesterol in early and late-compartments (white arrows). (B) The plot compares the relative amount (% versus 12–14 DIV) of BODIPY-cholesterol taken by 12–14 DIV and 23–28 DIV primary cortical neurons in a 24 h period, calculated by the intracellular fluorescent signal (Ext 495 nm/Em 507) of BODIPY-cholesterol (nmol of cholesterol where from a BODIPY-cholesterol standard curve) and normalized by the total protein in the sample (% versus 12–14 DIV; n = 9 independent cultures). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t test (** P < 0.01; P -value: 0.0046). (A, C) Plot showing the percentage of the total EEA1-positive (n = 1,536) or LIMP-I positive (n = 1,269) compartments that accumulate BODIPY-cholesterol, calculated from confocal images as the ones shown in panel (A) (N = 3 independent experiments). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed Mann–Whiitney Test (**** P < 0.0001). (D) Western blot analysis of total lysates of 12–14 DIV, 18–21 DIV, and 23–28 DIV rat cortical neurons shows a pronounced decrease in NPC1 protein levels in 18–21 DIV neurons that is maintained in 23–28 DIV neurons. An antibody against beta-actin was used to normalize the protein levels. Below, a plot with the quantification of the bands of Western blot experiments as the one shown. Protein levels are relative to 12–14 DIV. The graph shows the mean ± SEM. Statistical significance was analyzed by one-way ANOVA ( P -value: 0.0001). Comparison of the age-groups 18–21 DIV and 23–28 DIV to 12–14 DIV was performed by Dunnet’s multiple comparisons tests (n = 6 for 12–14 DIV and 18–21 DIV; n = 5 for 23–28 DIV, *** P < 0.001; P -value for 18–21 DIV to 12–14 DIV comparison: 0.0002; P -value for 23–28 DIV to 12–14 DIV comparison: 0.0004). (E) Western blot analysis of lysates of neuronal cultures at different aging points. The blot was tested with an antibody against the cholesterol transporter ABCA1 and actin. Below, a plot compares the relative ABCA1 protein levels between 12–14 DIV, 18–21 DIV and 23–28 DIV neuronal cultures. The bands corresponding to ABCA1 were quantified from blots such as the one shown above. Statistical significance was analyzed by Kruskal–Wallis Test ( P -value 0.0022). Comparison of the age-groups 18–21 DIV and 23–28 DIV to 12–14 DIV was performed by Dunn’s Test (n = 9 independent lysates per group; ** P < 0.01; P -value for 23–28 DIV to 12–14 DIV comparison: 0.0036).

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques: Confocal Microscopy, Staining, Marker, Two Tailed Test, Western Blot

Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: (A) Representative transmission electron microscopy (TEM) images of multivesicular bodies (MVBs) from untreated (control) or 4 µg/μl U18666A-treated 14 DIV cortical neurons. Scale bars = 200 nm. (A, B) Plot showing the number of ILVs normalized by the area (nm 2 ) of the MVB that contains them, between untreated (control) or 4 µg/μl U18666A (U18)-treated 14 DIV neuronal cultures, quantified from TEM images such as the ones shown in panel (A) (n = 121 MVBs; N = 2 experiments). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed Mann-Whitney Test (**** P < 0.0001). (C) The graph shows the quantification of extracellular vesicles by Nanosight from the medium of untreated 14 DIV neurons (control) or treated with increasing concentrations of U18666A. The graph shows single experiments, the mean ± SEM. Statistical significance was analyzed by one-way ANOVA ( P -value: 0.009). Comparison of 4 µg/μl U18666A (U18)-treated neurons to control group was performed by Dunnet’s multiple comparisons test (** P < 0.001; n = 6 for control and 3 µg/μl; n = 5 for 2 and 4 µg/μl). (D) Western blot analysis of a total lysate of 14 DIV neuronal culture and total lysates of small extracellular vesicles isolated from the medium of 14 DIV untreated (Cnt) or U18-treated neurons. The blot was tested with an antibody against CD81, a marker for exosomes, and calnexin (an ER protein used as a negative marker for exosomes). (E) Western blot experiment of total lysates of 14 DIV neuronal cultures transduced with a lentivirus expressing a scrambled shRNA as a control (Cnt) or an shRNA against NPC1. Blots were probed with an antibody against NPC1 or actin as a loading control. (F) Representative TEM images of 14 DIV neuronal cultures transduced with a scrambled shRNA (shRNA-cnt) or an shRNA against NPC1 (shRNA-NPC1). (G) The plot compares the average number of ILVs normalized by the area (nm 2 ) of the MVB containing them, between shRNA-cnt and shRNA-NPC1 transduced neurons. (F) Data were obtained by quantification of TEM images as the ones shown in panel (F) (n = 100 MVBs for shRNA-cnt; n = 108 for shRNA-NPC1; N = 2 independent cultures). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed Mann–Whitney test (**** P < 0.0001).

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques: Transmission Assay, Electron Microscopy, Two Tailed Test, MANN-WHITNEY, Western Blot, Isolation, Marker, Transduction, Expressing, shRNA

Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: (A, C, D) Representative transmission electron microscopy images of multivesicular bodies (MVBs) from non-infected 21 DIV neurons (control; subpanels [a and c]) or infected (NPC overexpression, subpanels [c and d]) with a Sindbis virus expressing GFP-tagged NPC1 (GFP-NPC1). (B) Western blot analysis of total lysates of 21 DIV control neurons (cnt) or overexpressing GFP-NPC1 (NPC1). Blots were tested with an antibody against NPC1 and Actin. (C) The plot compares the number of ILVs normalized by the area of the MVBs that contained them (nm 2 ), between control and GFP-NPC1-overexpressing (NPC1) neurons. (A) Quantifications were performed on transmission electron microscopy images such as the ones shown in panel (A) (n = 181 MVBs for control; n = 161 MVBs for NPC1; N = 3 independent cultures). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed Mann–Whitney test (**** P < 0.0001).

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques: Transmission Assay, Electron Microscopy, Infection, Over Expression, Expressing, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: (A) Western blot analysis of total lysates of neuronal cultures at different aging points (10, 13, 15 and 17 DIV). The blot was tested with antibodies against NPC1, Akt phosphorylated at serine 473 (pAKT), total Akt, the mTOR substrate S6 Kinase (S6K) phosphorylated at threonine 389 (pS6K), total S6K, and actin. (A, B) The plot compares the levels of NPC1 normalized to actin, during neuronal aging in vitro. The protein levels were quantified from the bands of Western blot experiments as the ones shown in panel (A) (n = 4 independent cultures). The graph shows the mean ± SEM. The means for 10, 13, 15 and 17 DIV were significantly different when compared by ANOVA ( P -value: 0.0219). Dunnet’s multiple comparisons tests were used for the post hoc analysis of the data (** P < 0.01; P -value for the 10 DIV to 17 DIV comparison: 0.0049). (C) Alterations of the AKT-mTORC1 pathway during neuronal aging in vitro. (A) The plots show changes in the levels of pAKT (normalized to total AKT; upper plot) and pS6K (normalized to total S6K; lower plot) at the same aging points of neuronal cultures as in panel (A). (A) The protein levels were quantified from the bands of Western blot experiments as the ones shown in panel (A) (n = 3 independent cultures). The graph shows the mean ± SEM. The means for 10, 13, and 15 DIV (for pAKT, upper blot) and the means for 10, 15 and 17 DIV (for pS6K, lower blot) were significantly different when compared by ANOVA ( P -value for pAKT: 0.0308; P -value for pS6K: 0.0320). Dunnet’s multiple comparisons tests was used for the post hoc analysis of data (* P < 0.05; P -value for 15 DIV to 10 DIV comparison in pAKT: 0.0199; P -value for 15 DIV to 10 DIV comparison in pS6K: 0.0362; P -value for 17 DIV to 10 DIV comparison in pS6K: 0.0370). (D) Western blot analysis of the total lysates of N2A cells untreated or treated with 130 nM insulin growth factor for 24 h. The blot was tested with antibodies against NPC1, pAKT (ser473) and actin. Below, a plot comparing the NPC1 levels between untreated and Insulin Growth Factor-treated N2A cells. Quantifications were carried out on Western blot experiments as the one shown above (n = 4 independent cultures). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t test (* P < 0.05; P -value: 0.0231). (E) Western blot analysis of total lysates of 14 DIV neuronal cultures treated with DMSO (control) or 5 µM of the pan-Akt activator SC79 for 24 h. The blot was tested with antibodies against NPC1, pAKT (ser473), and actin. Below, a plot comparing the NPC1 levels between DMSO-treated and SC79-treated neurons. Quantifications were carried out on Western blot experiments as the one shown above (n = 5 independent cultures). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t test (* P < 0.05; P -value: 0.027). (C, F) N2A cells treated with DMSO (control) or Akt inhibitor VIII for 48 h were analyzed as in panel (C) (n = 3 independent cultures). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t test (** P < 0.01; P -value: 0.0049). (F, G) Lysates of 17 DIV cortical neurons treated with DMSO (control) or the selective Akt inhibitor MK-2206 (2 µM) for 48 h were analyzed by Western blot with the same antibodies as in panel (F). Below, a plot comparing the NPC1 levels between DMSO-treated and MK-2206-treated neurons (n = 3 independent cultures). Statistical significance was analyzed by two-tailed unpaired t test (** P < 0.01; P -value: 0.0052). (H) Lysates from 17 DIV cortical neurons treated with DMSO (control) or the mTOR inhibitor rapamycin (2.5 µM) were analyzed by Western blot. The blot was tested with antibodies against NPC1, pS6K (thr389), and actin. Below, the plot compares the NPC1 levels between DMSO-treated and 2.5 µM rapamycin-treated neurons. Quantification of the bands were carried out on blots such as the ones shown above (n = 3 independent cultures). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t test (** P < 0.01; P -value: 0.0053). (I) Comparison of the concentration (upper plot) and size (lower plot) of vesicles in the medium of 48 h DMSO (Cnt)- or 2.5 µl/ml rapamycin-treated 18 DIV neuronal cultures determined by Nanosight (n = 5 independent cultures). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t test (* P < 0.05, ns: non-significant; P -value for concentration: 0.0134; P -value for size: 0.5072).

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques: Western Blot, In Vitro, Two Tailed Test, Concentration Assay

Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: (A) 14 DIV cortical neurons were treated with the NPC1 inhibitor U18666A (2 μg/ml) for 24 h or left untreated. Western blot analysis of total lysates show decrease mTOR-dependent phosphorylation of its substrate S6K. Actin was used to normalize for total protein. (B) The plot compares the levels of S6K phosphorylation between untreated (Control) or U18666A-treated (U18) neurons. (A) Quantification of pS6K levels from Western blot experiments as the one shown in panel (A) (n = 3). The graph shows the mean ± SEM and individual data points. Statistical significance was analyzed by two-tailed unpaired t test (* P < 0.05).

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques: Western Blot, Two Tailed Test

Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: (A) The plot compares the miR33 levels (normalized by the housekeeping RNA U6) between 13 DIV, 16–17 DIV and 21 DIV neuronal cultures (fold change versus DIV). The microRNA levels were determined by RT–PCR (n = 5 independent culture). The graph shows the mean ± SEM. Statistical significance was analyzed by one-way ANOVA ( P -value: 0.0410). Dunnet’s multiple comparisons test was used to compare the 21 DIV and the 16–17 DIV to the 13 DIV group (ns: non-significant; * P < 0.05; P -value for the 21 DIV to 13 DIV comparison: 0.0258). (B) Western blot analysis of lysates of N2A cells transfected with 40 nM of a non-targeting control mimic or 40 nM miR-33 mimic (miRNA-33). The blots were analyzed with antibodies against NPC1, ABCA1 and actin. (C) Plot comparing the relative protein levels of NPC1 between N2A cells transfected with 40 nM of a non-targeting control mimic (n = 4) or 40 nM miR33 mimic (miR-33; n = 4). (B) Blots were quantified from Western blot experiments as the one shown in panel (B). The graph shows the mean ± SEM and individual data points. Statistical significance was analyzed by two-tailed unpaired t test (** P < 0.05). (C, D) The plot compares the ABCA1 protein levels in the same experimental conditions as in panel (C). The graph shows the mean ± SEM and individual data points (n = 3). Statistical significance was analyzed by two-tailed unpaired t test (** P < 0.05). (E) Western blot analysis of brain lysates from 2 mo old WT or miR33 KO mice. Blots were tested with antibodies against NPC1 and HSP90. (F) Plots comparing the relative protein levels for NPC1 in the brain of WT (n = 5) and miR33 KO (n = 7) mice. (C) Protein levels were quantified from Western blot experiments as the one shown in panel (C). The graph shows the mean ± SEM and individual data points. Statistical significance was analyzed by two-tailed unpaired t test (* P < 0.05).

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Two Tailed Test

Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: (A) Western blot analysis of brain lysates from 2 mo old WT or miR33 KO mice (same animals as in ). Blots were tested with antibodies against ABCA1 and HSP90 (same HSP90 blot as in ). (B) Plots comparing the relative protein levels for NPC1 in the brain of WT (n = 3) and miR33 KO (n = 3) mice. (A) Protein levels were quantified from Western blot experiments as the one shown in panel (A). The graph shows the mean ± SEM and individual data points. Statistical significance was analyzed by two-tailed unpaired t test (*ns, non-significant). Although the statistical analysis did not reach significance, two of the WT mice show lower levels of ABCA1 in than the KO mice.

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques: Western Blot, Two Tailed Test

Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: (1) The activation of the AKT-mTOR pathway during neuronal aging triggers the degradation of the intracellular cholesterol transporter Niemann Pick disease, type C protein (NPC1), what induces the accumulation of cholesterol in endosomal compartments. (2) This in turn induces multivesicular bodies (MVBs) to generate a higher number of intraluminal vesicles (ILVs, called exosomes when they are secreted) in their lumen. (3) Decreased levels of NPC1 provoke a low endosome-to-ER cholesterol transport, what triggers the activation of the Sterol regulatory element-binding proteins through the Golgi and its translocation to the nucleus. There, sterol regulatory element-binding protein activates the transcription of the microRNA miR-33 located in the intronic region of the Srebf2 gene, after binding its sterol response element (SRE). (4) miR-33 blocks the synthesis of NPC1 by targeting its mRNA, what aggravates the accumulation of endosomal cholesterol and the formation of ILVs.

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques: Activation Assay, Binding Assay, Translocation Assay

Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: Information on the antibodies used in this study.

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques:

Journal: Genome biology

Article Title: zMAP toolset: model-based analysis of large-scale proteomic data via a variance stabilizing z-transformation.

doi: 10.1186/s13059-024-03382-9

Figure Lengend Snippet: Fig. 5 Applying reverse-zMAP to an HCC TMT data set. a Performing PCA on all samples of the HCC data set, with the z-statistics (derived by reverse-zMAP) of hypervariable proteins as features. b Venn diagram showing the overlap among PC1-correlated proteins, prognosis-related ones, and hypervariable ones. c Plotting the log-hazard ratio of each protein against the PCC between its z-statistics and the PC1 scores of samples. Colors indicate the BH-adjusted p-values for identifying prognosis-related proteins. d Hierarchical clustering of all patients based on the PC1 scores of their tumor samples. The patients were accordingly classified into three groups. The p-value associated with each clinical feature was derived by applying ANOVA to comparing PC1 scores. e Kaplan–Meier curves for the three groups of HCC patients. The p-value assessed the survival difference across the groups and was derived by applying log-rank test. f Violin plots showing the z-statistics of NPC1 in all NAT samples and different groups of tumor samples. The p-values were derived by applying t-test. g Dividing all patients into two groups based on the median expression of NPC1 in their tumor tissues and assessing the survival difference between the groups. h Using western blot to measure the expression of NPC1 in HepG2 cell line under different conditions. i, j Using MTT assay, transwell migration assay, and plate clone formation experiment to quantitatively assess the influence of NPC1 knock-down on the cell proliferation, migration, and colony forming ability of HepG2 cell line, respectively. Three biological replicates were generated for each experiment. k Heat map showing the GSVA scores of biological pathways associated with differential activity across sample groups. The average GSVA score of each pathway in each sample group is also displayed. The differential pathways were identified by applying limma to comparing GSVA scores (BH-adjusted p-value < 0.05)

Article Snippet: The first antibodies were UBE2C (SantaCruz), NPC1 (SantaCruz), TSC2 (Abclonal), HSP90 (CST), Actin (Proteintech).

Techniques: Derivative Assay, Expressing, Western Blot, MTT Assay, Transwell Migration Assay, Knockdown, Migration, Generated, Activity Assay

Journal: Virology

Article Title: Chinese hamster ovary cell lines selected for resistance to ebolavirus glycoprotein mediated infection are defective for NPC1 expression.

doi: 10.1016/j.virol.2012.05.018

Figure Lengend Snippet: Fig. 6. Resistant cell lines have mutations in NPC1. (a) Schematic of NPC1 protein. Location of insertions in R1 (star) and R2 (circle), and deletion (triangle) and substitution (diamond) in L2 indicated in diagram at top and in sequence at bottom. (b) Expression of NPC in WT, M12, and resistant cells transduced with pLenti CMV NPC1 Hygro or pLenti CMV GFP Hygro. (c) Resistant cells stably expressing NPC1 or GFP were challenged with VSV EboGP mCherry and infection was quantitated by flow cytometry. Bars represent average of means from three separate experiments done in triplicate; errors bars represent standard error means.

Article Snippet: The rLuc gene was removed from VSV-XN2 rLuc by cleavage with MluI and NheI and replaced with the GP-KpnIjunction-XhoI-mCherry sequence. pLenti CMV NPC1 Hygro was constructed by removing the GFP gene from pLenti CMV GFP Hygro and inserting into its place the NPC1 cDNA (Origene). pSport6 Axl (clone ID 5205825) was from Open Biosystems. pcDNA3.1 furin and pcDNA3.1-ACE2 were previously described (Simmons et al., 2005; Wool-Lewis and Bates, 1999).

Techniques: Sequencing, Expressing, Transduction, Stable Transfection, Infection, Cytometry

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: Generation of NPC1 KO cell line using CRISPR/Cas9 system MNT-1 cells were co-transfected with CRISPR/Cas9 KOspecific plasmids from Santa Cruz Biotechnology (NPC1 CRISPR/Cas9 KO Plasmid (h) (sc-403252) and NPC1 HDR Plasmid (h) (sc-403252-HDR), according to manufacturer’s instruction.

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: Generation of NPC1 KO cell line using CRISPR/Cas9 system MNT-1 cells were co-transfected with CRISPR/Cas9 KOspecific plasmids from Santa Cruz Biotechnology (NPC1 CRISPR/Cas9 KO Plasmid (h) (sc-403252) and NPC1 HDR Plasmid (h) (sc-403252-HDR), according to manufacturer’s instruction.

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: Generation of NPC1 KO cell line using CRISPR/Cas9 system MNT-1 cells were co-transfected with CRISPR/Cas9 KOspecific plasmids from Santa Cruz Biotechnology (NPC1 CRISPR/Cas9 KO Plasmid (h) (sc-403252) and NPC1 HDR Plasmid (h) (sc-403252-HDR), according to manufacturer’s instruction.

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: Generation of NPC1 KO cell line using CRISPR/Cas9 system MNT-1 cells were co-transfected with CRISPR/Cas9 KOspecific plasmids from Santa Cruz Biotechnology (NPC1 CRISPR/Cas9 KO Plasmid (h) (sc-403252) and NPC1 HDR Plasmid (h) (sc-403252-HDR), according to manufacturer’s instruction.

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: Generation of NPC1 KO cell line using CRISPR/Cas9 system MNT-1 cells were co-transfected with CRISPR/Cas9 KOspecific plasmids from Santa Cruz Biotechnology (NPC1 CRISPR/Cas9 KO Plasmid (h) (sc-403252) and NPC1 HDR Plasmid (h) (sc-403252-HDR), according to manufacturer’s instruction.

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: Generation of NPC1 KO cell line using CRISPR/Cas9 system MNT-1 cells were co-transfected with CRISPR/Cas9 KOspecific plasmids from Santa Cruz Biotechnology (NPC1 CRISPR/Cas9 KO Plasmid (h) (sc-403252) and NPC1 HDR Plasmid (h) (sc-403252-HDR), according to manufacturer’s instruction.

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: Generation of NPC1 KO cell line using CRISPR/Cas9 system MNT-1 cells were co-transfected with CRISPR/Cas9 KOspecific plasmids from Santa Cruz Biotechnology (NPC1 CRISPR/Cas9 KO Plasmid (h) (sc-403252) and NPC1 HDR Plasmid (h) (sc-403252-HDR), according to manufacturer’s instruction.

Techniques: Clinical Proteomics, Membrane, Mutagenesis