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: 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: 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: Nature Communications

Article Title: Tetrandrine regulates NAADP-mediated calcium signaling through a LIMP-2-dependent and sphingosine-mediated mechanism

doi: 10.1038/s41467-025-61565-9

Figure Lengend Snippet: A Quantification of Ca 2+ release in HEK293 cells by the indicated treatment. Cells were pre-loaded with Calbryte 520, followed by captured of microscope images every 3s for 5mins. Thirty seconds after starting of imaging, cells were treated with NAADP (1 μM), liposomes (10μl), NAADP-liposomes (10μl), and TPC2-A1-N (10 μM), respectively. ***, P < 0.001 compared to untreated cells by one-way ANOVA with Tukey post hoc test. B Quantification of NAADP-induced Ca 2+ release in the presence of the putative TPCs inhibitors. HEK293 cells were treated with DMSO, Tet (5 μM), AD-Tet (5 μM), SG-005 (5 μM), or SG-094 (5 μM), respectively, for 30minutes before the addition of NAADP-liposomes. ***, P < 0.001 compared to DMSO-treated cells by one-way ANOVA with Tukey post hoc test. C Quantification of NAADP-induced Ca 2+ release in cells transfected with the indicated siRNAs and in LIMP-2 knockout (LIMP-2-KO) HEK293 cells. HEK293 cells were transfected with the indicated siRNA for 72 h before the experiment. ***, P < 0.001, by one-way ANOVA with Tukey post hoc test. D Relative filipin intensity in LAMP1-positive vesicles of HeLa cells treated with Tet or transfected with siLIMP-2, with or without MβCD (1mM) treatment for 12 h. Cells were then immune-stained with LAMP1 antibody, followed by filipin staining. In each condition, fluorescence intensity of 20 randomly chosen cells were measured. ** p = (DMSO vs. Tet, 5 μM), ** p = 0.008 (Tet, 5 μM vs. Tet, 5 μM+MβCD), *** p < 0.001, by one-way ANOVA with Tukey post hoc test. E , F Quantification of NAADP-induced Ca 2+ release in HEK293 cells treated with Tet or transfected with siLIMP-2, with or without addition of MβCD (1mM) ( E ) or ectopic expression of NPC1 ( F ). Tet (5 μM) was added to the cells for 30min before Ca 2+ measurement. MβCD was added at to the cells 6 h before measurement. ***, p < 0.001 by one-way ANOVA with Tukey post hoc test. Ca 2+ level was presented as change in maximum fluorescence intensity [(F max -F 0 )/F 0 ]. In each condition, the fluorescence intensity of 40 randomly chosen cells were measured. Source data are provided in the Source Data file.

Article Snippet: LIMP-2 (ab176317), Anti-Mitochondria antibodies [113-1] (ab92824) were obtained from Abcam and were used at 1:1000 (for western blotting) and 1:500 (for immunofluorescence). β-glucosidase (sc-365745), NPC1 (sc-271334) antibodies were obtained from Santa Cruz Biotechnology and were used at 1:500 (for western blotting).

Techniques: Microscopy, Imaging, Liposomes, Transfection, Knock-Out, Staining, Fluorescence, Expressing