Journal: The Journal of Cell Biology

Article Title: A role for oxysterol-binding protein–related protein 5 in endosomal cholesterol trafficking

doi: 10.1083/jcb.201004142

Figure Lengend Snippet: Depletion of ORP5 impairs the transport of CI-MPR and TGN46, but not STxB. (A) HeLa cells were transfected with the indicated siRNAs for 72 h, followed by double-labeling with antibodies to CI-MPR and giantin. Confocal images are shown to indicate a more dispersed distribution of CI-MPR in cells treated with siORP5 rather than with siNPC1. Individual fluorescent profiles in merged images were quantified using the Olympus FluoView version 2.0 Viewer software and are shown on the right. (B) Quantification of cells with dispersed CI-MPR in A. Values are the percentage of cells with dispersed localization of CI-MPR (means + SD [error bars], n > 100; *, P < 0.05; **, P < 0.01). (C) HeLa cells were transfected with the indicated siRNA for 72 h, incubated on ice for 10 min, and chased with mouse monoclonal antibodies to CI-MPR for 30 min on ice. Antibody internalization was performed at 37°C for 60 min. Cells were then fixed by 4% paraformaldehyde, and internalized CI-MPR was detected using Alexa Fluor 488–conjugated goat anti–mouse antibodies. (D) HeLa cells were transfected with the indicated siRNAs for 72 h, followed by labeling with antibodies to TGN46. Confocal images are shown to indicate a dispersed distribution of TGN46 in cells treated with siORP5. (E) Quantification of cells with dispersed TGN46 in D. Values are the percentage of cells with dispersed localization of TGN46 (means + SD [error bars], n > 100; **, P < 0.01). Data are representative of three independent experiments with similar results. (F) HeLa cells, transfected with the indicated siRNA for 72 h in medium A, were incubated with Cy3-conjugated STxB in DME, 1% BSA, and 25 mM Hepes, pH 7.4, for 15 min at 37°C. Cells were washed in PBS and incubated for 1 h at 37°C in medium A before fixation by 4% paraformaldehyde, followed by labeling with antibodies to giantin. Bars, 10 µm.

Article Snippet: Antibodies used were goat polyclonal to ORP5 (Abcam) and β-1,4-galactosyltransferase I (β-1,4-GT1; Santa Cruz Biotechnology, Inc.), rabbit polyclonal to the C-terminal region of human NPC1 and golgin 97 (Abcam), GFP (Invitrogen), syntaxin 5 , calnexin (Cell Signaling Technology), DsRed (Takara Bio, Inc.), giantin (Covance), and TGN46 (AbD Serotec), mouse monoclonal to the Xpress tag (Invitrogen), actin (Abcam), GST, Lamp-1 and GFP (Santa Cruz Biotechnology, Inc.), and GM130 (BD).

Techniques: Transfection, Labeling, Software, Incubation, Bioprocessing

Journal: The Journal of Cell Biology

Article Title: A role for oxysterol-binding protein–related protein 5 in endosomal cholesterol trafficking

doi: 10.1083/jcb.201004142

Figure Lengend Snippet: Rescue effects of ORP5 expression. (A) HeLa cells grown in medium A were transfected with siORP5 for 48 h, followed by transfection with EGFP alone or RNAi-resistant GFP-OPR5 (GFP-ORP5-siR) for 6 h. Cells then received medium D supplemented with 50 µg/ml LDL for 18 h followed by processing for filipin staining and fluorescence microscopy. Images are shown to indicate that cholesterol accumulation was significantly alleviated in GFP-ORP5-siR–expressing cells (71 ± 2%) but not in GFP-expressing cells (5 ± 3%). (B) HeLa cells grown in medium A were transfected with siORP5 for 48 h, followed by transfection with RNAi-resistant GFP-OPR5 for 24 h. Cells were fixed and labeled with antibodies to TGN46. Confocal images are shown to indicate that the dispersed distribution of TGN46 was rescued in cells expressing GFP-ORP5. (C) HeLa cells grown in medium A were transfected with siORP5 for 48 h, followed by transfection with RNAi-resistant mCherry-OPR5 for 24 h. Cells were fixed and labeled with antibodies to CI-MPR. Confocal images are shown to indicate that the dispersed distribution of CI-MPR was rescued in cells expressing mCherry-ORP5. Bars, 10 µm.

Article Snippet: Antibodies used were goat polyclonal to ORP5 (Abcam) and β-1,4-galactosyltransferase I (β-1,4-GT1; Santa Cruz Biotechnology, Inc.), rabbit polyclonal to the C-terminal region of human NPC1 and golgin 97 (Abcam), GFP (Invitrogen), syntaxin 5 , calnexin (Cell Signaling Technology), DsRed (Takara Bio, Inc.), giantin (Covance), and TGN46 (AbD Serotec), mouse monoclonal to the Xpress tag (Invitrogen), actin (Abcam), GST, Lamp-1 and GFP (Santa Cruz Biotechnology, Inc.), and GM130 (BD).

Techniques: Expressing, Transfection, Staining, Fluorescence, Microscopy, Labeling

Journal: Traffic (Copenhagen, Denmark)

Article Title: Spatiotemporal Resolution of Rab9 and CI-MPR Dynamics in the Endocytic Pathway.

doi: 10.1111/tra.12357

Figure Lengend Snippet: Figure 2: Rab9Q66L induces dispersion of TGN46 to early endosomes. HeLa cells transfected with (A) GFP-Rab9 or (B) GFP-Rab9Q66L were stained for TGN46 and p230 as indicated. Cells with a predominantly dispersed, non-perinuclear localization of TGN46 were classified as dispersed; all others were classified as non-dispersed. Results are presented (C) as percentage of cells counted, n = 60 cells from three independent experiments. D) Pixel based colocalization using FLUOVIEW 1000, is shown as a percentage of total TGN46, based on three independent experiments using 20 cells per experiment. **p < 0.01, ***p < 0.001, Welch t-test. Scale bar 10 μm.

Article Snippet: For double labeling, sections were first labeled with sheep anti-human TGN46 AHP 500 (Serotec) at 1:30, rabbit anti-sheep IgG H&L at 1:500 and protein A gold 10 nm (Cell Microscopy Center, UMC) at 1:50, followed by rabbit anti-GFP (Thermo Fisher) at 1:25 and protein A gold 5 nm (Cell Microscopy Center, UMC) at 1:50.

Techniques: Dispersion, Transfection, Staining

Journal: Traffic (Copenhagen, Denmark)

Article Title: Spatiotemporal Resolution of Rab9 and CI-MPR Dynamics in the Endocytic Pathway.

doi: 10.1111/tra.12357

Figure Lengend Snippet: Figure 6: Rab9Q66L causes dispersion of CI-MPR. A) HeLa cells were treated with control RNA or siRNA against Rab9 and stained for endogenous CI-MPR (Alexa-555) and TGN46 (Alexa-488), or they were trans- fected with GFP-Rab9Q66L and stained for endogenous CI-MPR (Alexa-555). B) Cells transfected with control RNA (scr) or siRNA against Rab9 were lysed, and ana- lyzed using western blotting with antibodies against Rab9 to confirm the silencing, and against tubulin to verify equal loading. C and D) Confocal images were analyzed using IMAGEJ, where 60 cells were quantified per condition, from three independent experiments. Circles centered on the pixel(s) with the highest intensity were drawn, and the intensity of CI-MPR inside the rings was quantified relative to the total intensity in the whole cell. Pixel distances of the concentric rings are shown. **p < 0.01 Welch’s t-test. Scale bar 15 μm.

Article Snippet: For double labeling, sections were first labeled with sheep anti-human TGN46 AHP 500 (Serotec) at 1:30, rabbit anti-sheep IgG H&L at 1:500 and protein A gold 10 nm (Cell Microscopy Center, UMC) at 1:50, followed by rabbit anti-GFP (Thermo Fisher) at 1:25 and protein A gold 5 nm (Cell Microscopy Center, UMC) at 1:50.

Techniques: Dispersion, Control, Staining, Transfection, Western Blot

Journal: Molecular Therapy

Article Title: ATP7A Gene Addition to the Choroid Plexus Results in Long-term Rescue of the Lethal Copper Transport Defect in a Menkes Disease Mouse Model

doi: 10.1038/mt.2011.143

Figure Lengend Snippet: Characterization of C57BL/6-Atp7amo-br mouse model of Menkes disease. (a) Mutant (gray coat color) and wild-type (black coat color) 12-day pups with a heterozygote female mother (mottled coat color). (b) Conservation among eukaryotic species of amino acid residues 799 (alanine) and 800 (leucine), which are deleted in Atp7amo-br. (c) On copper-deficient solid media, yeast transformation indicated partial complementation of the S. cerevisiae copper transport mutant, ccc2δ, by Atp7amo-br. Plating pattern (clockwise from 12 O'clock) of the S. cerevisiae copper transport mutant ccc2δ transformed with: wild-type human ATP7A (sections 1 and 2); an empty vector (sections 3 and 4); rsATP7A used as the adeno-associated virus serotype 5 (AAV5) transgene (sections 5 and 6); Atp7amo-br (sections 7 and 8); and wild-type mouse Atp7a (sections 9 and 10). The Atp7amo-br allele showed faint growth consistent with partial complementation. The rsATP7A allele restored growth nearly as well as either the wild type mouse or the wild type human copper ATPase. (d) Yeast complementation growth assay. Growth of various ATP7A alleles expressed in ccc2δ, and cultured in copper-deficient media. The wild-type ATP7A allele complemented ccc2δ efficiently, as did the rsAP7A used as the AAV5 transgene in brain-directed treatments of mo-br mice. The rsAP7A showed copper transport capacity ~80% compared to wild type in this growth assay. In contrast, the Atp7amo-br allele, which lacks two highly conserved amino acids, showed <15% copper transport function compared to wild type. Transformation with an ATP7A allele harboring deletion of exons 20–23 (del ex20-23), included as a negative control, did not complement ccc2δ. Mock-transformed ccc2δ also failed to grow, as expected. (e) Intracellular localization of Venus-tagged Atp7a (upper row) and Atp7amo-br (lower row) following transfection of human embryonic kidney-293 (HEK-293T) cells. Both alleles show trans-Golgi localization as noted by overlap of the ATP7A signal with TGN46, a trans-Golgi marker (merge + DAPI column). DAPI, 4',6 diamidino-2-phenylindole, dihydrochloride nuclear counterstain. (f) Kaplan–Meier survival curve indicating failure of intraperitoneal (i.p.) copper chloride (dose = 10 µg/g body weight on postnatal day 7) to rescue mo-br males.

Article Snippet: DAPI nuclear counterstain (Electron Microscopy Sciences, Fort Washington, PA) and an anti-TGN46 antibody (Novus Biologicals, Littleton, CO) were used according to the manufacturers' instructions.

Techniques: Mutagenesis, Transformation Assay, Plasmid Preparation, Virus, Growth Assay, Cell Culture, Negative Control, Transfection, Marker