(A–E) HeLa cells transfected with NgBR-HA were fixed and stained with antibodies against HA, NPC2, Cathepsin D, and PDI using Bouin's fixative for detection of endogenous NPC2 (scale bar = 10µm).

(A) CHO cell lines (see Fig. 1D) were transfected with NPC2, incubated for 24 hours followed by treatment with 5ug/ml tunicamycin (TN) for 6 hours. (B) WT NPC2-myc or NPC2^N58Qmyc was transfected into CHO cell lines and levels of NPC2-myc assessed by Western blotting. (C) After 48hrs of treatment with Ctrl RNAi or RNAi directed against NgBR, EA.hy cells were treated for the indicated times with 1% (final concentration, vol/vol) concentrated conditioned medium from CHO cells transfected with NPC2-myc. A subset of Ctrl RNAi-treated cells were pre-treated with U18666A (1µM, 8hrs) prior to addition of exogenous NPC2. (D) CHO cells stably expressing a Vector control (Vector) or full-length NgBR (NgBR) were transfected with myc-tagged NPC2. 24hrs post-transfection, cells were treated with the indicated amounts of brefeldin A for 8hrs, lysed, and immunoblotting was performed for detection of NPC2 levels.

(A) SREBP-2 cleavage assay. EA.hy cells were treated with Ctrl RNAi or NgBR RNAi for 12hrs followed by addition of FBS or lipoprotein-deficient serum (LPDS) for a further 48hrs. Cells either remained in LPDS or were treated with 20µg/ml nLDL for 10hrs prior to lysis. (B) Cholesterol biosynthesis in HepG2 cells. HepG2 were treated with Ctrl RNAi or NgBR RNAi for 48 hours. Cholesterol biosynthesis was examined as described in Experimental Procedures. (C–D) EA.hy926 cells were treated with Ctrl RNAi or NgBR RNAi and LDL uptake and surface LDL binding measurements were performed as described in Experimental Procedures. (E) EA.hy926 cells treated with Ctrl RNAi or NgBR RNAi were subjected to analysis of LDL-R expression as described in Experimental Procedures. (F) HepG2 cell line stably expressing full-length NgBR. Ctrl RNAi or NgBR RNAi was incubated with HepG2 cells stably expressing either empty vector (V) or an RNAi-resistant NgBR construct (NGBR). (G) Surface LDL-R expression was analyzed in HepG2 stable cell lines after treatment with NgBR RNAi as described in Experimental Procedures. (H) EA.hy926 cells were treated with RNAi for 24 hours, followed by incubation with lipoprotein-deficient serum supplemented with nLDL in the absence or presence of 2.5µM 25-hydroxycholesterol (25-HC) for 24 hours. Surface LDL-R expression (H) was analyzed by flow cytometry as described in Experimental Procedures. (*p < 0.05, **p < 0.01).

(A) NgBR interacts with NPC2 in mammalian cells. CHO cell lines stably expressing either vector alone (Vector), HA-tagged, full-length NgBR (NgBR), or an HA-tagged, truncated form of NgBR lacking the C-terminal 158 amino acids (NgBRΔC) were transfected with myc-tagged NPC2. HA-tagged proteins were immunoprecipitated from lysates and subjected to Western blot analysis using anti-myc and anti-HA antibodies. (B) The same CHO cell lines in (A) were transfected with increasing amounts of plasmids encoding NPC2-myc or β-gal (0.5ug–2ug). (C) Conditioned media from CHO-Vector and CHO-NgBR transfected with NPC2-myc was collected and NPC2-myc was immunoprecipitated from the media using anti-myc antibodies. (D) Pulse-Chase analysis of NPC2. CHO cell lines were transfected with NPC2-myc and subjected to ³⁵S-Met/Cys incorporation for half-life determination of NPC2 in the absence and presence of NgBR expression. Error bars represent the ± S.E. of pooled data from 3 independent experiments. (E) NgBR knockdown and NPC2 levels. HepG2 and EA.hy926 cells were incubated with RNAi directed against NgBR for 48 hours. Cells were lysed and lysates were blotted for endogenous NgBR, NPC2, and Cathepsin D. (F) Analysis of NPC2 degradation. NgBR knockdown was performed as in (E) in the absence or presence of proteasome inhibitor (MG132, 5µM, 24hrs; or lactacystin, 5µM, 24hrs). After 48hrs of RNAi treatment, cells were lysed and lysates blotted for endogenous NgBR, NPC2, and Cathepsin D.

(A,B) ER membrane fractionation with iodixanol was performed as described by Radhakrishnan et al (see Supplementary Experimental Procedures). ER vs. lysosomal localization was assessed by fractionation of HEK-293T cells over an iodixanol gradient. Cells in (A) contain endogenous levels of NPC2 and NgBR; cells in (B) contain endogenous NPC2 but overexpressed NgBR. Fraction (1) is defined as a mixture of lysosomes, peroxisomes, and ER, and Fraction (2) is defined as purified ER (Radhakrishnan et al., 2008). (C–F) COS cells transfected with control plasmid (C), WT NgBR-HA (D), NgBRΔC-HA (E), or DHHC3-HA (F) were fixed with Bouin’s fixative and stained with anti-NPC2, anti-PDI, and anti-HA antibodies followed by secondary antibodies as described in Experimental Procedures (scale bar = 10µm).

(A) EA.hy926 cells or (B) NgBR^+/− fibroblasts (MEFs) were incubated with non-silencing RNA (Ctrl RNAi) or small interfering RNA directed against NgBR (NgBR RNAi) for 48 hours. U18666A (1µM, 8hours) was used as a positive control for inhibition of cholesterol trafficking. Cells were fixed and stained for free cholesterol with filipin as described in Experimental Procedures (scale bar = 20µm). (C) EA.hy926 cells were infected with an adenoviral construct expressing GFP or with adenovirus encoding NgBR (Ad-NgBR) for 24 hours, followed by incubation with Ctrl RNAi or NgBR RNAi for 48 hours. Cells were then fixed and stained for free cholesterol as in (A–B). (D) Conditioned medium from CHO cells transfected with NPC2-myc was concentrated and added to EA.hy926 cells incubated with Ctrl RNAi or NgBR RNAi for 24 hours. (E) EA.hy926 cells treated with Ctrl RNAi or NgBR RNAi were incubated with 2.5µM 25-hydroxycholesterol (25-HC) for 18 hours followed by fixation and staining with filipin. (*p < 0.05).