1. Oxysterol-Binding Protein-Related Protein 1L Regulates Cholesterol Egress from the Endo-Lysosomal System 
Kexin Zhao, Neale D. Ridgway 
Cell Reports 
Volume 19, Issue 9, Pages (May 2017)
DOI: /j.celrep
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2. Cell Reports 2017 19, 1807-1818DOI: (10.1016/j.celrep.2017.05.028)
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3. Figure 1
CRISPR Knockout of ORP1L in HeLa Cells Reduces Cholesterol Esterification in the ER
(A) Domain organization of ORP1L and ORP1S and location of the guide RNA (gRNA)-targeting site in OSBPL1 (Ank, ankyrin motif).
(B) RT-PCR analysis of ORP1L mRNA expression.
(C) Immunoblot analysis of ORP1L and ORP1S expression in HeLa and ORP1L-null cells.
(D and E) [3H]Oleate incorporation into cholesterol ester (D) and TAGs (E) in HeLa, ORP1L-null, and ORP1L-null cells transiently expressing an ORP1L cDNA (ORP1L rescue) and cultured in FBS or LPDS for 16 hr (∗p < ).
(F) Lysates of cells from (D) were immunoblotted for ORP1.
(G) Cholesterol esterification in cells cultured in FBS and treated with no addition (NA, solvent) or 25-hydroxycholesterol (25OH, 6 μM) for 2 hr (∗p < 0.05 and ∗∗p < 0.001).
(H) Lysates from cells cultured in FBS (+) or LPDS (−), as described in (A), were probed with an antibody against human ACAT1.
(I) HeLa and ORP1L-null cells were cultured as described in (A) and received no addition (NA, solvent) or brefeldin A (BFA, 5 μg/mL) for 1 hr prior to measuring cholesterol esterification (∗p < 0.01 and ∗∗p < ). Results of experiments in (D), (E), (G), and (I) are the mean and SD of three to four experiments.
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4. Figure 2 Cholesterol Synthesis Is Elevated in ORP1L-Null Cells
(A) The qPCR analysis of SREBP1, HMGCR, and LDLR expression in cells cultured as described in Figure 1A. Results are expressed relative to HeLa cells cultured in FBS and are the mean and SD of four experiments (∗p < 0.05).
(B–D) [3H]Acetate incorporation into cholesterol (B), lanosterol (C), and fatty acids (D) was measured in HeLa and ORP1L-null cells cultured in FBS or LPDS. Results are the mean and SD of three experiments (∗p < 0.005).
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5. Figure 3 ORP1L Deficiency Causes Cholesterol Sequestration in LELs
(A) Filipin localization of cholesterol in HeLa, ORP1L-null, and NPC1 null cells (scale bar, 20 μm) cultured in medium containing LPDS and LDL (50 μg/mL).
(B) Filipin fluorescence intensity was quantified in cells cultured as described in (A). Results are expressed as box and whisker plots (boxes indicate the interquartile range with bars at the median and whiskers at the fifth and 95th percentiles) for three experiments (100–150 cells total, ∗p < ).
(C) Filipin-positive puncta were counted using the analyze particle command in ImageJ. Results are from three experiments (110–140 cells total, ∗p < ).
(D) HeLa and ORP1L-null cells transiently expressing mCherry-D4H (Ch-D4H) and GFP-PLCD1-PH (GFP-PLCD1) were cultured in medium containing FBS (scale bar, 20 μm).
(E and F) Total cholesterol (E) and unesterified cholesterol (F) were measured in cells cultured in medium containing LPDS, FBS, or LPDS plus LDL (50 μg/mL) for 16 hr. Results are the mean and SD of three to six experiments (∗p < 0.05 and ∗∗p < 0.001).
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6. Figure 4 LEL Distribution in ORP1L-Null Cells
(A and B) LELs in cells cultured in FBS were visualized by immunostaining for LAMP1 (A) in fixed cells or with LysoTracker Green (B) for 30 min at 37°C in live cells (scale bar, 20 μm). The perinuclear index (I<5-I>10) of fluorescence intensity for the two markers was quantified in three experiments (35–50 cells, ∗p < 0.001).
(C) Immunostaining and line plots for LAMP1 and TGN46 in cells cultured in FBS. Nuclei were visualized with DAPI (scale bar, 20 μm).
(D) Immunostaining and line plots of NPC1 and LAMP1 in HeLa and ORP1L-null cells (scale bar, 30 μm).
(E) Immunoblot analysis of LEL-associated proteins in HeLa and ORP1L-null cells (results are representative of four separate experiments).
(F) Lysates prepared from HeLa or ORP1L-null cells transfected with RILP-FLAG or vector control were immunoprecipitated with a control mouse antibody (mIgG) or FLAG antibody, and they were immunoblotted for the indicated proteins. Input represents 10% of the immunoprecipitated lysate. Results were repeated two other times with similar results.
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7. Figure 5
Cholesterol Regulatory Phenotypes of NPC1 Null and ORP1L-Null Cells
(A) Whole-cell lysates of HeLa, NPC1 null, and ORP1L-null cells were immunoblotted for NPC1, ORP1, and actin.
(B) Quantification of ORP1L and ORP1S protein expression in NPC1 null cells from (A).
(C) ACAT activity in HeLa, ORP1L-null, and NPC1 null cells cultured in fetal calf serum (FCS) for 16 hr.
(D) ACAT activity was measured in mock-transfected HeLa cells and NPC1 null cells transiently transfected with vector (mCherry), mCherry-ORP1L, or ORP1S-mCherry for 24 hr and cultured in FBS.
(E) NPC1 null cells cultured in FBS were transiently transfected as described in (D), incubated with filipin, and imaged by spinning-disc confocal microscopy (scale bar, 20 μm). Filipin staining in a HeLa cells is shown for comparison. Results in (B)–(D) are the mean and SD of three to four experiments each (∗p < 0.005, ∗∗p < 0.001, and ∗∗∗p < compared to control HeLa cells).
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8. Figure 6 ORP1L Is a Sterol- and PI-4P-Binding Protein
(A) SDS-PAGE of recombinant ORP1L (0.5 μg) purified from Sf21 cells.
(B) Specific binding of [3H]25-hydroxycholesterol (25OH) by ORP1L (40 pmol/assay).
(C) Specific binding of [3H]cholesterol by ORP1L (40 pmol/assay).
(D) Extraction of [3H]cholesterol from liposomes by increasing concentrations of ORP1L.
(E) Extraction of [3H]cholesterol by ORP1L (80 pmol) from liposomes containing 0, 2, or 4 mol% PI-4P and PI-4,5P2.
(F) Extraction of [3H]cholesterol, [32P]PI-4P, or [32P]PI-4,5P2 from liposomes by ORP1L (100 pmol).
(G) Binding of ORP1L to phosphatidylinositol (PI), phosphatidylinositol-3-phosphate (PI-3P), PI-4P, phosphatidylinositol-5-phosphate (PI-5P), PI-4,5P2, phosphatidylinositol-3,4-bisphosphate (PI-3,4P2), phosphatidylinositol-3,5-bisphosphate (PI-3,5P2), and phosphatidylinositol-3,4,5-trisphosphate (PIP3) (100–300 pmol) immobilized on a Hybond-C membrane (representative experiment). Results in (B)–(F) are the mean and SD of three to four separate experiments.
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9. Figure 7
Sterol- and PI-4P-Binding Activities Are Required for ORP1L Function
(A) ORP1L-null cells were transiently transfected with empty vector or cDNAs encoding the indicated mCherry-tagged ORP1L proteins. After 16 hr in medium containing FBS, cholesterol esterification was assayed by [3H]oleate incorporation (mean and SD of three experiments, ∗p < 0.001). Transfected cells were immunoblotted with an ORP1L polyclonal antibody to confirm expression.
(B) HeLa and ORP1L-null cells were transiently co-transfected with empty vector or mCherry-ORP1L constructs and LAMP1-GFP for 24 hr in FBS-containing medium. Imaging of live cells was performed by spinning-disc confocal microscopy (scale bar, 20 μm).
Cell Reports  , DOI: ( /j.celrep )
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