1. Volume 19, Issue 13, Pages 2823-2835 (June 2017)
The GARP Complex Is Involved in Intracellular Cholesterol Transport via Targeting NPC2 to Lysosomes 
Jian Wei, Ying-Yu Zhang, Jie Luo, Ju-Qiong Wang, Yu-Xia Zhou, Hong-Hua Miao, Xiong-Jie Shi, Yu-Xiu Qu, Jie Xu, Bo-Liang Li, Bao- Liang Song 
Cell Reports 
Volume 19, Issue 13, Pages (June 2017)
DOI: /j.celrep
Copyright © 2017 The Author(s) Terms and Conditions

2. Cell Reports 2017 19, 2823-2835DOI: (10.1016/j.celrep.2017.06.012)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1 Selection of Cells with Cholesterol Trafficking Defects
(A) Schematic of the screen strategy. Amph. B, amphotericin B.
(B) Expression of the NPC1 protein in CHO-7 and CHO-NPC1 cells.
(C) Colocalization of stably expressed NPC1-RFP (red) and transiently transfected LAMP1-EGFP (green) in CHO-NPC1 cells. Scale bar, 20 μm.
(D) Strategy for determining the optimal LDL delivery period. Cells were cultured in cholesterol starvation medium for 16 hr and refed with LDLs for the indicated periods (0, 2, 4, and 6 h). Cells were then treated with amphotericin B for 30 min and stained with crystal violet. U18666A-treated cells were used as positive controls.
(E) Mut1 and Mut2 cells survived after five rounds of amphotericin B selection as shown in (A).
(F) Mut1 and Mut2 cells displayed robust filipin staining. Scale bar, 20 μm.
See also Figure S1A.
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4. Figure 2 VPS53 Is Required for Intracellular Cholesterol Trafficking
(A) RNA-seq analysis revealed a frameshift mutation in the Vps53 gene (NW_ ) of mutant cells.
(B and C) Expression of the VPS53 protein in mutant cells (B) and VPS53 knockdown cells (C). The asterisk in (B) denotes nonspecific bands.
(D) Confocal images of filipin (red) and LAMP1 (green) staining of control and VPS53 knockdown cells. The boxed areas are shown at a higher magnification on the right. Scale bar, 10 μm.
(E) Confocal images of filipin (blue) and LysoTracker (red) staining of control and VPS53 knockdown cells. Scale bar, 10 μm.
(F and G) Quantification of the relative fluorescence intensity of filipin (F) and LysoTracker (G) staining as shown in (E). Data are normalized to control cells and represented as mean ± SEM (n = 20). ∗∗∗p < , unpaired two-tailed Student’s t test.
(H) Confocal images of filipin (red) staining of VPS53 knockdown cells transiently transfected with the shRNA-resistant plasmid encoding VPS53-EGFP (green). A cell co-expressing VPS53 and EGFP is outlined. Scale bar, 20 μm.
(I) Analysis of SREBP2 cleavage using control and VPS53 knockdown cells. pSREBP2, precursor of SREBP2; nSREBP2, nuclear form of SREBP2.
See also Figures S1B–S1E.
Cell Reports  , DOI: ( /j.celrep )
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5. Figure 3
Missorting of NPC2 Results in Cholesterol Accumulation in VPS53 Knockdown Cells
(A and B) Expression of the NPC1 and NPC2 proteins in the lysates and culture media of the mutant cells (A) and VPS53 knockdown cells (B). WCL, whole cell lysate.
(C) Confocal images of NPC2 (red) and LAMP1 (green) staining of control, VPS53 knockdown, and NPC2 knockdown cells. Scale bar, 20 μm.
(D) The mRNA level of NPC2 in control and VPS53 knockdown cells. Data are normalized to control cells and represented as mean ± SEM (n = 3). ns, not significant; unpaired two-tailed Student’s t test.
(E) Left: confocal images of filipin (red) staining of control and U18666A-treated cells. Scale bar, 20 μm. Right: expression of the NPC2 protein in the lysates and culture media of control and U18666A-treated cells.
(F) Left: confocal images of filipin (red) staining of control and VPS53 knockdown cells treated with or without HPCD. Scale bar, 20 μm. Right: expression of the NPC2 protein in the lysates and culture media of control and VPS53 knockdown cells treated with or without HPCD.
(G) Confocal images of filipin (red) staining of the mutant cells transiently co-transfected with plasmids encoding NPC2 and EGFP (green). Cells co-expressing NPC2 and EGFP are outlined. Scale bar, 20 μm.
(H) Confocal images of filipin (red) staining of VPS53 knockdown cells transiently co-transfected with plasmids encoding NPC2 and EGFP (green). A cell co-expressing NPC2 and EGFP is outlined. Scale bar, 20 μm.
(I) Confocal images of filipin (red) staining of VPS53 knockdown cells transiently transfected with the plasmid encoding LAMP1-NPC2 (green). A cell expressing LAMP1-NPC2 is outlined. Scale bar, 20 μm.
See also Figures S2 and S3.
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6. Figure 4
Depletion of the GARP Complex Leads to NPC2 Missorting and Cholesterol Accumulation
(A) Cartoon of the GARP complex consisting of VPS51, VPS52, VPS53, and VPS54.
(B) Expression of the GARP complex subunits in control and VPS53 knockdown cells.
(C) The mRNA levels of the indicated genes in cells transfected with the corresponding siRNAs. Data are normalized to control cells and represented as mean ± SEM (n = 3).
(D) Expression of the NPC2 protein in the lysates and culture media of cells transfected with the indicated siRNAs.
(E) Confocal images of filipin staining of cells transfected with the indicated siRNAs. Scale bar, 20 μm.
(F) Quantification of the relative fluorescence intensity of filipin staining in (E). Data are normalized to control cells and represented as mean ± SEM (n = 20). ∗∗∗p < 0.0001, one-way ANOVA.
(G) Confocal images of LysoTracker staining (red) of cells transfected with the indicated siRNAs. Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm.
(H) Quantification of the relative fluorescence intensity of LysoTracker staining in (G). Data are normalized to control cells and represented as mean ± SEM (n = 20). ∗∗∗p < , one-way ANOVA.
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7. Figure 5
The GARP Complex Directs Lysosomal Targeting of NPC2 by Modulating CI-MPR Retrieval
(A) Confocal images of CI-MPR (green) and P230 (red) staining of control and VPS53 knockdown cells. Nuclei were counterstained with DAPI (blue). Cell contours are outlined. Scale bar, 20 μm.
(B) Percentage of CI-MPR colocalizing with P230 in (A). Data are represented as mean ± SEM (n = 20). ∗∗∗p < , unpaired two-tailed Student’s t test.
(C) Expression of CI-MPR and CD-MPR proteins in control and VPS53 knockdown cells. The asterisk denotes nonspecific bands.
(D) Confocal images of CI-MPR (green) and Rab7 (red) staining of control and VPS53 knockdown cells. Nuclei were counterstained with DAPI (blue). Boxed areas are shown at a higher magnification on the right. Scale bar, 20 μm.
(E) Overlap coefficient between CI-MPR and Rab7 in (D). Data are represented as mean ± SEM (n = 20). ∗∗∗p < , unpaired two-tailed Student’s t test.
(F) The dispersed CI-MPR colocalized with LysoTracker in VPS53-depleted cells. Shown are confocal images of CI-MPR (green) and LysoTracker (red) staining of control and VPS53 knockdown cells. Boxed areas are shown at a higher magnification on the right. Scale bar, 20 μm.
(G) Confocal images of expressed CD-MPR-EGFP (green) and endogenous P230 (red) staining of control and VPS53 knockdown cells. Nuclei were counterstained with DAPI (blue). Cell contours are outlined. Scale bar, 20 μm.
(H) Percentage of CD-MPR-EGFP that colocalized with P230 in (G). Data are represented as mean ± SEM (n = 20); Unpaired two-tailed Student’s t test.
(I) Expression of CI-MPR and NPC2 proteins in the lysates and culture media of control and CI-MPR knockdown cells.
(J) Confocal images of filipin staining of control and CI-MPR knockdown cells. Scale bar, 20 μm.
(K) Quantification of the relative fluorescence intensity of filipin staining in (J). Data are normalized to control cells and represented as mean ± SEM (n = 20). ∗∗∗p < 0.0001, unpaired two-tailed Student’s t test.
See also Figures S4 and S5.
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8. Figure 6 Cholesterol Homeostasis Is Disturbed in Vps54wr/wr Mice
(A) Expression of the indicated proteins in cervical spinal cord extracts from WT (+/+) and wobbler (wr/wr) mice. The asterisk denotes nonspecific bands.
(B) Expression of the NPC2 protein in the bloodstream of WT and wobbler mice.
(C) Confocal images of filipin staining in cervical spinal cord slices from WT and wobbler mice. Scale bar, 100 μm.
(D) Confocal images of filipin and LysoTracker staining of tail tip fibroblasts from WT and wobbler mice. Scale bar, 20 μm.
(E and F) Quantification of the relative fluorescence intensity of filipin (E) and LysoTracker (F) staining as shown in (D). Data are normalized to control cells and represented as mean ± SEM (n = 20). ∗∗∗p < , unpaired two-tailed Student’s t test.
Cell Reports  , DOI: ( /j.celrep )
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