Monitoring the itinerary of lysosomal cholesterol in Niemann-Pick Type C1-deficient cells after cyclodextrin treatment

Niemann-Pick type C (NPC) disease is a lipid storage disorder that is caused by mutations in the genes encoding NPC proteins and results in lysosomal cholesterol accumulation. 2-Hydroxypropyl- β -cyclodextrin (CD) has been shown to reduce lysosomal cholesterol levels and enhance sterol homeostatic responses, but CD’s mechanism of action remains unknown. Recent work provides evidence that CD stimulates lysosomal exocytosis, raising the possibility that lysosomal cholesterol is released in exosomes. However, therapeutic concentrations of CD do not alter total cellular cholesterol, and cholesterol homeostatic responses at the endoplasmic reticulum (ER) are most consistent with increased ER membrane cholesterol. To address these disparate findings, here we used stable isotope labeling to track the movement of lipoprotein cholesterol cargo in response to CD in NPC1-deficient U2OS cells. Although released cholesterol was detectable, it was not associated with extracellular vesicles. Rather, we demonstrate that lysosomal cholesterol traffics to the plasma membrane (PM), where it exchanges with lipoprotein-bound cholesterol in a CD-dependent manner. We found that in the absence of suitable extracellular cholesterol acceptors, cholesterol exchange is abrogated, cholesterol accumulates in the PM, and re-esterification at the ER is increased. These results support a model in which CD promotes intracellular redistribution of lysosomal cholesterol, but not cholesterol exocytosis or efflux, during restoration of cholesterol homeostatic responses. To address these disparate findings, we used stable isotope labeling to track the movement of lipoprotein cholesterol cargo in response to CD in NPC1-deficient cells. Our data support a model in which CD promotes redistribution of lysosomal cholesterol to the PM from where it is exchanged with cholesterol carried by extracellular acceptors, and to the ER where it directs cholesterol homeostatic responses. For this study, we directly monitored movement of lysosomal cholesterol cargo after CD treatment. Prior studies have relied on filipin staining to quantify residual lysosomal cholesterol or radiolabeled oleate to monitor total cholesterol esterification. The former is uninformative with respect to the post-lysosomal trafficking of cholesterol, and the latter is indirect, since it quantifies all cholesterol esterification, rather than esterification of molecules originating from the lysosome. Moreover, these approaches are confounded by the asynchronous export of cholesterol from the lysosome. By contrast, our stable isotopic approach allowed for specific loading of the lysosomal compartment with labeled cargo and synchronous release of labeled cholesterol through the use of a reversible LAL inhibitor. Using sensitive identification of deuterated products by mass spectrometry and selective enzymatic modification approaches (e.g., treatment with cholesterol oxidase), we followed the post-lysosomal trafficking of the pool of cholesterol that accumulates aberrantly in lysosomes in NPC1. These techniques provide powerful tools with which to more precisely delineate the itinerary of cholesterol and to investigate mechanisms that underlie its post-lysosomal trafficking.


INTRODUCTION
Cholesterol is an essential component of mammalian cell membranes that plays a major role in tuning membrane fluidity, thickness, and permeability to regulate membrane function and support the needs of specific organelles. Different cellular membranes vary widely in cholesterol content, ranging from the cholesterol-rich plasma membrane (PM) and endosomes to the cholesterol-poor endoplasmic reticulum (ER) and mitochondria (1)(2)(3). Due to its hydrophobicity, cholesterol does not transit between membranes through the aqueous phase. Rather, cholesterol transfer is facilitated by lipid-binding proteins or through membrane fusion events (1). Although a number of  (4). In the presence of functional LAL, NPC1, and NPC2, cholesterol is efficiently trafficked to the PM and ER as well as other cellular membranes. At the PM, excess cholesterol is effluxed through ABCA1, ABCG1, and SRB1 to ApoA1/HDL particles (5). In the ER by guest, on May 6, 2020 www.jlr.org Downloaded from membrane, cholesterol serves as a critical regulator of sterol homeostasis through the SREBP transcription factors, and excess cholesterol is esterified by ER-resident protein ACAT for storage in lipid droplets (6).
Interruption of the intra-lysosomal cholesterol trafficking network (NPC1, NPC2, or LAL) results in abnormal cholesterol homeostasis and lysosomal dysfunction. Mutations in NPC1 or NPC2 cause Niemann-Pick Type C disease, a fatal neurodegenerative disorder. Both cholesterol trafficking and homeostatic regulation are disrupted in NPC1-deficient cells, in which accumulation of free cholesterol in the lysosome is accompanied by elevated expression of cholesterol uptake and synthesis genes and decreased cholesterol esterification (7,8). There is currently no FDAapproved therapy for NPC disease, but 2-hydroxypropyl-beta-cyclodextrin (CD) has shown great promise in animal models and in human clinical trials (9). CD is a cyclic oligosaccharide frequently used as an excipient in drug formulations because of its ability to solubilize hydrophobic molecules. At concentrations >1 mM, CD can extract cholesterol from cultured cells (10). At lower concentrations, in the range of effective doses in vivo, CD enhances cholesterol trafficking from lysosomes without changing total cellular cholesterol, and neither increases in serum cholesterol nor cholesterol excretion are observed (11). In fact, in cell and animal models, CD treatment reduces expression of SREBP2 gene targets and stimulates cholesterol esterification, consistent with a model in which lysosomal cholesterol is redistributed to the ER membranes (7,8). While the mechanism of CD action remains unknown, recent studies provide evidence that CD promotes lysosomal exocytosis (2,12,13). However, if release from the cells of cholesterol-laden exosomes is responsible for the beneficial effects of CD treatment, this would be predicted to lower cellular cholesterol, a change inconsistent with the observed suppression of SREBP2 gene targets or with enhanced re-esterification by ACAT.

Cells and media: U2OS cells expressing the human scavenger receptor A (U2OS-SRA) and
U2OS-SRA cells with shNPC1 knockdown (U2OS-SRAshNPC1) were a gift from the Maxfield Laboratory (14). U2OS-SRAshNPC1 cells were transduced with a lentiviral vector for expression of TMEM192-HA-RFP as described (15). Cells were cultured in McCoy's medium containing 10% FBS, 1.2 g/L sodium bicarbonate, and 1 mg/mL G418. Lipoprotein-deficient medium (LPDM) contained lipoprotein-depleted FBS instead of regular serum. Media depleted of extracellular vesicles was prepared as described (16). Unless otherwise specified, experiments were performed using a standard protocol: On day 1, 2 x 10 5 cells were plated in 6-well dishes and grown overnight. On day 2, growth media was replaced with medium containing 10 µM lalistat-1, an inhibitor of LAL (Tocris Bioscience), with 25 µg/mL acLDL or acLDL reconstituted with d7 cholesteryl ester (d7-acLDL), and cells were incubated for an additional 18 h. For re-esterification experiments, 36 µg/mL d9 oleate complexed to BSA was included during this period. On day 3, cells were washed twice with PBS and then incubated in fresh growth media containing 500 µM CD (Janssen Pharmaceuticals) or vehicle (H2O) for 6 h. Reconstituted acLDL was prepared as described (17). Lipoproteins were obtained from Alfa Aesar or Kalen Biomedical. Human apoA1 was obtained from Millipore Sigma.
Lysosomal Isolation: U2OS-SRAshNPC1 cells expressing TMEM192-HA-RFP were plated at 12 x 10 5 in 10cm dishes, loaded with d7-acLDL and treated with CD according to the standard protocol. Lysosomes were isolated as described (15) using Pierce TM Anti-HA Magnetic Beads (Thermo Fisher) and were eluted from the beads with a 5-minute incubation in 50 mM NaOH. Eluate was neutralized immediately with 100 mM Tris pH 6.8. Cholesterol and cholesteryl ester content was measured in cell lysate, post-nuclear supernatant, or elution fractions by liquid chromatography-  Table   S1 for antibody sources and dilutions. plates (10 cm) per condition, loaded with 10 µg/mL d7-acLDL in the presence of 10 µM lalistat, and incubated overnight to load the lysosomal compartment. Cells were washed twice with PBS, then incubated in fresh media (depleted of EVs) with or without 500 µM CD for 6 h. EVs were isolated from conditioned media by differential ultracentrifugation as described (16). Cholesterol content was quantified in media, EVs, and media following depletion of EVs by LC-MS/MS. EV proteins were separated on 4-12% Bis-Tris precast gels, transferred to PVDF membranes and probed for nuclear (Lamin B1) and EV (CD63) markers.  Table S2. Additional assay parameters are reported in Supplemental Table S3. Data was analyzed using Xcalibur software. Calibration curves were constructed by plotting peak ratios of standard/internal standard versus analyte concentration.  (18). In the presence of lalistat, which prevents LAL mediated ester hydrolysis (19,20), cargo is retained in the compartment; upon inhibitor washout, cargo is released. We adapted this approach in order to specifically monitor the NPC1independent trafficking of lysosomal cholesterol after CD treatment. In NPC1-deficient mice, CD is rapidly cleared from the plasma in 3 hours and from the whole body in 6 hours (11) and CD is estimated to reach concentrations of 0.1-1 mM in vivo (21,22). Based on these studies we chose to analyze the effects of CD after 6 hours of treatment with 0.5 mM CD. U2OS-SRA cells with shRNA knockdown of NPC1 (U2OS-SRAshNPC1) were incubated with acLDL reconstituted with deuterated cholesteryl oleate (d7-acLDL) in the presence of lalistat (Fig. 1). After loading, d7-acLDL and lalistat were removed and replaced with media containing CD. Trafficking of deuterated cholesterol away from the lysosome or to the PM, ER, and culture media was monitored using LC-MS/MS-based biochemical trafficking assays.
Although previous studies have used filipin staining to demonstrate that total lysosomal cholesterol is decreased by CD treatment (8), these studies have not metabolically traced lipoprotein-derived cholesterol. We used U2OS-SRAshNPC1 cells expressing TMEM192-RFP-HA, a tagged lysosomal protein, to quantify changes in lysosomal cholesterol cargo derived from endocytosed lipoproteins. Lysosomes from U2OS-SRAshNPC1-TMEM192-RFP-HA cells contained LAMP1 and HEXA, membrane bound and soluble lysosomal proteins, respectively ( Fig. 2A). The isolated lysosomal fraction was depleted of contaminating membranes from the nucleus, and PM as indicated by histone H3 (HIS) and Na/K ATPase (NAK) markers respectively. While substantially depleted, ER marker calreticulin (CAL), and mitochondria marker, COXIV (COX) could still be detected in the isolated fractions. The lysosome-enriched fraction isolated from cells treated with CD had less d7 cholesterol relative to vehicle treated control (Fig. 2B). D7 cholesteryl oleate associated with the enriched fraction did not differ significantly between CD and vehicle treated cells (Fig. 2C). Some LAMP1-containing membranes were not immuno-isolated and were detected in the flow-through. These LAMP + membranes could represent lysosome-related organelles that do not contain TMEM197 or HEXA but may harbor cholesterol.
To trace the fate of lipoprotein cholesterol cargo to other cellular compartments, we analyzed cholesterol movement in U2OS-SRAshNPC1 cells. Similar to our findings in the lysosomes, total cellular d7 cholesteryl oleate was unchanged by CD treatment (Fig. 2D), suggesting LAL activity was not affected. To measure PM cholesterol, cells were fixed and treated with cholesterol oxidase and sphingomyelinase to oxidize d7 cholesterol to d7 cholestenone. Under vehicletreated conditions, d7 cholesterol trafficked to the PM (Fig. 2E). There was a trend for increased trafficking of lysosomal cholesterol to the PM following CD treatment, but this did not reach significance. In order to assess re-esterification, d9 oleate was included during loading to provide substrate for ACAT mediated re-esterification of d7 cholesterol to d16 cholesteryl ester. CD administration resulted in a 2-fold increase in the formation of d16 cholesteryl esters from cholesterol cargo originating in the lysosome (Fig. 2F). We also analyzed d7 cholesterol content of conditioned media to assess efflux. Most strikingly, compared to vehicle treatment, CD treatment increased the efflux of d7 cholesterol to the culture medium 28-fold (Fig. 2G). Overall, the magnitude of d7 cholesterol arriving at the ER was small when compared with the d7 cholesterol arriving at the PM and into the media.

Released d7 cholesterol is not detected in isolated extracellular vesicles (EVs)
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To further characterize the mechanism of release of d7 cholesterol into the culture medium, we performed a time course. D7 cholesterol was detectable in the culture medium as early as 3 hours after lalistat washout and CD treatment. Between 3 hours and 6 hours, d7 cholesterol in the media increased ~5 fold (Fig. 3A). Appearance of lysosomal cholesterol in the PM preceded appearance in the media (Fig. 3B). Similar levels of PM d7 cholesterol were measured after ionomycin or DMSO vehicle treatment. In contrast to CD, ionomycin did not promote release of the cholesterol into the media over 6 h. These data show that distribution of d7 cholesterol from the lysosome to the PM is an order of magnitude greater and occurs more rapidly than distribution into the media.
A recent report that CD induces lysosomal exocytosis in HeLa cells (13) provides a potential mechanism for the appearance of lysosomal cholesterol in the culture medium. To test whether d7 cholesterol arriving in the media was contained within membrane-bound structures released from the cells, we used differential centrifugation to isolate extracellular vesicles (EVs) from conditioned media from U2OS-SRAshNPC1 cells loaded with deuterated cholesterol and then treated with CD. The EV fraction was positive for the exosome marker CD63 but negative for nuclear marker laminin B (LMB) by western blot (Fig. 3C, top). Similar results were seen with EVs isolated from conditioned media from cells treated with ionomycin (Fig. 3C, bottom). While d7 cholesterol was readily detected in whole media and EV-depleted media from CD-treated cells, d7 cholesterol was not detected in EVs. Although EVs contained d0 cholesterol, the amount did not differ significantly between EVs isolated from the media of vehicle-and CD-treated cells (Fig.   3E). Together, these data indicate that EV release does not significantly contribute to the d7 cholesterol pool detected in the culture media.

CD-induced accumulation of cholesterol in the media is largely independent of canonical cell surface cholesterol transporters
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We next considered alternate mechanisms for release of lysosomal cholesterol following CD treatment. SRB1, ABCA1, and ABCG1 are cell surface sterol transport proteins. In order to test their contributions to CD-mediated accumulation of cholesterol in the media, we knocked down these proteins by si or shRNA and quantified delivery of d7 lysosome-derived cholesterol into the media. Two independent SRB1 siRNAs (siSRB1-1, siSRB1-2) robustly suppressed SRB1 protein expression (>95%) as shown by western blot (Fig. 4A). Despite this, CD-induced cholesterol accumulation in the media was not significantly different between siSRB1 and control siRNA (siCON)-treated cells (Fig. 4B). To test the effects of ABCA1 and ABCG1 protein depletion, stable shRNA knockdown cells were generated using target-specific (shABCA1-1 or shABCA1-2 and shABCG1-1 or shABCG1-2) or control (shCON) shRNAs. Using two different shRNAs for ABCA1, we isolated cell populations with 31 and 53% knockdown (Fig. 4C); for shABCG1, cell populations with 33 and 39% knockdown were isolated (Fig. 4D). Neither knockdown of ABCA1 nor knockdown of ABCG1 impaired d7 cholesterol accumulation in the media (Fig. 4E). Thus, CDinduced release of lysosomal cholesterol into the media is independent of SRBI and occurs even with substantial ABCA1 or ABCG1 knockdown.

CD-induced cholesterol accumulation in the media depends on serum lipoproteins
The majority of cholesterol found in serum is associated with serum lipoproteins. In order to test whether extracellular lipoproteins play a role in CD-induced release of lysosomal cholesterol into the media, the lipoprotein fraction was isolated from conditioned medium after CD treatment. LDL and HDL are the major lipoproteins present in FBS (23). In line with this, lipoprotein fractions 1 and 2 from conditioned culture medium of cells treated with CD or V contained two dominant species that co-migrated with either human LDL (Fig. 5A, lanes 1-4, upper band) or human HDL (Fig. 5A, lanes 1-4, lower band) on a native PAGE gel. Following CD treatment, the lipoprotein fractions contained >50% of the d7 cholesterol found in the medium (Fig. 5A, graph, right). When cells were treated with CD in lipoprotein-deficient medium (LPDM), d7 cholesterol was no longer detected in the medium (Fig. 5B). Supplementation of LPDM with 25 µg/mL HDL or LDL restored d7 cholesterol release. Equivalent concentrations of APOA1 or BSA did not support cholesterol release, suggesting that CD-mediated accumulation in the media requires lipidated lipoproteins.
Although the proportion of lysosomal cholesterol in the PM was not significantly different between vehicle and CD treated cells when delivered in medium containing FBS (Fig. 2E), lysosomederived cholesterol accumulated in the PM when lipoproteins were absent in the media (Fig. 5C).
Re-esterification of lysosome-derived cholesterol, detected as d16 cholesteryl ester, was enhanced in the absence of lipoproteins (Fig. 5D). This is likely due to increased delivery of d7 cholesterol to the ER for esterification, and not due to increased overall esterification, as the d9oleate pool (esterification product of non-labeled cholesterol) was not-significantly different between FBS and LPDM conditions (Fig. 5E). Together these findings support a model in which CD enhances the trafficking of lysosomal cholesterol to the PM where it is released into the media if lipoproteins are present, or further trafficked to the ER where it is esterified.

Bi-directional cholesterol exchange occurs at the PM
Despite release of lysosomal cholesterol from the cell, CD treatment in complete media did not alter total cellular cholesterol (Fig. 6A). Thus, we incubated cells with diI-labeled lipoproteins in order to test whether lysosome-derived cholesterol release was matched by re-uptake of lipoprotein cholesterol from the media during CD treatment. Only 1% per mg protein of the diI-LDL was taken up under both vehicle and CD conditions (Fig. 6B). This was blunted by the dynamin inhibitor, dynasore (DYN), consistent with a mechanism of receptor-mediated endocytosis. Uptake of HDL was negligible. Based on these observations, we hypothesized that in the presence of CD, release of cholesterol from the PM to lipoproteins in the media occurs as an exchange with lipoprotein cholesterol. To test this, we treated cells with CD in media containing lipoproteins carrying deuterated cholesterol. CD treatment increased cell-associated deuterated cholesterol in the presence of LDL or HDL donors (Fig. 6C, left). Suppression of cellular uptake of lipoproteins with DYN did not inhibit CD-dependent transfer of LDL or HDL-derived cholesterol (Fig. 6C, left). CD increased transfer of cholesterol from LDL to cells 2.8-fold more than vehicle (Fig. 6C, right). Transfer of cholesterol from HDL to cells was less efficient. Together, our data support a model in which CD facilitates exchange of lysosome-derived d7 with lipoprotein acceptors in the media, with LDL providing the more efficient exchange.

DISCUSSION
The mechanism by which CD redistributes cholesterol from the lysosomal compartment in the setting of NPC1 deficiency is not well understood. In order to specifically investigate the lysosomal cholesterol pool, we loaded lysosomes with isotopically labeled cholesteryl esters in reconstituted lipoproteins prior to CD treatment and tracked the itinerary of the labeled cholesterol pool. We observed increased esterification and release of lysosome-derived cholesterol into the media.
The cholesterol released into the media was largely associated with lipoprotein particles, but release was independent of SRBI and did not decrease with ABCA1 and ABCG1 knockdown. In the absence of lipidated lipoproteins, release did not occur, and lysosome-derived cholesterol accumulated in the PM. In the presence of lipoproteins, total cellular cholesterol was maintained through the bidirectional movement of cholesterol between lipoproteins and the cell surface.
Together these data support a model by which CD enhances trafficking of lysosomal cholesterol to the PM where it is available for exchange with extracellular lipoproteins or is routed internally for esterification (Fig. 7).
There is evidence that CD exerts its effects on cholesterol homeostasis through stimulation of lysosomal exocytosis, a hypothesis supported by localization of lysosomal markers with the PM and release of a lysosomal lipid, lysobisphosphatidic acid, into the media in NPC1-deficient cells treated with CD (2,12,13). Furthermore, TRPML1, a lysosomal ion channel that has been implicated in lysosomal exocytosis, autophagy, and vesicular trafficking (24)(25)(26), is required for CD-mediated correction of lysosomal cholesterol accumulation in NPC1-deificent cells (13). Relocalization of lysosomes to the PM in a TPRML1-dependent matter could also provide a mechanism by which CD increases trafficking of lysosomal cholesterol to the PM. However, these studies did not directly quantify movement of bulk or lysosome-derived cholesterol out of the cells. shows that lysosomal cholesterol accumulates in the media following CD treatment, the mechanism is one of exchange and not net efflux, and it is inconsequential with respect to overall cholesterol trafficking to the ER. The exchange mechanism is consistent with observations, in vivo, that CD treatment does not decrease total cellular cholesterol or increase cellular cholesterol clearance (11). Taken together, these data indicate that CD facilitates cholesterol exchange but does not support net efflux to CD as a functional pathway.  (U2OS-SRAshNPC1) were incubated in medium containing acLDL reconstituted with d7 cholesteryl oleate (d7-acLDL, red oval-chain) to load the lysosomal compartment (LYS). Lalistat was included during loading to prevent cleavage of labeled esters by LAL and release from the lysosome. d9 oleate (blue chain) was included during loading to provide cells with labeled oleate for detection of re-esterification product. B: Following removal of acLDL and lalistat, cells were incubated with CD. Washout of lalistat enables hydrolysis of cholesteryl esters at the lysosome. Upon arrival at the ER, d7 cholesterol was re-esterified with d9 oleate to form d16 cholesteryl ester, which can be distinguished from loaded d7 cholesteryl ester. Arrival of cholesterol at the PM was detected by sphingomyelinase and cholesterol oxidase treatment, which converts d7 cholesterol to d7 cholestenone. Efflux of lysosomal cholesterol was monitored by appearance of d7 cholesterol in the media.  C-E: EVs were isolated from conditioned media at 6 h by differential centrifugation. Immunoblot analysis of total cell lysate (cell), and EVs for exosomal marker CD63 and nuclear marker lamin-B (LMB, C). Total d7 cholesterol in 27 ml conditioned media (input), 27 ml EV-depleted media, and EVs (from 27 ml conditioned media) at 6 h (D). Total EV cholesterol, normalized to EV protein (E). Means + SE for n = 3. *, P < 0.05 by unpaired t-test; NS, not significant by two-way ANOVA; ND, not detected.  A, B), or with CON or ABCA1-or ABCG1-targeting shRNAs (C-E). Following loading with d7-acLDL in the presence of lalistat, cells were treated for 6 h with CD or vehicle (V), and effluxed d7 cholesterol quantified by LC-MS/MS. A: Representative immunoblot for SRB1 with GAPDH loading control (above) and quantification for n = 3 (below). B: Moles released d7 cholesterol relative to moles d7 cholesteryl ester loaded expressed as efflux (relative units, RU). C, D: Representative immunoblots for ABCA1 (C) and ABCG1 (D) with GAPDH loading control (above) and quantification for n = 3 (below). E: Relative efflux. Means + SE for n = 3. *, P < 0.05 for siRNA or shRNA vs. CON by unpaired t-test. siRNA or shRNA vs. CON by two-way ANOVA was not significant for V and CD conditions. Following treatment in media containing FBS, lipoproteins were isolated from conditioned media and assayed for d7 cholesterol content. Sypro Ruby stained native PAGE gel analysis (left) and d7 cholesterol content (right) of conditioned media and its lipoprotein fractions (F1 and F2) and remaining material (F3) after lipoprotein isolation. Pure human LDL (hLDL) and HDL (hHDL) shown as markers. B: Media-associated d7 cholesterol quantified following 6 h treatment in FBS, lipoprotein-deficient media (LPDM), or LPDM supplemented with 25µg/mL APOA1, HDL, LDL, or BSA. Efflux reported relative to efflux with FBS. C: PM-localized lysosomal cholesterol after 6 h CD treatment in FBS or LPDM reported as percent of total cholestenone pool that was deuterated. D: Re-esterification of lysosomal cholesterol after 6 h CD treatment in FBS or LPDM. E: esterification of non-deuterated cholesterol (d9 CE 18:1) after 6 h CD treatment in FBS or LPDM. Means + SE for n = 3-5. *, P<0.05 by unpaired t-test or two-way ANOVA for comparisons indicated; NS, not significant. Cellular uptake of diI-labeled LDL or HDL after 6 h with CD or V and in the presence or absence of dynasore (DYN) to inhibit dynamin activity. Data expressed by the ratio of cell associated fluorescence relative to fluorescence in media at baseline per mg of total protein. C: Cells were incubated with d7 cholesterol loaded LDL or HDL for 6 h in the presence or absence of CD and in the presence or absence of DYN. Graph on left reports ratio of cell-associated d7 cholesterol relative to d7 cholesterol in media at baseline. Graph on right reports cell-associated cholesterol for each lipoprotein incubation in the presence of CD relative to V (no DYN). Means + SE for n = 3-5. *, P < 0.05 by unpaired t-test or two-way ANOVA for comparisons indicated; ns, not significant.