![Figure 1. Chemical and genetic cholesterol manipulations and LEs. (A) Modulation of intracellular cholesterol levels. Mel JuSo cells were cultured in normal medium (FCS), cholesterol-depleted medium supplemented with lovastatin (statin), or normal medium supplemented with U-18666A, as indicated. Fixed cells were stained with filipin to detect cholesterol. A color look up table (LUT) shows fluorescence intensities. n > 100 for each condition. (B) Effects of intracellular cholesterol manipulation (control [CTRL], U-18666A, or statin treatment) on LE positioning in RILP- or ORP1L-silenced Mel JuSo cells. Cells were stained for the LE marker CD63 (red), endogenous ORP1L (green), and actin (blue). The position of the CD63-positive vesicles relative to the nucleus (radial distribution) was determined for the various conditions (>50 cells per condition), binned, and plotted (right). The distributions were statistically different according to the Jonckheere-Terpstra test for control siRNA (P = 1.52 × 10−5 and 7.76 × 10−6 for CD63 and ORP1L, respectively) only. No difference was detected for siRILP (P = 0.20 and 0.54 for CD63 and ORP1L, respectively) and for siORP1L (P = 0.29 for CD63). (C) Dynein motor activity and clustering of LEs in NPC1-silenced cells. Mel JuSo cells were transfected with siRNA for NPC1 for 72 h and, after 48 h, transfected with an expression construct for p50dynamitin to disrupt the dynein–dynactin motor. Fixed cells were stained for CD63 and p50dynamitin to mark the overexpressing cells. n > 100. Bars: (A and C)10 µm; (B) 20 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/185/7/10.1083_jcb.200811005/7/jcb_200811005_rgb_fig1.jpeg?Expires=1771602394&Signature=UWvuyv4oLftaNlyYUeW0IbXWZURzc3IL21LH51lzqRjnvHRYheKWXoRVsPM48rrY2NNtLu56Utuf6tY3xfdY6pUn~hpSHwtKgqAsR3aFNREZWjtjxj4sfOJpniy529HBghBV5e4zh5dHYNmM4xqY2aVrSfypdxzPiHUONfYfDKLsLocSdbgok4R~oAnBVLuvflkpFvfv-SE-5QptQoRhWWrbEHs76mNs-w~orWIUmI7zU42VSh21alVK-8g~TSN06p7zbqXoop17JuP1HBKwbRb2kny2xfrl7~dvBalF8-bjzSCk4IpaTTz0Zhnb5IZ2bGwaErcB-jAwWIHRPZ~Ifw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Chemical and genetic cholesterol manipulations and LEs. (A) Modulation of intracellular cholesterol levels. Mel JuSo cells were cultured in normal medium (FCS), cholesterol-depleted medium supplemented with lovastatin (statin), or normal medium supplemented with U-18666A, as indicated. Fixed cells were stained with filipin to detect cholesterol. A color look up table (LUT) shows fluorescence intensities. n > 100 for each condition. (B) Effects of intracellular cholesterol manipulation (control [CTRL], U-18666A, or statin treatment) on LE positioning in RILP- or ORP1L-silenced Mel JuSo cells. Cells were stained for the LE marker CD63 (red), endogenous ORP1L (green), and actin (blue). The position of the CD63-positive vesicles relative to the nucleus (radial distribution) was determined for the various conditions (>50 cells per condition), binned, and plotted (right). The distributions were statistically different according to the Jonckheere-Terpstra test for control siRNA (P = 1.52 × 10−5 and 7.76 × 10−6 for CD63 and ORP1L, respectively) only. No difference was detected for siRILP (P = 0.20 and 0.54 for CD63 and ORP1L, respectively) and for siORP1L (P = 0.29 for CD63). (C) Dynein motor activity and clustering of LEs in NPC1-silenced cells. Mel JuSo cells were transfected with siRNA for NPC1 for 72 h and, after 48 h, transfected with an expression construct for p50dynamitin to disrupt the dynein–dynactin motor. Fixed cells were stained for CD63 and p50dynamitin to mark the overexpressing cells. n > 100. Bars: (A and C)10 µm; (B) 20 µm.
