Figure 5.
OSBP regulates polarized cholesterol distribution. (A) MDCK cells stably expressing D4H-EGFP were transfected with control or OSBP-silencing siRNA and reseeded into Matrigel 24 h later. Cysts were left to develop for 72 h, fixed, and imaged by confocal microscopy. D4H-EGFP signal on the cell surface significantly dropped upon OSBP silencing. Open circles on the superplot correspond to the mean intensity of one cyst, while filled triangles show the mean of all the analyzed cysts from each independent experiment. Violin plots indicate the distribution of single-cell intensity values across all experiments. N = number of independent experiments presented. Mean cyst intensities were compared by paired t test. More cysts are presented in Fig. S4 A. Bar = 15 μm, the diameter of the insets = 30.8 μm. (B) MDCK cells stably expressing D4H-EGFP were seeded into Matrigel. 72 h later, cysts were treated overnight with SWG. Following treatment, cysts were fixed and prepared for confocal imaging. D4H-EGFP signal on the cell surface significantly dropped upon OSBP inhibition. Open circles on the superplot correspond to the mean intensity of one cyst, while filled triangles show the mean of all the analyzed cysts in each independent experiment. Violin plots indicate the distribution of single cell intensity values across all experiments. N = number of independent experiments presented. Mean cyst intensities were compared by paired t test. More cysts are presented in Fig. S4 B. Bar = 15 μm, diameter of the insets = 30.8 μm. (C) MDCK cells were seeded in Matrigel, left to form cysts for 72 h, and then treated with SWG for indicated times. Cysts were fixed, extracted from Matrigel, and stained with Filipin to quantify plasma membrane cholesterol levels by confocal microscopy. Filled symbols on the superplot correspond to the mean intensity of one cyst, while open circles indicate single-cell intensities. N = number of independent experiments presented. Symbols correspond to cyst data obtained from independent experiments. Mean cyst intensities were compared by two-way ANOVA followed by Sidak’s multiple comparison test. Arrowhead points to apical surface. Bar = 15 μm, the diameter of the insets = 30.3 μm. (D and E) 5 h ORPphilin-treated (D) or OSBP-silenced (E) MDCK cells were stained with the plasma membrane polarity dye NR12A, and confocal images were taken using emission windows corresponding to 550–600 and 600–650 nm. Ratio values are shown in the pseudocolored micrographs. N = number of independent experiments presented. Histograms show the mean ± SEM of four fields, each containing at least 8–10 cells. Triangles point to mean values. The three independent experiments are shown in Fig. S4, C–E and Fig. S4, F–H. Bar = 25 μm. (F) MDCK cells were filter-polarized and then treated with SWG for 5 h. Apical and basolateral surfaces were stained with the solvatochromic dye NR12A, then confocal images were taken using emission windows corresponding to 550–600 and 600–650 nm. The obtained ratiometric images were analyzed to obtain intensity histograms corresponding to apical and basolateral cell surfaces. N = number of independent experiments presented. Histograms show the mean ± SEM of five fields, each containing at least 8–10 cells. Triangles point to mean values. The three independent experiments are shown in Fig. S4, I–K. Bar = 10 μm.

OSBP regulates polarized cholesterol distribution. (A) MDCK cells stably expressing D4H-EGFP were transfected with control or OSBP-silencing siRNA and reseeded into Matrigel 24 h later. Cysts were left to develop for 72 h, fixed, and imaged by confocal microscopy. D4H-EGFP signal on the cell surface significantly dropped upon OSBP silencing. Open circles on the superplot correspond to the mean intensity of one cyst, while filled triangles show the mean of all the analyzed cysts from each independent experiment. Violin plots indicate the distribution of single-cell intensity values across all experiments. N = number of independent experiments presented. Mean cyst intensities were compared by paired t test. More cysts are presented in Fig. S4 A. Bar = 15 μm, the diameter of the insets = 30.8 μm. (B) MDCK cells stably expressing D4H-EGFP were seeded into Matrigel. 72 h later, cysts were treated overnight with SWG. Following treatment, cysts were fixed and prepared for confocal imaging. D4H-EGFP signal on the cell surface significantly dropped upon OSBP inhibition. Open circles on the superplot correspond to the mean intensity of one cyst, while filled triangles show the mean of all the analyzed cysts in each independent experiment. Violin plots indicate the distribution of single cell intensity values across all experiments. N = number of independent experiments presented. Mean cyst intensities were compared by paired t test. More cysts are presented in Fig. S4 B. Bar = 15 μm, diameter of the insets = 30.8 μm. (C) MDCK cells were seeded in Matrigel, left to form cysts for 72 h, and then treated with SWG for indicated times. Cysts were fixed, extracted from Matrigel, and stained with Filipin to quantify plasma membrane cholesterol levels by confocal microscopy. Filled symbols on the superplot correspond to the mean intensity of one cyst, while open circles indicate single-cell intensities. N = number of independent experiments presented. Symbols correspond to cyst data obtained from independent experiments. Mean cyst intensities were compared by two-way ANOVA followed by Sidak’s multiple comparison test. Arrowhead points to apical surface. Bar = 15 μm, the diameter of the insets = 30.3 μm. (D and E) 5 h ORPphilin-treated (D) or OSBP-silenced (E) MDCK cells were stained with the plasma membrane polarity dye NR12A, and confocal images were taken using emission windows corresponding to 550–600 and 600–650 nm. Ratio values are shown in the pseudocolored micrographs. N = number of independent experiments presented. Histograms show the mean ± SEM of four fields, each containing at least 8–10 cells. Triangles point to mean values. The three independent experiments are shown in Fig. S4, C–E and Fig. S4, F–H. Bar = 25 μm. (F) MDCK cells were filter-polarized and then treated with SWG for 5 h. Apical and basolateral surfaces were stained with the solvatochromic dye NR12A, then confocal images were taken using emission windows corresponding to 550–600 and 600–650 nm. The obtained ratiometric images were analyzed to obtain intensity histograms corresponding to apical and basolateral cell surfaces. N = number of independent experiments presented. Histograms show the mean ± SEM of five fields, each containing at least 8–10 cells. Triangles point to mean values. The three independent experiments are shown in Fig. S4, I–K. Bar = 10 μm.

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