Figure 2.
OSBP inhibition affects apical and basolateral trafficking routes. (A–D) MDCK cells were cotransfected with various RUSH model cargoes (CDH1- (A), DSG2- (B), EGFP-GPI- (C), GP135- (D)) and the OSBP MCS reporter mCherry-OSBPHHK>AAA. Cargo secretion was initiated by biotin addition. Cells were then kept at 19.5°C for 1.5 h to accumulate cargoes at the TGN. Following this, cells were transferred to 37°C to perform time-lapse live imaging. DMSO or OSW-1 was added to the cells 5 min following the start of the imaging. Intensity values of Golgi areas corresponding to both EGFP and mCherry channels were quantified and plotted. Bold lines with the shaded areas indicate mean ± SEM and indicated P values were calculated by two-way ANOVA followed by Sidak’s multiple comparison test. N = number of independent experiments presented, n = number of cells used to calculate mean and error. The three independent experiments are shown in Fig. S2, A–D. Line plots indicate fluorescence intensities of each corresponding cargo and mCherry-OSBPHHK>AAA at the indicated areas. Bar = 10 μm. (E) MDCK cells were seeded into Matrigel to form cysts for 72 h. Cysts were then treated with SWG overnight. Representative cysts stained for apical/basolateral markers are shown. More cysts are shown in Fig. S2 E. Bar = 10 μm, diameter of the insets = 14.5 μm. (F) MDCK cells stably expressing EGFP were seeded into 12 well plates in low density and left to form cell colonies for 72 h. Following the addition of DMSO or SWG, colonies were analyzed by time-lapse fluorescence imaging. Cell scattering was quantified by measuring the colony perimeters. Each line corresponds to one colony and bold lines with the shaded area indicate mean ± 95% confidence interval. N = number of independent experiments presented, n = number of colonies analyzed. P values were calculated by two-way ANOVA followed by Sidak’s multiple comparison test. Bar = 150 μm.

OSBP inhibition affects apical and basolateral trafficking routes. (A–D) MDCK cells were cotransfected with various RUSH model cargoes (CDH1- (A), DSG2- (B), EGFP-GPI- (C), GP135- (D)) and the OSBP MCS reporter mCherry-OSBPHHK>AAA. Cargo secretion was initiated by biotin addition. Cells were then kept at 19.5°C for 1.5 h to accumulate cargoes at the TGN. Following this, cells were transferred to 37°C to perform time-lapse live imaging. DMSO or OSW-1 was added to the cells 5 min following the start of the imaging. Intensity values of Golgi areas corresponding to both EGFP and mCherry channels were quantified and plotted. Bold lines with the shaded areas indicate mean ± SEM and indicated P values were calculated by two-way ANOVA followed by Sidak’s multiple comparison test. N = number of independent experiments presented, n = number of cells used to calculate mean and error. The three independent experiments are shown in Fig. S2, A–D. Line plots indicate fluorescence intensities of each corresponding cargo and mCherry-OSBPHHK>AAA at the indicated areas. Bar = 10 μm. (E) MDCK cells were seeded into Matrigel to form cysts for 72 h. Cysts were then treated with SWG overnight. Representative cysts stained for apical/basolateral markers are shown. More cysts are shown in Fig. S2 E. Bar = 10 μm, diameter of the insets = 14.5 μm. (F) MDCK cells stably expressing EGFP were seeded into 12 well plates in low density and left to form cell colonies for 72 h. Following the addition of DMSO or SWG, colonies were analyzed by time-lapse fluorescence imaging. Cell scattering was quantified by measuring the colony perimeters. Each line corresponds to one colony and bold lines with the shaded area indicate mean ± 95% confidence interval. N = number of independent experiments presented, n = number of colonies analyzed. P values were calculated by two-way ANOVA followed by Sidak’s multiple comparison test. Bar = 150 μm.

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