Figure S1.
Effects of PI4KIIIβ inhibition and OSBP silencing on cargo sorting, trafficking, and cyst morphology. (A) MDCK cells were transfected with mCherry-OSBP-expressing plasmid and mounted for epifluorescence time-lapse microscopy the next day. PIK93 was added to the cells for 5 min at 500 nM concentration. PIK93 treatment reduced the TGN-associated OSBP pool by 75%. N = number of independent experiments presented, n = number of cells analyzed. Bar = 10 μm. (B) Additional images showing the effect of PIK93 treatment on MDCK cyst morphology. (C) MDCK cells were nucleofected with mCherry-OSBP-expressing plasmid and time-lapse fluorescence imaging was performed on the next day. PI4KIIIbeta-IN-10 was added to the cells after 5 min at 100 nM concentration. PI4KIIIbeta-IN-10 reduced the TGN-associated Golgi pool by ∼70%. N = number of independent experiments presented, n = number of cells analyzed. Bar = 10 μm. (D and E) MDCK cells were transfected with basolateral CDH1 and apical EGFP-GPI RUSH model cargo-expressing constructs. 24 h later, biotin was added to the samples, and then cells were kept at 19.5°C for 1.5 h to synchronize cargoes at the TGN. Following this, cells were kept at 37°C for 40 min in the absence or presence of 100 nM PI4KIIIbeta-IN-10. After the treatment, cells were fixed, then confocal microscopy was performed. Line-plots indicate fluorescence intensities of each cargo at the corresponding areas and arrowheads point to post-Golgi vesicles positive for single or double cargoes. (E) Distribution of each single- and double cargo-containing post-Golgi vesicles in control and PI4KIIIbeta-IN-10-treated cells. At least 9 cells/condition were quantified; in each cell, at least 30 post-Golgi vesicles were counted. N = number of independent experiments presented, n = number of cells/sample used for the analysis. Error bars show SEM and P values were calculated by two-way ANOVA followed by Sidak’s multiple comparison test. Bar = 10 μm, diameter of the insets = 9 μm. (F and G) MDCK cells were seeded into Matrigel matrix and cysts were left to develop for 72 h in the absence or the presence of 25 nM PI4KIIIbeta-IN-10. (I) The number of cysts with luminal defects increased upon PI4KIIIbeta-IN-10 treatment. N = number of independent experiments. P value was calculated by unpaired t test. Bar = 10 μm, diameter of the insets = 14.5 μm. (H) Western blot analysis to test the efficacy of OSBP silencing following transfection with RUSH cargoes. (I) Results of three independent RUSH experiments using CDH1 and EGFP-GPI model cargoes upon OSBP silencing. Thin lines indicate individual cells. Bold lines correspond to means, while shaded areas indicate SEM. n = number of cells analyzed/experiment to obtain mean and SEM. P values were calculated by two-way ANOVA followed by Sidak’s multiple comparison test. ‡ indicates the representative experiments shown in Fig. 1, J and K, respectively. (J) Additional cysts to show the effect of OSBP silencing on MDCK cyst development. Bar = 10 μm. Source data are available for this figure: SourceData FS1.

Effects of PI4KIIIβ inhibition and OSBP silencing on cargo sorting, trafficking, and cyst morphology. (A) MDCK cells were transfected with mCherry-OSBP-expressing plasmid and mounted for epifluorescence time-lapse microscopy the next day. PIK93 was added to the cells for 5 min at 500 nM concentration. PIK93 treatment reduced the TGN-associated OSBP pool by 75%. N = number of independent experiments presented, n = number of cells analyzed. Bar = 10 μm. (B) Additional images showing the effect of PIK93 treatment on MDCK cyst morphology. (C) MDCK cells were nucleofected with mCherry-OSBP-expressing plasmid and time-lapse fluorescence imaging was performed on the next day. PI4KIIIbeta-IN-10 was added to the cells after 5 min at 100 nM concentration. PI4KIIIbeta-IN-10 reduced the TGN-associated Golgi pool by ∼70%. N = number of independent experiments presented, n = number of cells analyzed. Bar = 10 μm. (D and E) MDCK cells were transfected with basolateral CDH1 and apical EGFP-GPI RUSH model cargo-expressing constructs. 24 h later, biotin was added to the samples, and then cells were kept at 19.5°C for 1.5 h to synchronize cargoes at the TGN. Following this, cells were kept at 37°C for 40 min in the absence or presence of 100 nM PI4KIIIbeta-IN-10. After the treatment, cells were fixed, then confocal microscopy was performed. Line-plots indicate fluorescence intensities of each cargo at the corresponding areas and arrowheads point to post-Golgi vesicles positive for single or double cargoes. (E) Distribution of each single- and double cargo-containing post-Golgi vesicles in control and PI4KIIIbeta-IN-10-treated cells. At least 9 cells/condition were quantified; in each cell, at least 30 post-Golgi vesicles were counted. N = number of independent experiments presented, n = number of cells/sample used for the analysis. Error bars show SEM and P values were calculated by two-way ANOVA followed by Sidak’s multiple comparison test. Bar = 10 μm, diameter of the insets = 9 μm. (F and G) MDCK cells were seeded into Matrigel matrix and cysts were left to develop for 72 h in the absence or the presence of 25 nM PI4KIIIbeta-IN-10. (I) The number of cysts with luminal defects increased upon PI4KIIIbeta-IN-10 treatment. N = number of independent experiments. P value was calculated by unpaired t test. Bar = 10 μm, diameter of the insets = 14.5 μm. (H) Western blot analysis to test the efficacy of OSBP silencing following transfection with RUSH cargoes. (I) Results of three independent RUSH experiments using CDH1 and EGFP-GPI model cargoes upon OSBP silencing. Thin lines indicate individual cells. Bold lines correspond to means, while shaded areas indicate SEM. n = number of cells analyzed/experiment to obtain mean and SEM. P values were calculated by two-way ANOVA followed by Sidak’s multiple comparison test. ‡ indicates the representative experiments shown in Fig. 1, J and K, respectively. (J) Additional cysts to show the effect of OSBP silencing on MDCK cyst development. Bar = 10 μm. Source data are available for this figure: SourceData FS1.

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