OSBP targeting affects surface proteome dynamics. (A) MDCK cells were treated with SWG for 0, 6, and 12 h. Surface proteins were then labeled and subsequently analyzed using quantitative mass spectrometry. Heat map showing the temporal dynamics of proteins that displayed a significant change in abundance on the cell surface at least at one time point compared to time 0. 4-color coding indicates k-means clusters. (B) K-means clusters show the temporal dynamics of each cluster. Red lines indicate cluster centroids. The bar plot indicates the distribution of the significantly changed surface proteins across the four k-means clusters. (C) Surface proteins with highly basolateral and apical localizations were highlighted among the SWG-perturbed proteins. The distribution of these hits indicates that most of the highly basolateral cargoes are grouped in cluster 1, whereas the majority of the apical proteins were gathered in cluster 3. (D–F) MDCK cells stability expressing βGalT1-BFP were treated with ORPphilins for 4 h and endogenous E-cadherin was immunolabeled and observed by confocal microscopy (D). Superplots showing E-cadherin enrichments in the plasma membrane (E) and in the TGN (F) upon ORPphilin treatments. N = number of independent experiments to calculate means (filled circles). Means were compared by one-way ANOVA with Dunnett’s post hoc test. Bar = 20 μm. (G and H) Confluent MDCK cells were kept in a calcium-free medium overnight and then placed in a regular culture medium in the absence or presence of ORPphilins. Cells were allowed to recover cell–cell junctions for 2 h, fixed, immunolabeled for endogenous E-cadherin and then imaged by confocal microscopy. N = number of independent experiments to calculate means (filled circles). Means were compared by one-way ANOVA with Dunnett’s post hoc test. Bar = 20 μm.