SNB19 cells were pretreated with either DMSO, 2.5 μM clomiphene, or 1 μM toremifene for 1 hour, followed by the addition of VLP-GP1,2 as described in Materials and Methods. The impact of clomiphene or toremifene treatment on trafficking was assessed with confocal microscopy to determine the localization of VLP-GP in relation to LAMP1. (A) Ten random image fields per sample were analyzed with the JACoP plugin on ImageJ. Auto-thresholds were subtracted for each component 8-bit channel. Colocalization of VLPs (red, marked by mCheery-VP40) with LAMP1 (green) is reported as the average Mander’s overlap coefficient. Error bars represent SD. (B) Representative images (enlarged regions from an image field) from each treatment are shown with the green channel thresholds raised to 255 to better reveal areas of colocalization. This adjustment was made uniformly in all three images. White arrows indicate examples of colocalization.