Cellular cholesterol manipulation does not interfere with CD4 conventional secretion from cells. (A and B) CD4-GFP translocation efficiency under conditions of either increased (A) or decreased (B) cellular cholesterol levels. CHO cells stably expressing CD4-GFP in a doxycycline-dependent manner were imaged by single molecule TIRF microscopy as described previously (Dimou et al., 2019). Following treatment with either Cholesterol:Methyl-β-Cyclodextrin to increase cellular cholesterol levels (A), or 5 μM mevastatin and 50 μM mevalonate in the presence of de-lipidized serum to decrease cholesterol levels (Cheng et al., 2006; B), cells were incubated on ice for 30 min with Alexa-Fluor-647-labeled anti-GFP nanobodies (for details, see Materials and methods). For each condition, a widefield image and the corresponding TIRF image is shown (A and B, subpanel a; Scale bar = 6 μm). Quantification of CD4-GFP transport to the plasma membrane in CHO cells for all conditions indicated is shown in subpanel b of both A and B. Single nanobody particles were analyzed using the Fiji plugin TrackMate (Tinevez et al., 2017). The number of nanobody particles detected per cell was normalized for the surface area of the corresponding cell. The mean values for each condition are shown in brackets, with the mock condition set to 1. Data are shown as mean ± SD (n = 4). The statistical analysis was based on a two-tailed unpaired t test. Data distribution was assumed to be normal, but this was not formally tested.