Cells were imaged on a Thermo Fisher high-content imaging platform, either a Cellomics Arrayscan Vti or a Cell Insight CX7, using a 40× or a 20×0.6 NA objective. Data were acquired from 250 cells per well, with the smoothened and intensity-thresholded whole cell stain image used to define the cells, using the spot detector or colocalisation bio-application in the Thermo HSC Studio software and sequential acquisition of the three- or four-colour images with multi-line filters. Relevant field average parameters were exported and analysed in Origin software (OriginLab Corporation, Northampton, MA, USA). For most experiments presented, biological replicates were analysed in multiple (usually 4) wells, and the statistics shown represent one-way ANOVA analysis for the average values obtained from these replicates and calculated in Origin software. For the Rab7-GTP staining, a single well with 250 cells was analysed for each cell line and condition (control or TBC1D5 KD). For the Glut1-Lamp1 colocalisation analysis images acquired on a Zeiss inverted microscope were analysed in Volocity software (PerkinElmer, Waltham, MA, USA). Images were intensity-thresholded before colocalisation analysis.
