HeLa cells stably expressing mCherry-Rab25 were previously generated within the lab (Teo et al., 2016) and were maintained in DMEM (Gibco) supplemented with 10% (v/v) FCS (Bovogen) and 2 mM L-glutamine (Invitrogen) in a humidified air/atmosphere (5% CO2) at 37°C. Cells were transfected at 70% confluence with pcDNA3.1-N-eGFP plasmid constructs using Lipofectamine 2000 as per manufacturer’s protocol (Invitrogen) and examined 18–24 hr later. The HeLa cell line used in this study was from America Type Culture Collection (#ATCC CCL2). Parental and stable cells lines were negative for mycoplasma by DAPI staining, and authenticated by STR profiling (Cell Bank Australia). For inhibitor treatments, cells were treated with either 100 nM wortmannin or 1 µM Vps34-IN1 for 1 hr.