GFP-Vps34 (A–I) or GFP-UV-RAG (J–R) knock-in HEK293 cells were serum-starved overnight and treated as indicated with or without VPS34-IN1 (1 µM) and/or GDC-0941 (0.5 µM) for 60 min prior to stimulation with IGF1 for 15 min. Cells were subsequently fixed with 4% (v/v) paraformaldehyde and GFP distribution was visualised using chicken anti-GFP primary and anti-chicken Alexa Fluor 488 to enhance the GFP signal. Co-localisation of hVPS34 or UV-RAG with an early endosomal EEA1 marker was visualised using rabbit anti-EEA1 primary and anti-rabbit Alexa Fluor 594 secondary antibody. The histograms display quantitation of the sum of intensity of fluorescent signal from GFP-Vps34 (I) or GFP-UV-RAG (R), co-localising with the EEA1 marker ± SEM following the various treatments.