IGF1 stimulation enhances endosomal PtdIns(3)P and recruitment of GFP-SGK3 to the endosomes.
HEK293 cells were serum-starved overnight then treated as indicated with or without VPS34-IN1 (1 µM) and/or GDC-0941 (0.5 µM) for 60 min prior to stimulation with 50 ng/ml IGF1 for 15 min. Cells were subsequently fixed with 4% (v/v) paraformaldehyde and stained with recombinant wild-type (WT) PtdIns(3)P-binding probe FYVE domain of HRS[147–223] conjugated to Alexa Fluor 488 conjugate (green channel) (A, C, E and G) or with the mutant non-PtdIns(3)P-binding FYVE domain of HRS[147–223, H176A/H177A] (red channel, Alexa 594). (B, D, F and H). (I) The histogram displays a representative quantitation of the sum fluorescence intensity of PX domain staining on endosomal structures ± SEM (increase in PtdIns(3)P levels upon IGF1 stimulation ranged from 30 to 50% compared with serum-starved cells). (J–Q) GFP-SGK3 knock-in HEK293 cells were serum-starved overnight and treated as above prior to IGF1 stimulation. Cells were 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 (upper panel). SGK3 co-localisation with the early endosomal marker EEA1 was visualised using rabbit anti-EEA1 primary and anti-rabbit Alexa Fluor 594 secondary antibody (lower panel). (R) The histogram displays a representative quantitation of the sum of intensity of fluorescent signal from GFP-SGK3, co-localising with the EEA1 marker at the endosomes ± SEM following the various treatments (increase in GFP-SGK3 levels at the endosomes ranged from 30 to 60% upon the addition of IGF1 compared with serum-starved conditions).