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. 2014 Oct 10;463(Pt 3):413–427. doi: 10.1042/BJ20140889

Figure 4. VPS34-IN1 rapidly reduces GFP–2×FYVEHrs probe localization on endosomes.

Figure 4

(A) The U2OS cell line stably expressing GFP–2×FYVEHrs was recorded for approximately 1 min, before adding either no inhibitor (top panel), 1 μM VPS34-IN1 (second panel), 0.5 μM GDC-0941 (third panel) or a combination of 1 μM VPS34-IN1 and 0.5 μM GDC-0941 (bottom panel). Images were taken starting at 1.5 min from the time that the inhibitor was added (the first time point we could reliably measure) and subsequently at 0.5 min intervals up to a period of 1 h. Time-lapse microscopy was performed on Zeiss 710 microscope using ×63 objective. Similar results were obtained in at least two separate experiments. Scale bar, 20 μm. (B) As in (A) except the U2OS cell line stably expressing GFP–PHAkt1 was starved of serum overnight and treated with either no inhibitor (left panel), 1 μM VPS34-IN1 (middle panel) or 0.5 μM GDC-0941 (right panel) for 1 h before stimulation with IGF (100 ng/ml for 15 min). The cells were fixed with 4% paraformaldehyde and images were taken using Zeiss 710 microscope at ×63 objective. Representative images are shown and similar results were obtained in two experiments. Scale bar, 20 μm.