Quantitation of GLUT4 plasma membrane insertion.
(A) Representative TIRF microscopy images and fluorescence intensity profiles for the indicated line scans (marked in yellow in the merged images) of GLUT4-GFP and Alexa647-anti-myc staining under basal and compound-treated conditions. CHO-K1 hIR/GLUT4-myc-GFP cells were grown over night in 96-well plates (35,000 cells/well), starved for 3 hours in HBSS buffer, stimulated for 10 minutes, fixed with 4% para-formaldehyde, and stained using an Alexa647 labeled anti-myc antibody. Fluorescence in the evanescent field was recorded for GFP and Alexa647 at 488 and 640 nm, respectively. Scale bar = 20 μm. (B) Quantitation of fluorescence signals in stimulated cells with respect to untreated cells (n > 120 cells). Fluorescence was normalized to the values prior stimulation. Error bars are based on the standard error of the mean. ***P < 0.001 and ****P < 0.0001, significant increase with respect to starved cells. (C) Binding probability determined by fluorescence-guided AFM measurements under starved and compound treated conditions. Cells were grown on standard 30 mm glass slides (350,000 cells/slide) over night, starved for 3 hours in HBSS buffer, stimulated with the indicated substances for 10 minutes, and fixed with 4% para-formaldehyde. Error bars are based on the standard error of the mean. ****P < 0.0001, significant increase with respect to starved cells. (D) Linear correlation regression analysis between myc-staining and AFM measurements. (E) AFM mapping experiments of starved, insulin, purslane, ginger, and tindora treated cells, respectively. Force curves were acquired on a surface area of 250 nm x 250 nm with a step size of approximately 7–8 nm. White pixels represent positive unbinding events, whereas black pixels depict no binding.