Figure 9

PI3K mediates F-actin disruption in GT1-7 cells A, Cultured GT1-7 cells were incubated in the absence and presence of leptin (10 nM) ± wortmannin (10 nM) or LY 294002 (10 μM) for 30 minutes. Following treatment cells were fixed and permeabilized, as described in the Methods, incubated with Alexa 488 conjugated phalloidin and Alexa 594 conjugated DNase I and subsequently processed for visualising F- and G-actin respectively by confocal microscopy. B, Plot of average alexa 488-phalloidin fluorescence intensity (green) and alexa 594-DNase 1 (red) in fixed cells treated with 10 nM leptin (L) alone or cells treated with leptin and jasplakinolide (100 nM; J + L), LY294002 (10 μM; L + L) or wortmannin (10 nM; W + L) relative to cells untreated (C) with drug (n = 8 separate experiments, with 8 cells measured under each condition for each experiment). C, GT1-7 cells were incubated with PBS only (C), 10 nM leptin (L) or leptin and jasplakinolide (100 nM; J + L), LY294002 (10 μM; L + L) or wortmannin (10 nM; W + L). Cells were treated to extract actin pools as described in Methods and equal amounts of pool lysate were subjected to SDS-PAGE and transferred to nitrocellulose membrane. The levels of actin were detected by immunoblotting with an actin monoclonal antibody. D, Plot of average Triton-X-100 soluble (G, red), Triton-X-100 insoluble (F, green) and total actin (gray) concentration from live cells, relative to control untreated cells (n = 4 separate experiments), for data as shown in C. Error bars indicate s.e.m. and * significance of P < 0.01.