Fig. 7. Effect of KHC^mut on glucose-stimulated human growth hormone (hGH) release from MIN6 and INS-1 cells. Cells were co-transfected with 0.5 µg hGH-encoding plasmid pXGH5 together with 1 µg KHC^mut-pAdTrack-CMV or the corresponding empty vector pAdTrack-CMV. (A) For immunocytochemistry, cells were fixed and co-stained with guinea-pig ployclonal anti-insulin (1:500) and mouse monoclonal anti-hGH (1:150) antibodies and then visualised with Alexa 488 goat anti-guinea pig and Alexa 568 goat anti-mouse secondary antibodies. (a) Alexa 488 and (b) Alexa 568 fluorescence (488 and 568 nm excitations, respectively). (c) Overlay of a and b. Overlap appears as yellow. (d-f) High magnification of boxed regions in a-c. Bars, 2.5 µm (a-c); or 0.4 µm (d-f). (B,C) For hGH assay, cells were cultured for 2 days in complete growth medium and then starved in DMEM medium containing 3 mM glucose 12 hours before stimulation. INS-1 (B) or MIN6 (C) cells were incubated first in the presence of 3 mM glucose for 20 minutes and then at 16 mM or 30 mM glucose for 20 or 90 minutes. Following stimulation, the cells were lysed in 0.5% Triton X-100 and assayed for total hGH using a colorimetric sandwich ELISA method. hGH release was expressed as a percentage of the total hGH and was compared with values obtained with the empty vector (pAdTrack-CMV) at 3 mM glucose. Number of transfections for each condition are indicated in the columns. ^**P<0.001 compared with basal (empty vector at 3 mM glucose).