Figure 1.

Visualisation of PI3Kγ in liver and detection of down-stream pAkt signalling. (a,b) Immunoblot analyses of liver extracts from control mice and mice subjected to sepsis by PCI (a) and quantitation of phospho-Akt (pAkt)/Akt levels normalised to sham control (b). (c,d) Anti-pAkt/Akt immunoblotting analyses (c) and quantitative analyses of Hepa1-6 cells stimulated with 100 nM insulin (5 min) and of Hepa1-6 cells stimulated with insulin after 1 h preincubation with the general PI3K inhibitor Wortmannin (WM, 100 nM) and the PI3Kγ inhibitor AS605240 (AS, 1 µM), respectively (d). Note that insulin-mediated pAkt signalling is completely dependent on PI3K and that a considerable portion of this signalling also is dependent on the PI3Kγ isoform in Hepa1-6 cells. (e–g) Anti-PI3Kγ immunofluorescence analysis of liver sections of wild-type (WT) (e) and PI3Kγ KO mice (f) and secondary antibody control (g) of a WT liver section. (h–j) Confocal images of anti-PI3Kγ (shown in red in merges) immunohistostainings of WT liver sections together with i) anti-albumin immunostaining (green in merge h) highlighting hepatocytes (h), with ii) phalloidin staining (shown in green in merge i) highlighting F-actin-rich structures (i), and with iii) an immunostaining for a marker for endothelial cells (CD31; green in merge j) (j). Insets in (i) show enlargements of boxed areas in (i). DAPI in blue in merges. Arrows mark examples of F-actin and PI3Kγ-positive putative canalicular structures (1–2 µm in width). Arrowheads mark examples of perpendicularly and longitudinally cut large structures of about 6 µm in diameter (larger bile ducts or sinusoidal structures). Bars, 20 µm (e–j). Data, mean ± standard error of the mean (SEM). n = 4 each. Kruskal–Wallis + Dunn’s posttest (b); 1way ANOVA + Tukey’s posttest (d). *P < 0.05; ***P < 0.001.