Figure 8.

OSF-2 stimulates the Akt/PKB pathway via integrin-dependent PI3-Kinase activation. (A,B) β1-integrin, but not α5 integrin is induced by the addition of rOSF-2 (10, 100 ng/mL). Integrin receptor expression was analyzed by Western blot in HNSCCUM-02T cells. Actin served as the loading control. Representative results (n = 2) are shown. Densitometric analysis of detected bands is shown in (B). (C) Specific phosphorylation of Akt1/PKBα on Ser473 was detected in HNSCCUM-02T cells. Insulin and serum-containing medium (+FCS) served as positive, serum deprivation (−FCS), and PI3K-inhibitor (LY294002; +FCS) treatment as negative controls for staining. Scale bar, 5 µm. (D) Quantification of p-Akt positive cells. At least 200 cells from three separate images were examined, visually inspected, and counted as p-Akt positive or negative. Ratio of p-Akt positive cells (= #pAkt positive/all counted cells) is shown for indicated treatments. All treatments, except PI3K-inhibitor, and +FCS, were performed under serum starvation conditions. (E) Akt1/PKBα phosphorylation was confirmed by Western blot analysis using HNSCCUM-02T cells. Akt1/PKBα phosphorylation was induced by recombinant OSF-2 (rOSF-2) and conditioned medium under serum-free conditions. OSF-2-induced phosphorylation was prevented by PI3K inhibition. Actin served as the loading control. Representative results (n = 2) are shown. (F) HNC cancer cell (Fadu) survival was reduced after treatment with r-OSF-2 and a PI3K-inhibitor (LY294002). ns: p > 0.05; * p ≤ 0.05. The uncropped western blot figures were presented in Figure S13.