Fig. 8

PI3K/Akt/mTOR pathway activation and PTEN loss in SN-MM. A Western blots showing RICTOR, PTEN, NDRG1 and pNDRG1(Thr346) expression in SN-MM cell lines cultured in complete RPMI medium; ACTIN and GAPDH are used as housekeeping control. B Immunohistochemistry for PTEN and pNDRG1(Thr346) on parental tumor biopsies (left panels) and pNDRG1(Thr346) on cell blocks (right panels) from SN-MM samples. Lack of PTEN expression is shown in SN-MM2 and SN-MM3 parental tumors; a significant PTEN reduction is observed also in SN-MM5. pNDRG1 is strongly expressed in SN-MM2, SN-MM3 and SN-MM4 cell lines, and correspondingly in SN-MM2 and SN-MM4 parental tumors. Sections are counterstained with haematoxylin. Magnification 200X; scale bar 100 µm. C Viability of SN-MM cell lines treated with the PI3K inhibitor measured by MTS assay. Histograms represent the percentage of viable cells after administration of LY294002 (50 µM) for 24 h, 48 h, and 72 h, relative to untreated SN-MM cells (n = 3–5). Two-way ANOVA statistical analysis and Bonferroni’s multiple comparison post-test have been performed. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. D Western blots showing Akt expression and its phosporylation p-Akt(Ser473) and p-Akt(Thr308) in SN-MM cell lines cultured in complete RPMI medium ± LY294002 (50 µM) for 1 h and 24 h. GAPDH is used as housekeeping control