Journal: Breast Cancer Research : BCR

Article Title: Frequent PTEN genomic alterations and activated phosphatidylinositol 3-kinase pathway in basal-like breast cancer cells

doi: 10.1186/bcr2204

Figure Lengend Snippet: Up-regulated phosphatidylinositol 3-kinase (PI3K) signalling pathway in human basal-like breast cancers . Akt is activated in basal-like carcinomas (BLCs). The expression of (a) total Akt and (b) phosphorylated/active form of Akt (phospho-Akt (S473)) were measured by reverse phase protein array (RPPA) as well as Akt activity determined as the (c) ratio 'phospho/total' in human BLCs and human epidermal growth factor receptor overexpressing (HER2+) carcinomas. mTOR is activated in BLCs. Box plots show the (d) expression of mTOR and (e) its form phosphorylated via the PI3 kinase/Akt signalling pathway (phospho-mTOR (S2448)) determined by Western blotting as well as (f) mTOR activity (phospho/total ratio) in human BLCs and HER2+ carcinomas. Outliers are shown for BLCs (solid circles) and HER2+ carcinomas (open circles). The y axes represent logarithmic transformed relative quantifications. p values (* p < 0.05) are represented (Mann-Whitney test). Data are representative of four and two separate experiments for RPPA and Western-blot, respectively.

Article Snippet: Akt phospho-Akt (S473), PTEN and stathmin antibodies (Cell Signaling Technology, Ozyme, Saint Quentin en Yveline, France) were utilised at a dilution of 1:1000, 1:250, 1:200 and 1:100, respectively.

Techniques: Expressing, Protein Array, Activity Assay, Western Blot, Transformation Assay, MANN-WHITNEY

Journal: Breast Cancer Research : BCR

Article Title: Frequent PTEN genomic alterations and activated phosphatidylinositol 3-kinase pathway in basal-like breast cancer cells

doi: 10.1186/bcr2204

Figure Lengend Snippet: Phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitors inhibit basal-like cell line proliferation whereas apoptosis is induced only by PI3K inhibition . (a) Akt activation is associated with low/lack of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression in human basal-like cell lines. The expression of Akt, phospho-Akt (S473) and PTEN were analysed by Western blotting in four basal-like (BT20, HCC38, HCC1937 and MDA-MB-468), one human epidermal growth factor receptor overexpressing (HER2+) (SKBr3) and one luminal (MDA-MB-453) human breast cell lines as well as in epidermal growth factor stimulated (EGF) or not (-) A431 cells. (b) PI3K and mTOR inhibition induce cell growth arrest of basal-like cell lines. BT20 (blue triangle), HCC1937 (red square) and MDA-MB-468 (green diamond) cells were exposed continuously for seven days to increasing concentrations of LY294002 (upper panel) or rapamycin (lower panel). Growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) dye conversion and presented as the percentage of control cell growth inhibition obtained from DMSO-treated cells. The x axes represent logarithmic transformed concentration of drugs. (c,d) The inhibition of PI3K, but not mTOR, induces apoptosis in basal-like cell lines. BT20, HCC1937 and MDA-MB-468 were exposed to varying concentrations of LY294002 (0 to 100 μM) or rapamycin (0 to 100 nM) for 24 hours and apoptosis was detected by measuring (c) caspase3/7 activity and the (d) cleavage of PARP (cPARP). (c) Caspase 3/7 activity was normalised by caspase 3/7 activity from vehicle-treated cells. (d) Actin was used as a loading control. The data represented the (b,c) average of three separate experiments performed in triplicates or are representative of (a,d) three separate experiments. Error bars represent standard deviation and p values (** p < 0.01, *** p < 0.001) were calculated by using Student's t test.

Article Snippet: Akt phospho-Akt (S473), PTEN and stathmin antibodies (Cell Signaling Technology, Ozyme, Saint Quentin en Yveline, France) were utilised at a dilution of 1:1000, 1:250, 1:200 and 1:100, respectively.

Techniques: Inhibition, Activation Assay, Expressing, Western Blot, Transformation Assay, Concentration Assay, Activity Assay, Standard Deviation

Journal: PLoS ONE

Article Title: Hepatic Glucose Intolerance Precedes Hepatic Steatosis in the Male Aromatase Knockout (ArKO) Mouse

doi: 10.1371/journal.pone.0087230

Figure Lengend Snippet: Protein phosphorylation analyses of Akt and AMPK levels were performed on protein extracted from insulin stimulated ( a ) gonadal adipose tissue, ( b ) gastrocnemius muscle, ( c ) liver in insulin stimulated liver of 6 month-old male wildtype (WT) and aromatase knockout (KO) and 2.5 µg/day estrogen-replaced KO (KOE) mice. Expression data from 6–8 samples per genotype are shown, and presented from replicate analysis as the mean ± SD. *p<0.05, **p<0.01, ***p<0.001 versus expression in age-matched WT samples and # p<0.05, ### p<0.001 versus KO samples.

Article Snippet: Primary antibodies diluted in 1% BSA in Tris Buffered Saline with 1% Tween were incubated overnight at 4°C (mouse anti-Phosphor-Akt (Ser473), rabbit anti-Akt, rabbit anti-phosphor-AMPK (Thr172), rabbit anti-AMP alpha (Cell Signaling Technologies, Beverly MA, USA).

Techniques: Knock-Out, Expressing