Journal: PLoS ONE

Article Title: WAVE3-NFκB Interplay Is Essential for the Survival and Invasion of Cancer Cells

doi: 10.1371/journal.pone.0110627

Figure Lengend Snippet: Representative histograms using flow cytometry of control shRNA (ctrl-sh, green)- or sh-WAVE3-expressing (red) MDA-MB-231 cells after TNFα treatment stained by Annexin V for apoptosis (A) and by Propidium Iodide for cell death (B). (C) Representative confocal images of Ctrl-sh and sh-W3 MDA-MB-231 cells stained Annexin V (Green) and cleaved caspase3 (Red) before and after TNFα treatment (50 ng/μl for 15 min). The bright field images in the right panels indicate healthy cells. High resolution enlarged images are shown in the insets. (D & E) Quantification of Annexin V staining levels (D) and Caspase 3 staining levels. (F) Western blot analysis with the indicated antibodies of cell lysates form the Ctrl-sh and sh-W3 cells after treatment with TNFα at the indicated times. The numbers below the p-AKT and the p-p38 panels indicate their respective fold change with respect to the untreated Ctrl-sh cells. All data are representative of 3 independent experiments, or are the mean (±SE; n = 3; *, p <0.05; Student's t-test).

Article Snippet: Rabbit anti-WAVE3 (1∶1000), rabbit anti-MMP9 (1∶1000), rabbit anti-MMP2 (1∶1000), rabbit anti-p38 (1∶1000), rabbit anti phospho p38 (1∶1000), rabbit phospho ERK (1∶1000), rabbit ERK (1∶1000), rabbit anti phospho AKT (Ser473; 1∶1000), rabbit anti panAKT (1∶1000), rabbit anti Phospho JNK (1∶1000), rabbit anti-JNK (1∶1000), mouse anti-phospho p65 (Ser536) (1∶1000), rabbit anti-Cortatcin are from Cell Signaling Technologies. (Danvers, MA); mouse anti-GFP, mouse anti-WAVE2 (1∶3000), mouse anti-WAVE1 (1∶3000) are from Santa Cruz Biotechnology Inc (Santa Cruz, CA); rabbit anti-NFκB p65 (1∶5000) from Biolegend (San Diego, CA), rabbit anti NAP1 (1∶2000), rabbit anti ABI1 (1∶5000), rabbit anti CYFIP2 (1∶3000), mouse anti-actin (1∶5000), rabbit anti-Myc (1∶1000) are from Sigma-Aldrich (St. Louis, MO); goat horseradish peroxidase-conjugated anti-mouse IgG (1∶5000) and goat horseradish peroxidase-conjugated anti-rabbit IgG (1∶5000) from Calbiochem; and Alexa 488-conjugated anti-rabbit IgG and Alexa 568-conjugated anti-mouse IgG, Alexa 568-conjugated phalloidin are from Invitrogen.

Techniques: Flow Cytometry, shRNA, Expressing, Staining, Western Blot

Journal: PLoS ONE

Article Title: Prognostic Significance of Erythropoietin in Pancreatic Adenocarcinoma

doi: 10.1371/journal.pone.0023151

Figure Lengend Snippet: Primary antibodies.

Article Snippet: Phospho-Akt (ser473) , Rabbit polyclonal , IB, 1∶1000 , Cell Signaling Technology, Inc., Danvers, MA,USA.

Techniques:

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: EGFR (clone 31G7, 1:40 dilution, Zymed, Invitrogen, Cergy-Pontoise, France), CK5/6 (clone D5/16B4, 1:50 dilution, Dako, Trappes, France), CK14 (clone LL002, prediluted, Biogenex, San Ramon, CA, USA) and phospho-Akt (S473) (clone 736E11, 1:50 dilution, Cell Signaling Technology, Ozyme, Saint Quentin en Yveline, France) antibodies were used.

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: EGFR (clone 31G7, 1:40 dilution, Zymed, Invitrogen, Cergy-Pontoise, France), CK5/6 (clone D5/16B4, 1:50 dilution, Dako, Trappes, France), CK14 (clone LL002, prediluted, Biogenex, San Ramon, CA, USA) and phospho-Akt (S473) (clone 736E11, 1:50 dilution, Cell Signaling Technology, Ozyme, Saint Quentin en Yveline, France) antibodies were used.

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

Journal: Radiation Oncology (London, England)

Article Title: NVP-BEZ235 and NVP-BGT226, dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitors, enhance tumor and endothelial cell radiosensitivity

doi: 10.1186/1748-717X-7-48

Figure Lengend Snippet: BGT226 and BEZ235 attenuate oncogenic signaling and reduce clonogenic survival after radiation . SQ20B cells were exposed to the indicated drugs for 1 h and evaluated by Western blotting for phosphorylation of mTOR (Ser2448), Akt (Ser-473) and S6 protein (ser-235/236). β-actin was used as a loading control. A , Dose response of the PI3K/mTOR pathway in SQ20B cells after 1 h exposure to the indicated drug. B , Time-course of PI3K/mTOR inhibition to indicated drugs at maximal effective doses. C-D , Response of PI3K/mTOR pathway 1 h after 4 Gy. Cells were treated with 5 nmol/L BGT226 (C) or 50 nmol/L BEZ235 (D) for 1 h before and 1 h after irradiation. E , Plating efficiency of SQ20B,T24 and FaDu cells after exposure to indicated drugs for 1 h before up to 17 h post-irradiation (n = 3). F , Clonogenic survival of indicated cell lines after 18 h treatment with BEZ235 (50 nmol/L) and BGT226 (5 nmol/L), as described in "Methods" (n = 3). P < 0.05; **, P < 0.01 over DMSO-treated control.

Article Snippet: Phospho-mTOR (ser-2448), phospho-Akt (ser-473) and phospho-S6 (ser-235/236) primary antibodies (Cell Signaling) were used at 1:1,000 dilution. β-actin clone AC-15 (Sigma) was used at 1:4,000 dilution.

Techniques: Western Blot, Inhibition, Irradiation

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Garlic-Derived S-Allylmercaptocysteine Ameliorates Nonalcoholic Fatty Liver Disease in a Rat Model through Inhibition of Apoptosis and Enhancing Autophagy

doi: 10.1155/2013/642920

Figure Lengend Snippet: Addition of SAMC reduced NAFLD-induced hepatic apoptosis via modulating the LKB1/AMPK and PI3 K/Akt pathways. Protein expressions of phosphorylated and total (a) LKB1, (b) AMPK, (c) PI3 K, and (d) Akt were measured by Western blot and then quantified by ImageJ software. Data presented are expressed as mean ± SEM ( n = 7), and experimental groups marked by different letters represented significant differences between groups at P < 0.05 (Kruskal-Wallis test followed by Dunn's post hoc test). N + S: NAFLD + SAMC cotreatment.

Article Snippet: Antibodies of phosphorylated liver kinase B1 (LKB1) at Ser428, total LKB1, phosphorylated AMP-activated protein kinase (AMPK) at Thr172, total AMPK, phosphorylated p53 at Ser15, total p53, phosphorylated Akt at Ser473, total Akt, total PI3 K (p85 subunit), cytochrome c, TNF-related apoptosis-inducing ligand (TRAIL), Fas, Fas-associated protein with death domain (FADD), cleaved caspase-3, cleaved caspase-8, phosphorylated mTOR at Ser2448, mTOR, beclin 1, Atg12, LC3 II, and p62 were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Software

Journal: International Journal of Nanomedicine

Article Title: Styrene maleic acid-encapsulated RL71 micelles suppress tumor growth in a murine xenograft model of triple negative breast cancer

doi: 10.2147/IJN.S148908

Figure Lengend Snippet: Tumor protein expression levels of EGFR, Akt, mTOR, 4EBP1, and NF-κB following treatment with SMA-RL71. Notes: Tumor-bearing mice were treated with SMA-RL71 (10 mg/kg, iv) or SMA control twice a week for 3 weeks. ( A ) Representative Western blots of the various proteins from individual mice. Scanning densitometry of Western blots of ( B ) EGFR, ( C ) Akt, ( D ) mTOR, ( E ) 4EBP1, and ( F ) NF-κB. Bars represent the mean ± SEM from eight mice per group. Significance was determined with a one-way ANOVA coupled with a Bonferroni post-hoc test. *Significantly different compared to SMA control, p <0.05. Abbreviations: ANOVA, analysis of variance; 4EBP1, 4E-binding protein-1; Akt, protein kinase B; EGFR, epidermal growth factor receptor; mTOR, mechanistic target of rapamycin; NF-κB, nuclear factor-kappa B; RL71, 3,5-bis(3,4,5-trimethoxybenzylidene)-1-methylpiperidine-4-one; p4EBP1, phosphorylated 4EBP1; pAkt, phosphorylated Akt; pEGFR, phosphorylated EGFR; pmTOR, phosphorylated mTOR; SMA, styrene maleic acid.

Article Snippet: 4E-binding protein-1 (4EBP1) phosphorylated 4EBP1 (p4EBP1), mTOR, phosphorylated mTOR (pmTOR), cleaved caspase-3, phosphorylated Akt (pAkt), EGFR, phosphorylated EGFR at amino acid Tyr1148 (pEGFR), NF-κB were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Western Blot, Binding Assay