Journal: British Journal of Cancer

Article Title: Disabled-2 downregulation promotes epithelial-to-mesenchymal transition

doi: 10.1038/sj.bjc.6605975

Figure Lengend Snippet: Downregulation of Dab2 leads to constitutive EMT. ( A ) Cell lysates were prepared from untreated or cells treated with 2.5 ng ml −1 TGF β for 48 h followed by western blot analysis for Dab2 expression as described in Materials and Methods. Equal protein loading is demonstrated by western blot analysis for HSP90. Arrows indicate the position of the p96 and p67 Dab2 isoforms; Asterisk indicates a non-specific band. ( B , D ) Detection of Dab2 or E-cadherin by qRT–PCR analysis in untreated and cells treated with 2.5 ng ml −1 TGF β for 48 h was performed as described in Materials and Methods. Quantitation is expressed relative to levels in untreated M1 Neg cells, which is set to 1.0. Shown is the mean expression value ±s.d. from an individual experiment; n =3 independent experiments. ( C ) Upregulation of EMT marker proteins following knockdown of Dab2. Cell lysates from unstimulated or cells treated with 2.5 ng ml −1 TGF β for 48 h were subjected to western blot analysis for N-cadherin (Ncad) and vimentin expression as described in Materials and Methods. HSP90 expression by western blot analysis demonstrates equal protein loading ( n =3 independent experiments).

Article Snippet: To further extend these observations, we probed the relative mRNA abundance of the p96 and p67 isoforms of Dab2 in 12 normal human breast tissue and 12 breast cancer samples using the Human Breast Cancer Rapid-Scan Panel (Origene).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Quantitation Assay, Marker

Journal: British Journal of Cancer

Article Title: Disabled-2 downregulation promotes epithelial-to-mesenchymal transition

doi: 10.1038/sj.bjc.6605975

Figure Lengend Snippet: Downregulation of Dab2 in HME5-cdk4 (K4) cells results in a constitutive EMT phenotype. ( A , B ) Cell lysates from unstimulated cells or cells treated with 2.5 ng ml −1 TGF β for 48 h were subjected to western blot analysis for Dab2, N-cadherin (Ncad) and vimentin expression as described in Materials and Methods. HSP90 expression by western blot analysis demonstrates equal protein loading ( n =3 independent experiments). Arrow indicates the position of the p96 Dab2 isoform; Asterisk indicates a non-specific band. ( C ) K4 Neg, K4 599 and K4 1059 cells were untreated or stimulated with TGF β (2.5 ng ml −1 ) for 48 h and subjected to western blot analysis for phospho-ERK1/2 (pERK1/2) and total ERK2 expression as described in Materials and Methods. ( D ) Elevated expression levels of TGF β isoforms exhibited by Dab2 knockdown cells. Transforming growth factor β 1, TGF β 2 and TGF β 3 mRNA expression was determined by qRT–PCR analysis in untreated K4 Neg and shDab2-expressing cells as described in Materials and Methods. Shown is mean±s.d. from assays performed in triplicate with expression in K4 Neg set to 1.0. ( n =3 independent experiments).

Article Snippet: To further extend these observations, we probed the relative mRNA abundance of the p96 and p67 isoforms of Dab2 in 12 normal human breast tissue and 12 breast cancer samples using the Human Breast Cancer Rapid-Scan Panel (Origene).

Techniques: Western Blot, Expressing, Quantitative RT-PCR

Journal: British Journal of Cancer

Article Title: Disabled-2 downregulation promotes epithelial-to-mesenchymal transition

doi: 10.1038/sj.bjc.6605975

Figure Lengend Snippet: Loss of Dab2 facilitates EMT. In the basal state, cytosolic Grb2 can be found in association with Dab2 or Sos. Loss of Dab2 expression, commonly observed in breast cancer, is postulated to increase the proportion of Grb2 associated with Sos, resulting in Ras activation (RAS * ), stimulation of MAPK signalling and phosphorylation of ERK (pERK). Activation of ERK stimulates increased transcription of the TGF β isoforms, in particular the TGF β 2 isoform. Increased TGF β stimulation induces transient EMT, and also initiates establishment of an autocrine TGF β signalling loop leading to a stable EMT phenotype exemplified by increased expression of EMT markers. Inhibition of TGF β signalling with SB431542 or MAPK signalling with U0126 decreases TGF β production and causes partial reversion of the EMT phenotype.

Article Snippet: To further extend these observations, we probed the relative mRNA abundance of the p96 and p67 isoforms of Dab2 in 12 normal human breast tissue and 12 breast cancer samples using the Human Breast Cancer Rapid-Scan Panel (Origene).

Techniques: Expressing, Activation Assay, Inhibition

Journal: Theranostics

Article Title: RNA interference screens discover proteases as synthetic lethal partners of PI3K inhibition in breast cancer cells

doi: 10.7150/thno.68299

Figure Lengend Snippet: Metap1, Metap2 or Usp7 knockdown sensitize murine breast cancer cells to PI3K inhibition. A : MTT viability as mean ± SD (n ≥ 3) of Dox treated (total 5 days) to uninduced, untreated controls of the BKM MTT viability assays in B. B : Log2 fold change (Fc) of BKM EC20 and EC50 values of protease targeted (Dox treated) PyB6-TA (B6), PyMG-TA (MG) or NMuMG/E9-TA (NMU) cells relative to uninduced cells based on MTT viability assay dose-response curves. Data presented as fold change of EC means (n ≥ 3). MiR-E expression was pre-induced for 3 days prior inhibitor treatment for 48 h. EC: effective concentration. C-E : Competitive growth flow cytometry assays. C : General setup. Flow cytometry-based analysis of competitive cell growth upon combined protease targeting and PI3K inhibition in miR-E-transduced PyB6-TA breast cancer cells mixed with non-fluorescent PyB6-TA controls cells. Cells cultured for 8 days treated as indicated. D : Representative flow cytometry histograms for miR-E-Usp7-2-transduced PyB6-TA cells mixed with non-fluorescent PyB6-TA cells treated with BEZ EC20 ± Dox. Fluorescent cells from living, single cells. Gates in %. E : Effect of combined protease targeting and PI3K inhibition on competitive cell growth. Circle radius: Mean depletion of fluorescent cells relative to DMSO-only or PI3K-inhibitor-only treated cells in %. Circle color: significance of circle radius; one-sample t-test to 0 (n = 5-6). Table with all mean values ± SD in Supplementary . For simplification miR-Es are indicated by sh. Note that the EC values were determined in 48 h MTT assays.

Article Snippet: Metap2; rabbit , 1:1000 , 12547; Cell signaling.

Techniques: Knockdown, Inhibition, MTT Viability Assay, Expressing, Concentration Assay, Flow Cytometry, Cell Culture

Journal: Theranostics

Article Title: RNA interference screens discover proteases as synthetic lethal partners of PI3K inhibition in breast cancer cells

doi: 10.7150/thno.68299

Figure Lengend Snippet: Primary antibodies for western blot

Article Snippet: Metap2; rabbit , 1:1000 , 12547; Cell signaling.

Techniques: Western Blot