Arf6 activation or inactivation interferes with E-cadherin expression and localization.

<p>CHO WT, CHO R749W and CHO E757K were transiently transfected with vectors expressing mutant forms of Arf6: ARF6 Q67L (constitutive active form) and ARF6 T27N (dominant negative form). (A) Arf6, E-cadherin and Actin were detected in whole cell lysates by Western Blot. Actin was used as a loading control. The intensity of the bands was quantified and normalized against the transfection control. The intensity average of three independent experiments is shown below the respective sample. (B) Cells were fixed and immunostained with anti-human E-cadherin antibody. Nucleus was counterstained with DAPI. The pictures were taken under a 40× objective. (C) Flow cytometry technique was used to assess E-cadherin cell surface expression. Each histogram represents surface E-cadherin in cells untransfected (yellow), transfected with ARF6 Q67L (blue) or with ARF6 T27N (red) and the transfection control (green). The black area in the histogram represents the cells that were not incubated with primary antibody; this sample was used as negative control. The results are representative of three independent experiments. (D) For each sample, the number of cells expressing surface E-cadherin was calculated. The mean fluorescence intensity was also quantified and normalized against the transfection control of WT expressing cells. The graphs show the average + SE, n = 3 (* represents p≤0.05).</p

DMSO improves E-cadherin interaction with PIPKIγ, β- and p120-catenins.

<p>CHO cells transduced with the empty vector (Mock) or with WT, R749W or E757K hEcadherin were treated with 2% DMSO or with Medium, and the interaction of E-cadherin with PIPKIγ (A), p120- (B) and β-catenins (C) was assessed by PLA. Cells were fixed and incubated with antibodies against E-cadherin and PIPKIγ or E-cadherin and p120 or E-cadherin and β-catenin. In the negative control, CHO WT cells were incubated only with the antibody against E-cadherin. Close proximity of oligonucleotide-ligated secondary antibodies allows a rolling-circle amplification and the detection of the rolling-circle amplification product by a fluorescently labelled probe. Nuclei were counterstained with DAPI. The pictures were taken under a 40× objective. The number of spots per cell was quantified in each condition. The graphs show the average of relative number of blobs per cell + SE, n = 3 (* represents p≤0.05).</p

Arf6 specific inhibition by siRNA leads to an increase of E-cadherin protein expression.

<p>Arf6 inhibition by siRNA was performed in CHO cells stably transduced with WT or E757K hEcadherin. (A) Cell lysates were extracted 48 h after transfection. E-cadherin, Arf6 and Actin were analyzed by Western Blot. Actin was used as a loading control. The intensity of the bands was quantified and normalized against the siRNA control. In the graph, bars represent the average + SE of E-cadherin or Arf6 protein expression of three independent experiments. (B) Flow cytometry technique was used to assess E-cadherin cell surface expression. Each histogram represents the cell surface expression of E-cadherin in WT or E757K cells treated with siRNA control (blue) or with specific siRNA for Arf6 (green). The black area in the histogram represents the cells that were not incubated with primary antibody; this sample was used as negative control. For each sample, the number of cells expressing surface E-cadherin as well as the mean fluorescence intensity (arbitrary units) was calculated. The graphs show the average + SE of three independent experiments (* represents p≤0.05). (C) Cells were fixed 48 h after transfection and immunostained with anti-human E-cadherin antibody. Nucleus was counterstained with DAPI. The pictures were taken under a 40× objective.</p

Endocytosis inhibition increases E-cadherin at the plasma membrane.

<p>CHO cells stably transduced with the empty vector (Mock) or with WT, R749W or E757K hEcadherin were treated with Dynasore or MiTMAB for 17h. (A) Clathrin, E-cadherin and Arf6 expression were analyzed in whole cell lysates by Western Blot. Actin was used as a loading control. The intensity of the bands was quantified and normalized against the untreated WT cells. The graphs show the average + SE of protein level, in three independent experiments. (B) After treatment, Transferrin 594 was added to cells. Nucleus was counterstained with DAPI. The pictures were taken under a 63× objective. (C) Cells were fixed and immunostained with anti-human E-cadherin antibody. Nucleus was counterstained with DAPI. The pictures were taken under a 40× objective. (D) Flow cytometry technique was used to assess E-cadherin cell surface expression. Each histogram represents the cell surface expression of E-cadherin in cells WT, R749W or E757K, treated with Dynasore (red) or MiTMAB (green) or untreated (blue). The black area in the histogram represents the cells that were not incubated with primary antibody, this sample was used as negative control. For each sample, the number of cells expressing surface E-cadherin was calculated. The mean fluorescence intensity was also quantified and normalized against the control of WT expressing cells. The graphs show the average + SE, n = 3 (* represents p≤0.05).</p

Epidermal growth factor receptor structural alterations in gastric cancer-1

<p><b>Copyright information:</b></p><p>Taken from "Epidermal growth factor receptor structural alterations in gastric cancer"</p><p>http://www.biomedcentral.com/1471-2407/8/10</p><p>BMC Cancer 2008;8():10-10.</p><p>Published online 16 Jan 2008</p><p>PMCID:PMC2244615.</p><p></p>ain of EGFR – missense mutation (2300 C>T) in exon 20, leading to the substitution of the Alanine 767 for a Valine. (B) Diffuse gastric carcinoma with EGFR increased copy number caused by chromosome 7 polysomy. (C) Neoplastic cells exhibiting gene amplification with the formation of clusters with numerous signals for EGFR

Epidermal growth factor receptor structural alterations in gastric cancer-0

<p><b>Copyright information:</b></p><p>Taken from "Epidermal growth factor receptor structural alterations in gastric cancer"</p><p>http://www.biomedcentral.com/1471-2407/8/10</p><p>BMC Cancer 2008;8():10-10.</p><p>Published online 16 Jan 2008</p><p>PMCID:PMC2244615.</p><p></p>ain of EGFR – missense mutation (2300 C>T) in exon 20, leading to the substitution of the Alanine 767 for a Valine. (B) Diffuse gastric carcinoma with EGFR increased copy number caused by chromosome 7 polysomy. (C) Neoplastic cells exhibiting gene amplification with the formation of clusters with numerous signals for EGFR