Fig. 2.

E-cadherin positively impinges EGFR activation. a Left panel: representative western blotting of three performed on lysates from OAW42 and OVCAR5 cells transiently transfected with a control (CO) siRNA or with two E-cadherin siRNAs, separately (E-cadh-1, E-cadh-2) or pooled (E-cadh-1/2). Cells were starved (−) for 24 h and then stimulated with EGF 20 ng/ml (+) for 15 or 30 min (OAW42 and OVCAR 5, respectively). β-actin was used as control for gel loading. Right panel: quantitative evaluation of E-cadherin, EGFR and P-EGFR on E-cadherin silenced cells. The graph reports the ratio between the target protein and β-actin from three different experiments performed on both OAW42 and OVCAR5 cells. b Left panel: western blotting on lysates from MCAs from HG-SOC patient #21 transiently transfected with a control (−) or a pool (+) of E-cadherin siRNAs (E-cadh-1/2). Cells were starved for 24 h and then stimulated with EGF 20 ng/ml overnight (+). Right panel: quantitative evaluation performed as Fig. 1a panel right. c Cell cycle analysis performed on OAW42 cells transiently transfected with a control (Control siRNA) or a pool of E-cadherin siRNAs (E-cadherin siRNA), starved 24 h and then stimulated with EGF 20 ng/ml for 48 h. Western blotting with anti-E-cadherin Ab to evaluate E-cadherin silencing on these experiments are reported in Additional file 2: Figure S2d. d Upper panel: confocal IF on fixed OAW42 cells performed with anti-E-cadherin (cadh, green) and -EGFR (red) Abs. Lower panel: Representative staining with anti-E-cadherin (cadh, green) and anti-EGFR (red) Abs on MCAs from HG-SOC patient #4. Enlarged detail in box is shown in the upper right side of the merge image. Bars, 10 μm. e Upper panel: IP performed with anti-EGFR or -E-cadherin (cadh) Abs on lysates from OAW42 cells. IPs were performed upon transient transfection of non silencing RNA or siRNA for E-cadherin or EGFR followed by IP with anti-EGFR or –E-cadherin, respectively, to test Abs specificity. Upon knockdown of the relevant protein, the complex E-cadherin/EGFR was not formed. Input, total cell lysates; Unbound, protein fraction not immunoprecipitated. Lower panel: IP performed with anti-EGFR Ab on lysates from 3D OVCAR5 and SKOV3 cells. Immunoprecipitated samples were analyzed by western blotting together with the unbound fraction. Immunoblottings were performed with Abs against the proteins reported on the left