Figure 4

Silencing of Grb2 in Tpr-Met-IEC-6 cells restores an epithelioid morphology, E-cadherin expression and anoikis sensitivity, while Shc knockdown decreases cell growth. (A) Protein levels of Grb2, Shc and Tpr-Met were assessed by IB analyses of lysates of serum starved Tpr-Met-IEC-6 cell populations stably expressing shRNA against Grb2 (TM-shGrb2) or Shc (TM-shShc), or a control non-targeting shRNA (TM-shCTRL). Densitometry analyses of IB revealed close to 60% reduction in Grb2 and Shc protein levels in TM-shGrb2 and TM-shShc cells, respectively, when compared to TM-shCTRL. (B) Photographs show typical morphology of the indicated shRNA-expressing Tpr-Met-IEC-6 cells when cultured in the presence of serum at either low or high density. (C) E-cadherin protein levels were determined by IB analyses in lysates of serum-starved cells. (D) Densitometric analysis of E-cadherin protein levels normalized to actin was performed. The bar graph shows the mean ± S.D. fold-change relative to TM-shCTRL cells calculated from at least 3 independent experiments. (E) Cell-count assays were performed at the indicated time after seeding the cells under adherent culture conditions and in presence of serum. Bar graph shows the mean number of cells ± S.E.M., from 3 independent experiments performed in triplicate. (F) Anoikis sensitivity of the indicated cell populations was evaluated. Cell viability was measured by XTT assays 18 hours after seeding the cells in suspension or adherent conditions. The bar graph shows in percentage the mean ± S.E.M. cell viability in suspension relative to in adherent state, normalized to TM-shCTRL cells calculated from 3 independent experiments performed in triplicate. (G) The bar graph shows in percentage the mean ± S.E.M. of cell viability in adherent condition expressed relative to Tpr-Met.