Figure 6
From: TGFβ engages MEK/ERK to differentially regulate benign and malignant pancreas cell function
pERK antagonizes TGFβ-induced CDK2/p21 association and is required for TGFβ-induced EMT in PANC1 cells. (a) PANC1, BXPC3, and ASPC1 cancer cells were pulsed with with 5–10ng/ml exogenous TGFβ1 and levels of pERK evaluated after 30 min. (b) PANC1 cells were pre-incubated with U0126 prior to TGFβ-treatment, and downstream signals evaluated by western blotting. (c) PanIN cells were again pre-incubated with U0126 prior to TGFβ-treatment and TGFBR1 isolated by immunoprecipitation. The association betweem TGFBR1 and pERK was then assessed by western blotting. (d) 24 h following administration of U0126 and/or TGFβ1, the association between p21and CDK2 was assessed by immunoprecipitation. (e) PANC1 cells were again starved of growth supplement and pre-incubated with U0126 prior to TGFβ-treatment. Cells were fixed after 72 h and evaluated by immunocytochemistry for SMAD4. (f) Cells were dual-stained for p21 and pERK. (g) Cells were next evaluated for the proliferation surrogate PCNA and the p21 target CDK2. (h and i) PANC1 cells were then either assessed by phase microscopy for changes in morphology, or stained for the epithelial marker E-Cadherin and the mesenchymal marker Vimentin, indicating that pERK is necessary for TGFβ-induced EMT.
