Figure 3.

A, effect of LY2109761 on L3.6pl cell apoptosis induced by the complete loss of adhesion (anoikis). To mimic the condition of complete loss of adhesion present during metastatic hematogenous dissemination, L3.6pl cells (1.0 × 10⁵ per well) pretreated for 24 h with LY2109761 (5 μmol/L) or with DMSO were trypsinized and incubated in suspension in Costar ultra-low attachment plates (Corning) for 2, 4, or 8 h (detached cells) still in the presence of LY2109761 or DMSO or were trypsinized and analyzed immediately (attached cells). The extent of apoptosis was determined by Annexin V and propidium iodide staining and flow cytometric analysis. Means and 95% confidence intervals of three independent experiments. *, P = 0.0004; #, P <0.0001;†, P =0.0005bytwo-tailedunpairedStudent’s t test.B,dotplotsofL3.6plcellsincubatedinsuspensionfor8hinthepresenceofLY2109761. LY2109761. C, effect of LY2109761 on the activation of Smad2-dependent and Smad2-independent intracellular pathways downstream of TGF-β1 types I and II serine/threonine kinase receptors. L3.6pl/GLT cells were cultured for 24 h in serum-free medium and washed in PBS, and the medium was changed to serum-free medium containing LY2109761 or DMSO (as control). After 24 h, 10% fetal bovine serum, 5 ng/mL TGF-β1, or both were added to the medium. Thirty minutes later, L3.6pl/GLT cells were lysed and the whole-cell lysates were subjected to Western blot analysis for examination of the expression of actin, and total and phosphorylated Smad2, extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun-NH₂-kinase (JNK). Phosphorylated Smad2/Smad2 band density ratios were calculated using the ImageQuant software.