Figure 5.

Pellino-1 promotes Slug and Snail stabilization in lung cancer cells. (a) BEAS-2B, H358 and PC9 cells (Pellino-1^Low) were transfected with Myc or Myc-Pellino-1. At 48 h post-transfection, the cells were harvested and subjected to immunoblotting for Pellino-1, E-cadhrin, Snail, Slug and actin. (b) H441, H1833, H1975 and HCC827 cells (Pellino-1^High) were transfected with shLuc, shPellino-1 or 3′-UTR shPellino-1. At 48 h post-transfection, the cells were harvested and subjected to immunoblotting for Pellino-1, E-cadhrin, Snail, Slug and actin. (c) A549 cells were transfected with Myc or Myc-Pellino-1 (WT; 0, 1, 3, 6 μg of plasmid). At 48 h post-transfection, the cells were harvested and subjected to immunoblotting for Myc, Snail, Slug and actin and (d) qRT-PCR analysis for SNAI1 (encoding to Snail) and SNAI2 (encoding to Slug). (e) A549 cells were transfected with Myc or Myc-Pellino-1 (ΔC; 0, 1, 3, 6 μg of plasmid). At 48 h post-transfection, the cells were harvested and subjected to immunoblotting for Myc, Snail, Slug and actin and (f) qRT-PCR analysis for SNAI1 and SNAI2. (g) A549 cells were transfected with shLuc or shPellino-1 and subsequently treated with the proteasome inhibitor, MG132 (10 μM) for 6 h. The cells were subsequently subjected to immunoblotting for Slug, Snail, Pellino-1 and actin. (h) A549 cells were transfected with GFP, GFP-Pellino-1 (FL, ring-like domain deletion (ΔC), ring-like domain (C)), shLuc, or 3′-UTR shPellino-1 plasmids. At 36 h post-transfection, the cells were harvested and immunoprecipitated with indicated antibodies. (i) A549 cells were transfected with Myc or Myc-Pellino-1. At 36 h post-transfection, the cells were treated with cycloheximide (CHX) for indicated times. The cell lysates obtained at each time point were subjected to immunoblotting for Snail, Slug, Myc and actin (top). The results were quantified through scanning densitometry of the Slug or Snail protein level with actin as an internal control (bottom). All data are shown as the means±S.D. of at least three independent experiments. The P-values were calculated using unpaired Student's t-test. *P <0.05; **P <0.01; ***P <0.005