Fig. 5

Mechanism of GFP-WR action. a Fractionation experiments reveal an overall decrease in TWIST1 and GFP protein expression in the cytoplasm as the level of GFP-WR co-expressed in cells increases. TWIST levels also decrease in the nucleus, but GFP-WR is not expressed in the nuclear fraction. This suggests that GFP-WR expression may lead to TWIST1 degradation. Histone H1 and alpha tubulin were used as nuclear and cytoplasmic markers, respectively. b Cycloheximide (CHX) treatment of cells co-transfected with TWIST1 and either GFP or GFP-WR. Left, representative western blot demonstrates more rapid turnover of TWIST1 in the presence of GFP-WR than GFP. Right, quantitation of duplicate experiments. c TWIST1 and GFP levels in the cytoplasmic fraction show a 2-fold increase upon MG132 treatment at 1x dose of GFP-WR (biological duplicate experiments, each condition normalized to its 0 GFP-WR control). d Dual luciferase assay demonstrates that MG132 treatment increases IL-8 driven FFluc expression. Graph represents firefly luciferase expression normalized to renilla luciferase for each condition. Error bars represent standard deviations of biological triplicate experiments. GFP without GFP-WR or MG132 was used as the basis for statistical comparisons. pGL3 lacking the IL-8 promoter was used as a negative control. Error bars, standard deviation. *, p < .05