PARP inhibition inhibits the acquisition of an EMT phenotype in malignant melanoma cells.

<p>Human melanoma G361 cells and murine B16- F10 melanoma cells (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003531#pgen.1003531.s003" target="_blank">Figure S3</a>) were used for these experiments. Cells were treated with either DPQ (40 µM), PJ-34 (10 µM) or KU0058948 (100 nM) for 22 hours. IF, western blot or qPCR assays were performed to evaluate the effects of PARP inhibition on EMT markers. PARP inhibition reduced the expression of vimentin and Snail1 and up-regulated E-cadherin in human melanoma cells as determined by immunoblotting (<b>A</b>), indirect immunofluorescence (<b>B</b>), and mRNA levels (<b>C</b>). (*<i>P<</i>0.05, ***<i>P<</i>0.001, PARP Inhibitor groups <i>versus</i> the control). β-actin was used as an internal control for protein loading. (<b>D</b>) Snail1 and E-cadherin promoter activity are regulated by PARP inhibitors. Luciferase activity was determined after transfecting the constructions into G361 cells. Firefly Luciferase was standarized to the levels of Renilla Luciferase. Cells were cotransfected with 0.5 µg renilla as a transfection control and 0.5 µg of Snail1 or E-cadherin using jetPEI cationic polymer transfection reagent according to the manufacturer's instructions. Cells were compared in the presence or absence of serum (***<i>P<</i>0.001 control <i>versus</i> PJ-34). The expression of both Firefly and Renilla luciferase was analyzed 48 h after transfection. Cloning of the human Snail1 promoter (−869/+59) into pGL3 basic (Promega) was described previously (41). The E-Cadherin promoter was cloned into pGL3-basic (Promega) to generate pGL3-E-cadherin (−178/+92). (<b>E</b>) Inhibitory effect of PARP on B16F10 motility. Treatment with the PARP inhibitor PJ-34 (10 µM) decreased cell migration in vitro. Migration was quantified as distance between Wound Healing limits (*** <i>P<</i>0.001 control <i>versus</i> DPQ).