Figure S1. (S1.a) mRNA levels of Snail and E-cadherin in human melanoma cells (G361) treated 22 hour with the PARP inhibitor, PJ-34. The level of Snail1 mRNA was down-regulated and E-cadherin mRNA was up-regulated after 22 hours of treatment with PJ-34. *P < 0.05 control versus PJ-34. (S1.b) Transcripitonal activities of the E-cadherin and Snail1 promoter. Luciferase activity was determined after transfecting the constructions into theG361 cells. Firefly Luciferase was standardized to the value of RenillaLuciferase. Cells were cotransfected with 0.5 μg Renilla as control of transfectiontogether with 0.5 μg of the Snail1 or E-cadherin plasmid using jetPEI cationicpolymer transfection reagent according to the manufacture´s instructions. Fold:cells with serum vs. cells without serum. ***P < 0.001 control versus PJ-34. Theexpression of Firefly and Renilla luciferases was analyzed 48 h aftertransfection, according of the manufacturer´s instructions. Cloning of thehuman Snail1 promoter (-869/+59) in pGL3 basic (Promega), was describedpreviously (Barbera et al., 2004). E-Cadherin promoter were cloned into pGL3-basic (Promega) to generate pGL3-E-cadh (-178/+92).
Figure S2. (S2.a) Indirect immunofluorescence showing the effect of PARP inhibitionon E-cadherin, Vimentin and Snail1 in G361 cells. G361 cells treated with the PARP inhibitor, PJ-34 or in untreated cells (control). Analysis of endogenous E-cadherin and Snail1 by immunofluorescence assay was performed using a previously published protocol (Dominguez et al., 2003). (S2.b) Western blot showing the subcellular localization of Snail1 after PJ-34 treatment. For subcellular protein extracts (nucleus/cytoplasm), cells were lysed with extraction buffer (10 mM Hepes pH 7.8, 1.5 mM MgCl2, 10 mM KCl and 0.5 mM DTT) and the supernatant was used as cytoplasmic extract fraction and the insoluble pellet as nuclear extract. Cytoplasmic extract was washed with buffer (0.3 M Hepes, 1.4 M KCl and 30 mM MgCl2) and nuclear extract was resuspended with buffer (20 mM Hepes pH 7.8, 25 % glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA and 0.5 mM DTT). Both extracts were rotated for 30 min at 4ºC, spined-down at 15,000xg at 4ºC for 15 min and the cytoplasmic and nuclear fractions (supernatants) were obtained. (S2.c) Confocal microscopy colocalisation of PARP-1 and Snail1. A375 cells were treated with PJ-34 (left panel) and G361 cells treated with PJ-34 or KU0058948 and colocalization was determined. *P 0.05. (S2.d)Co-IP of Snail1 and PARP-1 endogenous proteins in melanoma SK-Mel-28 cells and in HepG2 cells; an irrelevant antibody was used as negative control (middle lane).(S2.e) Confocal microscopy of distribution of E-cadherin and Snail1 in MCF7 cells.MCF7 cells treated with the PARP inhibitors (PJ-34 or KU0058948) or untreated cells (control) and the analysis of endogenous E-cadherin and Snail1 by immunofluorescence assay were performed using a previously published protocol protocol (Dominguez et al., 2003). Also the Knockdown of PARP-1 by siRNA was used. (S2.f) Effect of PARP inhibition on indirect immunofluorescence of E-cadherin, Snail1 and Vimentin in mesenchymal V12Ras-transformed MDCK-f3 cells. In Vimentin imagen quantification of circularity shown next to the images. Images were analyzed with the ImageJ. Circularity was measured with the ImageJ´s Measure command that calculates object circularity using the formula circularity=4π(area/perimeter2). Circularity value of 1.0 indicates a perfect circle. As the value approaches 0.0, it indicates an increasingly elongated polygon. ***P < 0.001. (S2.g) Hepatocyte Growth Factor (HGF)-induced cell scattering is prevented by PARP-1 inhibitors. MDCK cells were seed and treated with PJ-34 during 2 hours and then with HGF 5ng/ml during all the experiments.
Figure S3. mRNA levels of Snail in human melanoma cells A375 treated 0, 15, 30, 60 and 120 minutes by the PARP inhibitor, PJ-34 or 22 hours with KU0058948. **P < 0.01.
Figure S4. “In silico” results of the hypotetic PAR-binding sites in the C-terminal an N-terminal domain of Snail.