On targetplus smartpool sirna duplexes

For knock-down experiments, cells were transfected with ON-TARGETplus SMARTpool siRNA duplexes (Dharmacon, Lafayette, CO, US) targeting WRN (L-010378–00), MLH1 (L-003906–00) or MSH3 (L-019665–00) using Lipofectamine RNAiMAX reagent according to the manufacturer’s instructions (Invitrogen, Waltham, MA, US). For WRN knock-down, additionally an individual siRNA was used (J-010378–05). Chromosome spreads, immunoblotting, immunofluorescence and live cell imaging experiments were performed using a final siRNA concentration of 20 nM. Cell viability assays were performed using 10 nM siRNA in 96-well plates in a total volume of 100 µl per well. Viability was determined using CellTiter-Glo (Promega, Madison, WI, US). Seven days post-transfection, 100 µl CellTiter-Glo solution was added directly to the cell medium, mixed and incubated for 10 min prior to determination of the luminescence signal. Crystal violet staining was performed in 24-well format using a total volume of 1000 µl per well. Seven days post-transfection, cells were fixed with 4% formaldehyde in PBS and stained twice for 30 min with crystal violet (SIGMA, HT901). For co-depletion of p53 and WRN 10 nM of the respective siRNA duplexes each were used for immunoblot and viability assay.

Goat anti-human VE-cadherin polyclonal antibody, rabbit anti-human Flk-1 (VEGFR-2) polyclonal antibody, and rabbit anti-vav2 polyclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse anti-phosphotyrosine antibody was purchased from BD Bioscience (San Jose, CA). Mouse anti-HA antibody conjugated agarose beads and rabbit anti-HA antibody were purchased from Covance (Berkeley, CA). Anti-p120 catenin antibodies were purchased from Santa Cruz Biotechnology and BD Bioscience. Rabbit polyclonal anti-phospho-ERK1/2 (extracellular signal-regulated kinases 1/2) antibody was purchased from Cell Signaling (Beverly, MA). Specific inhibitors for VEGFR-2 tyrosine phosphorylation (SU1498) and Src (PP2) were obtained from Calbiochem (Indianapolis, IN). ON-TARGET plus SMART pool siRNA duplexes targeting the human P2Y₂ receptor, VE-cadherin and p120 were purchased from Dharmacon (Chicago, IL). VE-cadherin cDNA was a kind gift from Dr. Elisabetta Dejana (IFOM-IEO, Milan, Italy). All other reagents including nucleotides were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise specified.