X tremgene sirna transfection reagent

Cells seeded in six-well dishes were transfected with an siRNAs pool (50 nM) and 7 µL of Lipofectamine 3000 (Invitrogen) or X-tremGENE siRNA transfection reagent (Roche) on the following day. The medium was removed after 3 h of transfection and replaced with fresh medium. SETD2 (L-012448-00), containing four different sequences that target the SETD2 sequence, and non-targeting ON-TARGET (D-001810) SMARTpool siRNAs were purchased from Dharmacon (Supplementary Table 2). For SETD2 overexpression experiments, HeLa cells were transfected with 2 µg of a SETD2-containing plasmid (gift from Dr. Xiaobing Shi, MD Anderson Cancer Center, Houston, Texas, USA) or the corresponding pcDNA3.1 empty vector used as mock using 6 µl XtremeGene HP Reagent (Roche).

Evaluation of p53 acetylation in SMCs was done by a PathScan Acetylated p53 Sandwich ELISA kit (Cell Signaling 7236C), following manufacturer instructions. Briefly, total cell lysates were obtained, sonicated and protein concentration measured. Then, 100 μl of each sample containing equal amount of protein was added to a microwell coated with a p53 mouse monoclonal antibody and incubated at 4 °C overnight. The microwells were extensively washed, and an acetylated-lysine rabbit monoclonal antibody added for 1 h at 37 °C. After washing, an anti-rabbit horseradish peroxidase-linked antibody was added for 0.5 h at 37 °C. After washing, 100 μl of TMB substrate was added for 10 min at 37 °C. Then, 100 μl of STOP solution was added and absorbance at 450 nm read in a Synergy 2 Microplate Reader (BioTek). The magnitude of the absorbance is proportional to the quantity of acetylated p53. Triplicates for every genotype and condition were used every time. When indicated, cells were treated with 10 μM RO5963 (Calbiochem 44153), 50 μM of ICG001 (SelleckBio S2662) or vehicle (DMSO); or transfected with CBP siRNA (Santa Cruz sc-29243) or control siRNA (Santa Cruz sc-44231) using X-tremGENE siRNA transfection reagent (Roche 04476093001).