The coding regions of human wild-type K-Ras and H3 were amplified by PCR and were subcloned into pEGFP-N1 plasmid (Clontech Inc., Palo Alto, CA). The amplified PCAF and HDAC2 were subcloned into pcDNA6.0/HA-tag vector (Invitrogen, Carlsbad, CA, USA). The pEGFP-K-RasG12V/T35S construct was mutated using site-directed mutagenesis. The pEGFP-H3K9Q construct was constructed using the TaKaRa MutanBEST Kit (#D401, TaKaRa, Dalian, China). Two different sequences of siRNAs specific for HDAC2 (si-HDAC2–1 and si-HDAC2–2) were purchased from GenePharma (Shanghai, China). A non-targeting sequence was used as a negative control (si-con). MDM2-MU for expression of MDM2C464A was constructed and recombination into pIB/V5-His Vector (Invitrogen).
SW48 cells were seeded in 6-well plates with a density of 1 × 105 cells/well. When 50% confluence was researched, the cells were transfected with plasmids or siRNAs by using lipofectamine 3000 (Invitrogen). At 48 h of transfection, the culture medium was replaced by the complete medium to stop transfection. Transfection efficiency was confirmed by using Western blot and/or RT-qPCR.
SW48 cells were seeded in 6-well plates with a density of 1 × 105 cells/well. When 50% confluence was researched, the cells were transfected with plasmids or siRNAs by using lipofectamine 3000 (Invitrogen). At 48 h of transfection, the culture medium was replaced by the complete medium to stop transfection. Transfection efficiency was confirmed by using Western blot and/or RT-qPCR.