Materials and methods Cultured cells, and SPRR2A stable transfectants useful site The human intrahepatic cholangiocarcinoma cell line HuCCT 1 was maintained as reported. Methods to obtain stable transfectants with a SPRR2A expressing vector were previously published. Plasmids We used the C terminal His V5 tagged human SPRR2A expression vector previously described. A human Halo tagged p300 vector was purchased from Promega. Other plasmids, including luciferase re porter plasmids, were purchased from Addgene luc p21 promoter constructs. luc p53 wt. luc p53 mut. Ha p300. and Ha p300 CH3 deletion. Florescence imaging The SPRR2A sequence was cloned into a DsRed mam malian expression vector and transfected into HuCCT 1 cells grown on glass coverslips. 48 hours after transfection, the coverslips were fixed for 1 hour in 1% paraformaldehyde.

Nuclear staining was done with Hoechst dye. DsRed SPRR2A and Hoechst florescence was captured using an AxioImager M1 microscope with a 40X objective lens, NA 0. 95. Biotinylated oligonucleotide precipitation assays The probes for DNA pull down assays are shown in Figure 2A. The assays were carried out as described. Briefly, twenty four hours after transfection, cells were lysed with HKMG buffer containing protease and phosphatase inhibitors. Extracted proteins were pre cleared with ImmunoPure streptavidin agarose beads. Pre cleared lysates were then incubated 12 hours with 1 ug of the 5 biotinylated double stranded oligonucleotides and 10 ug of competitor DNA poly to eliminate non specific pro tein/DNA interactions.

Oligo specific bound proteins were collected with streptavidin agarose beads, separated Anacetrapib by SDS PAGE, and protein identification done by West ern blotting. Transfections and luciferase reporter assay Transfections with DNA plasmids or empty vector were done with Lipofectamine 2000 using the manufacturers recommended protocol for adherent cells. p300 and HDAC1 knock down transfections were done with target specific or negative control SilencerW Select siRNA using RNAiMAX. Luciferase assays were carried out with a Promega assay kit system 24 hours post transfection and mea sured on a luminometer. Western blotting Cell lysates were obtained using TNE buffer con taining protease inhibitors 48 hours after treatments. Cytosolic and nuclear proteins were separated using an NE PER extraction kit. Proteins were separated by SDS PAGE and visualized using enhanced chemiluminescence reagents. Antibodies used are the following p53, GAPDH, p300, and Ha HDAC1 . V5 . Halo . PCAF, acetylated lysine, and Ac K382 p53. Western blots were measured using imageJ software. Immunoprecipitation Cell lysates were obtained 48 hours post treatment using TNE buffer containing protease inhibitors.