Prl cmv renilla plasmid

The MSH2 and MSH6 luciferase reporter and NRIP1 expression vectors were previously described [26 (link),27 (link),28 (link),29 (link)]. HCT116 cells were transfected in 96-well plates (2.5 × 10⁴ cells per well) 24 h prior to DNA transfection with Jet-PEI (275 ng of total DNA). Increasing doses of pEF-c-myc-RIP140 or pEF-c-myc-RIP^MSI were cotransfected with the pGL3-MSH2-Luc or the Sp1 mutant pGL3-MSH2m1-Luc (kind gifts of E. Huang [28 (link)]). Similar experiments were performed with the pGL3-MSH6-Luc reporter vector and a Sp1 mutant pGL3-MSH6M1-2/7-Luc (kind gifts of R.D. Kolodner [29 (link)]). The pRL-CMV-renilla plasmid (Promega, Charbonnières-les-Bains, France) was used to normalize transfection efficiency. Firefly luciferase values were measured and normalized by the Renilla luciferase activity. Values were expressed as the mean ratio of luciferase activities.
ChIP assays for proteins at the MSH2 and MSH6 promoters were performed in HT29 cells using the CHIP-IT kit (Active Motif, Carlsbad, CA, USA). Sonicated chromatin was immunoprecipitated with antibodies against IgG (sc-3739, Santa Cruz Biotechnology, Inc, Heidelberg, Germany), H3pan (CC16310135, Diagenode, Liège, Belgium) and NRIP1 (ab42126, Abcam, Paris, France). Immunoprecipitated DNA was amplified by qPCR using the primers listed in Supplementary Table S1.

A day prior of transfection, 4.0–6.0x10⁴ DAOY and ONS-76 cells were seeded on 24-well plate at a density that reached 90% confluence at transfection. Cells were then cotransfected with 0.5μg of pCRMP1-full, pCRMP1-distal, or pCRMP1-proximal plasmids and 20pmol of siRNA targeting HMGA1 (Invitrogen, Carlsbad, CA, USA) or Silencer Negative Control #1 siRNA (Invitrogen) using Lipofectamine 2000 (Invitrogen). pRL-CMV renilla plasmid (Promega) was included as internal control to normalize transfection efficiency. After 48h incubation, the firefly luciferase activity was measured with Dual Luciferase Reporter Assay System (Promega). Relative luciferase unit (RLU) was calculated as the ratio of firefly to renilla luciferase activities. The experiments were repeated three times and three replicate measurements were taken in each test.