Pgl 4

The ORF of WDR5 and RbBP5 were amplified from cDNA of DU145 cells and cloned into pCMV (Clontech, Mountain View, CA, USA), and fused with Flag, and HA tag. The pGL 4.35-SNAI1 (9x UAS-SNAI1-Luc) was constructed base on pGL 4.35 (Promega, Madison, WI, USA): promoter of SNAI1 (-2000~-1) was amplified from genomic DNA of DU145 cell and cloned into pGL 4.35 with Hind III digestion. GAL4-RBBP5/WDR5 were constructed on the basis of pCMV (Clontech): GAL4-DBD(GAL4) was amplified from pGBKT7 (Clontech), and an IgA linker sequence was added to the 3′ site by primers; WDR5 and RBBP5 were amplified from cDNA of DU145 cells. Promoter of CDH1(-2000~-1) and VIM(-2000~-1) were amplified from genomic DNA of DU145 cell and cloned into pGL3-basic(luc) and pRL-null(Rluc) to form pGL3-CDH1 and pRL-VIM. All constructs were employed with In-Fusion® Clone Kit (Clontech).
Capped and tailed mRNA were generated by mMESSAGE mMACHINE® T7 ULTRA Transcription Kit (Ambion®), as per manufacturer's recommendations. Primer sequence are describing in Supplementary Table S3.

HEK293T or Mel888 cells were transfected in a 12-well plate with 500 ng (3 μg for Mel888) of pGL 4.35 (Promega) and different GAL4 constructs (GAL4, TCF7-GAL4 and ΔβCat-TCF7-GAL4), and 25 ng of RL-TK (75 ng for Mel888). After 24 h the cells were lysed with Passive Lysis Buffer and light intensity was measured on a Victor multilabel plate reader (PerkinElmer) using the Promega Dual-Luciferase® Reporter Assay System (catalog number: E1910) according to the manufacturer’s instructions. For each well, firefly luciferase activity was normalized to Renilla activity. In the experiments with PKF 118-310 the results were normalized to either GAL4 or the mutant Lambda B1 reporter because of interference of PKF with Renilla luciferase activity. Previously, other drugs have also been reported to interfere with luciferase activity⁵⁴ . The used inhibitors were CHIR-99021 (Selleck Chem—S1263) and PKF 118-310 (Sigma-Aldrich—K4394). The Mel888 cells were kindly provided by the lab of prof. L. Larue. The SPI1 luciferase reporter genes were kindly provided by the lab of prof. O. Bernard.

HepG2 cells were maintained at 37 °C in E-MEM containing 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin. The transfection experiments were performed using Fugene HD (Roche).
HepG2 cells were plated in a 6-well plate at 5 × 10⁵ cells/well. To evaluate the SHP transcriptional activity cells were transfected with 100 ng pFN26A-[SHPLBD/GAL4], 300 ng of the Promega reporter vector pGL4.35[luc2P/9XGAL4UAS/Hygro] and with 100 ng pGL4.70 (Promega), a vector encoding the human Renilla gene. 48 h post-transfection, cells were stimulated 18 h with 10 μM all trans retinoic acid ATRA, a SHP agonist, or with 10 μM compounds 1–7. After treatments, cells were lysed in 100 μl diluted reporter lysis buffer (Promega) and 10 μl cellular lysate was assayed for luciferase activity using the Luciferase Assay System (Promega). Luminescence was measured with the Glomax 20/20 luminometer (Promega). Luciferase activities were normalized for transfection efficiencies by dividing the relative light units (RLU) by relative renilla units (RRU).