TLE4 acts as a corepressor of Hes1 to inhibit inflammatory responses in macrophages

C57/BL6 mice were purchased from Jackson Laboratory. Hes1 mice were originally obtained from R. Kageyama (Imayoshi et al., 2008). Mice with inducible deletion of Hes1 (Hes1Mx1-Cre) have been described (Shang et al., 2016). TLE4 globally deficient (Tle4−−) and Tle4 mice were generated by CRISPR/Cas9 genome editing in Beijing Biocytogen Co., Ltd. Genetic heterozygous mice (Tle4−) were used for breeding pairs to generate Tle4, Tle4− and Tle4−− littermates. Mice with a myeloidspecific deletion of TLE4 (Tle4Lyz2-Cre) were generated by crossing Tle4 animals to Lyz2-Cre mice. Experiments on mice were performed at 6-8 weeks of age with gender matched littermates.

and U0126 were purchased from Calbiochem, and Bay11-7082 was purchased from Sigma.

Enzyme-Linked Immunosorbent Assay (ELISA)
Cytokine secretion was quantified with IL-6 and IL-12p40 ELISA kits from BD Biosciences according to the manufacturers' instructions.

Generation of TLE-deficient iBMDMs
Lentivirus vector lentiCRISPR containing the gRNAs of Tle1, Tle2, Tle3 and Tle4 (LentiCRISPR-TLE) were generated according to the instructions of the Zhang Lab at Harvard University (Shalem et al., 2014). HEK 293T cells were transfected with lentiCRISPR-TLE vectors together with packaging plasmids pVSVg (AddGene 8454) and psPAX2 (AddGene 12260) to make lentivirus using Fugene HD transfection reagent (Promega). iBMDMs were then infected with these lentiviruses and were selected with 2 μg/ml of puromycin (Invivogen) for 3 days. Single clone was selected by serial dilution and TLE gene deletion was confirmed by sequence.

Co-immunoprecipitation and Immunoblotting Analysis
pMx vectors expressing full-length Hes1 cDNA, a dominant-negative Hes1 (dnHes1), and Hes1 deleted with HLH domain or WRPW motif were generated as previously described (Shang et al., 2016). For co-immunoprecipitation, HEK 293T cells were transfected with pMx-GFP, pMx-Flag-Hes1 or pMx-Flag-Hes1 mutants using Fugene HD transfection reagent (Promega). 24 h post transfection, cells were lysed in a lysis buffer containing 10 mM Tris, 150 mM NaCl, 1% NP-40, 5 mM EDTA and 1 mM PMSF, 1 mM NaVO3, and the proteinase inhibitor cocktail (Roche). Cell lysates were incubated with anti-Flag antibody (F1804, Sigma) for 4 h and then incubated with protein A/G agarose beads (Santa Cruz Biotechnology) overnight at 4º C. Whole cell lysates were prepared as described previously (Hu et al., 2006). Whole cell lysates or immunoprecipitated extracts were then separated on 10% SDS-PAGE and transferred to PVDF membranes (Millipore) for immunoblotting with specific antibodies.

Luciferase Reporter Assay
Murine Il6 luciferase reporter plasmid containing a 1300 base pairs promoter sequences was generated as described previously (Hu et al., 2008). Murine Il12b luciferase reporter plasmid containing sequences from positions -356 to +55 was a gift from S.T.
Smale (Plevy et al., 1997). RAW 264.7 cells were co-transfected in duplicate with the Il6 or Il12b reporter plasmid and an expression plasmid encoding full length Hes1

Statistical Analysis
P values were calculated with a two-tailed paired or unpaired Student's t test. P values of 0.05 or less were considered significant.