Pgl3b

According to the results of CHIP, four, one and five putative KBSs, respectively, were predicted from the promoter regions of human BCL2 (positions −1201 to −1), BAX (positions −645 to −317) and BIK (positions −1200 to −1). The above promoter regions were amplified from genomic DNA and were subcloned into firefly luciferase reporter vector pGL3-Basic (pGL3b) or pGL4.1 (Promega, Madison, WI) to get pGL3b-BCL2, pGL4.1-BAX and pGL4.1-BIK. Constructs with mutations of the putative KBSs were generated using mutagenic oligonucleotide primers according to the manual of the GeneTailor Site-Directed Mutagenesis System (Invitrogen). HUVECs were cotransfected with firefly luciferase reporter constructs and Renilla luciferase reporter vector pRL-TK (E2241, Promega) together with pCDNA3.1-Kaiso or pCDNA3.1 empty vector using FuGENE HD Transfection Reagent (Roche, Mannheim, Germany). Then, 48 h after transfection, relative luciferase activity represented as the ratio of firefly to Renilla was measured with a Dual-Luciferase Reporter Assay System (Promega). All primers used for genomic DNA amplification and site-directed mutation are listed in Supplementary Tables S4–S5.

For LOXL4 promoter analysis, the different constructs (DR5-1; DR5-2) were obtained using PCR amplifications from BAC (RPCI-11 HS) (Fisher) (see Table 1), and cloned into the pGL3 basic vector containing firefly luciferase (pGL3b) (Promega). The mutagenesis of DR5 sites (four mutated bases in the core sequence) into DR5-1-pGL3b or DR5-2-pGL3b constructs was carried out by PCR (see Table 1). All the constructions were verified by DNA sequencing (GATC). Human RAR (α, β, γ) and RXRα expression plasmids were obtained from Pierre Chambon (IGBMC, Strasbourg, France). Co-transfection of pRL-tk vector (1/20) (Promega) containing Renilla luciferase allowed the normalization of the transfection efficiency.

All siRNA duplexes were purchased from Integrated DNA Technologies (Coralville, IA, USA). Primary human macrophages and RAW264.7 cells were transfected with concentration-matched pairs of scrambled siRNA or siRNA against target genes using HiPerFect transfectant (Qiagen, Valencia, CA, USA) as previously described51 (link) and lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, respectively. For overexpression, RAW264.7 cells were infected with the empty control virus (Ad-EV) or the adenovirus carrying the human TonEBP gene (Ad-TonEBP). A 1.6-kb fragment of the mouse IL-10 promoter (−1538/+64 pGL2B) and its 5′ deletion mutants were obtained from Addgene Inc. (Cambridge, MA, USA) and were subcloned into pGL3B (Promega, Madison, WI, USA). Sp1- or C/EBP-binding sites in −688/+64 promoter were mutated by a two-step PCR procedure using overlapping internal primers that contain a mutant sequence as published previously29 (link). All plasmids were purified using an endotoxin-free purification system (Qiagen) and transfected using lipofectamine 2000 (Invitrogen).