C maf

MAT1A promoter (−839 to +30) was described previously (17 (link)), c-Myc promoter was purchased from Addgene (Cambridge, MA), MafG and c-Maf promoters were purchased from GeneCopoeia. The wild type E-box-luc consisted of 3-repeat sequences: 5’-CCTGTTCTCACGTGCTCACA-3’, (−155 to −175 relative to ATG start site of the human MAT1A promoter), and core motif of E-box CACGTG was changed to CTCGTC for E-box mutant. The various promoter constructs, and a control Renilla luciferase expression vector (2.5 ug) were co-transfected into KMCH or H69 cells with Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. Luciferase assays were performed 24 hours later using the Dual Luciferase Reporter Assay System (Promega, Madison, WI) as directed by the manufacturer suggested protocol. Firefly luciferase activity was normalized to the Renilla luciferase activity.

Primary human MDMs were transfected with 100 nM scrambled or ON-TARGETplus SMARTpool siRNA against Twist1, Twist2, c-Maf, Bmi1, ATF4, C/EBPα, Runx1, Runx2, (Dharmacon, Lafayette, CO) (4 pooled siRNAs for each gene), or with 2 μg pT3-EF1a-Bmi1 (Addgene plasmid 31783 kindly deposited by X. Chen (23 (link))), c-Maf (GeneCopoeia, Rockville, MD) or empty vector using Amaxa nucleofector technology (Amaxa, San Diego, CA).