Pgl3 basic firefly luciferase vector

ChIP assay was adapted and performed as previously described [44 (link)] using anti-WT1 (ThermoFisher) or rabbit IgG antibodies. For luciferase reporter assay, JN-DSRCT genomic DNA was extracted by DNAzol Reagent (Invitrogen) and 2 kb SIK1 proximal promoter (P4) was PCR amplified from JN-DSRCT-1 genome then inserted into pGL3Basic firefly luciferase vector (Promega, Madison, WI) using Gibson Assembly Cloning kit (NE BioLabs, Ipswich, MA). Truncated promoter plasmids, P1-P3, were generated by PCR amplification followed by digestion and ligation into NheI and XhoI sites. Primers sequences can be found in Table S5. U2OS cells were seeded in 24-well plates and transfected with 0.5 μg SIK1 promoter constructs and 0.5 μg pcDNA3-E-KTS, pcDNA3-E+KTS or empty pcDNA3 vectors along with 0.1 μg of Renilla Luciferase plasmid using Lipofectamine 3000 (Invitrogen). Luciferase activities were measured at 48 h post-transfection using Dual Luciferase Reporter Assay (Promega).

To analyze activation of the hBD-3 promoter activity, a firefly luciferase plasmid containing 2484 bp of the human hBD-3 promoter was used. The hBD-3 promoter region was amplified using the primers 5′-AGC CTC GAG TGC AGT TCC AAG TGC TGT GAC-3′ and 5′-CAG AAG CTT GGA TGA AAA GGT GTG CTT GGT C-3′. Both primers contain recognition sites for the restriction enzymes XhoI and HindIII, respectively, to facilitate subsequent cloning into the promoterless pGL3-basic firefly luciferase vector (Promega, Madison, WI). Verification of the correct insertion of the hBD-3 promoter region into the pGL3-basic plasmid was done by sequencing. To analyze hBD-3 promoter activity, 0.5 μg of the hBD-3 firefly promoter plasmid was transfected together with 0.05 μg of an internal control Renilla luciferase expression plasmid (pGL4.74[hRluc/TK], Promega) in primary keratinocytes (70–90% confluence) using the transfection reagent FuGENE HD (Promega). After 6 hours, the transfection medium was removed and replaced by fresh medium. After additional 20–24 hours, cells were stimulated with PRGF (1 : 10 diluted in KGM-2) for 24 hours. Luciferase activity was analyzed using the Dual-Luciferase Assay System (Promega) on a TD-20/20 luminometer (Turner Design, Sunnyvale, CA). Relative promoter activity was calculated as the ratio between firefly and Renilla luciferase activity.

NIH3T3 and HeLa cell lines were cultured in DMEM with 10% FBS, L-glutamine, and antibiotics (Invitrogen, Carlsbad, CA). HCT116, SW620 and SW48 were cultured in McCoy's with 10% FBS, L-glutamine, and antibiotics (Invitrogen). For live-cell imaging, cells were cultured in DMEM medium without phenol red, supplemented with heat-inactivated FBS (Invitrogen). HCT116, SW620, SW48 and NIH3T3 cells were transfected using Lipofectamine-Plus reagent (Invitrogen) according to manufacturer's instructions. The pcDNA3.1-Hmga1b and pCEFL-HA-Hmga1b vectors were previously described, respectively [39 (link), 40 (link)]. The pLuc-Bub1b reporter plasmid and pT81-Mad2l1 promoters were gifts of Dr. P. Carbon [41 (link)] and Dr. CW Lee [42 (link)], respectively. The Bub1 promoter region was amplified by PCR from human genomic DNA using the following primers:

lucBub1Fw: 5′-AATTCTCGAGGCTTGAAGCTGT TTGACAGG-3′

lucbub1Re: 5′-AATTAAGCTTCACATTCCAAAC CCAGGAAG-3′

The forward and reverse primers contain, respectively, the recognition sites for the restriction enzymes Hind III and XhoI. The amplified fragment was cloned into pGL3-Basic Firefly luciferase vector (Promega, Fitchburg, WI, USA) at the XhoI and Hind III sites upstream the luciferase gene. Human Ttk promoter spanning from −524 to +72 relative to the transcription start was previously reported [43 (link)].