Pgl3 basic luciferase plasmid

The 2000 bp promoter region of NANOG was cloned into pGL3-Basic Luciferase plasmid (Promega, USA, #E1751) to generate pGL3-Basic-NANOG wild-type plasmid. Subsequently, a pGL3-Basic region-3 deletion of NANOG promoter plasmid was constructed using Fast Mutagenesis Kit (Vazyme, #C214-01). To access the luciferase activity, the Dual Luciferase Reporter Assay Kit (Vazyme, #DL101-01) was employed in HEK-293T cells transfected with the Firefly luciferase and Renilla luciferase plasmids (pRL-TK, Promega, #E2241).

293T cells were passaged 1 × 10⁵ per well in 48-well plate. In the following day, medium was changed and 0.6 µg of overexpression plasmids (pCMV-WRN/BLM, a gift from Dr. Vilhelm Bohr, NIA) were transfected using 0.8 µL Lipo8000 (Beyotime) in each well. After overnight incubation, medium was changed and transfected with 0.4 µg pGL3-basic luciferase plasmid (Promega) containing SHOX promoter sequences, 0.04 µg pRL-TK renilla plasmid and 0.6 µL Lipo8000 per well. Next day, cells were digested using Dual-Glo Luciferase Assay System (Promega) according to manufacturer’s protocol. Luciferase and renilla reading were taken by SpectraMax i3x (Molecular Devices) plate reader.

The 3’UTR of Rad52 and mutated controls (position 172-178bp mutant into AGCUGAA) were cloned into the pGL3-basic Luciferase plasmid (Promega, USA). miRNA mimics and pRL-TK Renilla luciferase report plasmid were then transfected into the HL-60 and U937 cell containing wild-type or mutant 3’UTR pGL3-basic plasmids. pRL-TK Renilla luciferase report plasmid was used as internal loading control. After cell lysis the luciferase activity was measured using Luciferase Reporter Assay System (Promega Corporation Madison, WI, USA) according to manufacturer’s protocol.

Anti-PELP1 antibody and anti-T7 antibody were purchased from Bethyl Laboratories, Inc. Vinculin was procured from Sigma–Aldrich and F4/80 (macrophage marker), anti-c-Rel, and β-actin antibodies from Santa Cruz Biotechnology. Anti-rabbit and antimouse horseradish peroxidase (HRP)-conjugated secondary antibodies were procured either from Santa Cruz Biotechnology or Rockland. Phospho p44/42 MAPK (catalog no.: 9106S), p44/42 MAPK (catalog no.: 9107S), Phospho STAT3 (catalog no.: 9134S), total STAT3 (catalog no.: 4904S), Phospho GSK3β (catalog no.: 5558S), and total GSK3β (catalog no.: 12456S) antibodies were purchased from Cell Signaling Technology. For IHC and immunofluorescence studies, anti-PELP1 antibody (IHC0013) was purchased from Bethyl Laboratories, and CD68 (AM416-5M) was purchased from Biogenex. Alexa Fluor 488, Alexa Fluor 546, and 4′,6-diamidino-2-phenylindole were procured from Life Technologies. Anti-GMCSF antibody was purchased from Abcam (catalog no.: Ab9741). The pGL3 Basic luciferase plasmid was purchased from Promega Corporation. Control siRNA and custom siRNA smart pool against PELP1 (L-041621-01-0005) and c-Rel (L-047122-00-0005 & EMU072641) were procured from GE Dharmacon and Sigma–Aldrich. Lentiviral particles (control [SHC001V] and PELP-1 targeting [TRCN0000159617]) for stable clone generation (MISSION shRNA) were purchased from Sigma–Aldrich.

PDCD4 promoter sequences were inserted into pGL3-Basic luciferase plasmid (Promega, Madison, WI, USA) to generate PDCD4 promoter reporter vector. Then, PDCD4 promoter reporter was transfected into ECA109/DDP cells using Lipofectamine 2000 (Invitrogen) along with phRL-TK vector (Promega) and (Vector or TUG1) or (si-con or si-TUG1). Luciferase Reporter assay system (Promega) was performed to detect luciferase activity in ECA109/DDP cells 48 h post-transfection.

HEK293T cells were transfected with the pGL3 basic luciferase plasmid (Promega) containing the T-bet promoter alone or the T-bet promoter in combination with an upstream enhancer region (Yang et al., 2007 (link)), or the empty pGL3 basic in combination with an internal control pRL-TK Renilla plasmid (Promega). The T-bet enhancer/promoter plasmid was described before (Hosokawa et al., 2013 (link)) and kindly provided by H. Hosokawa (Tokai University, Japan). In order to assess gene regulation by c-Maf, putative Maf responsive elements in the promoter and enhancer were mutated using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs). In addition to mutated reporter plasmids, cells were co-transfected with c-Maf coding sequence in pMSCV. Luciferase activity was measured on a SpectraMax i33 microplate reader (Molecular Devices) after 24 hr using dual luciferase assay system (Promega). Luciferase activity was determined relative to Renilla.