Pgl4.33 luc2p sre hygro

This was performed following a previously described method [49 (link)]. In brief, CHO cells were plated to 96-well white plates (REF3610, Corning Life Sciences, NY, USA) in DMEM (HyClone^TM, Cytivalifescience, Seoul, Korea) supplemented with 10% FBS and 1% penicillin at a density of 1.5 × 10⁴ cells/well. After 24 h of incubation, the cells were co-transfected using the Cignal PPAR Reporter (QIAGEN, Cignal PPAR (Luc) Reporter kit, CCS-3026L) and pCMV6-hFFAR4-GFP (RG201538, OriGene Technologies, Inc., Rockville, MD, USA) or with SRE plasmids (pGL4.33 (luc2P/SRE/Hygro), Promega, Madison, WI, USA) and FFAR4 (RG231184, OriGene Technologies, Inc., Rockville, MD, USA) using a Lipofetamine 3000 device (Invitrogen, Thermo Fisher Scientific Korea, Ltd., Seoul, Korea) according to the manufacturer’s instructions. After 6 h of incubation, the transfected CHO cells were treated with various concentrations of the compounds or the positive coFtablntrol. Control cells were treated with 0.1% DMSO. After incubation for another 24 h, luciferase activity was measured using the dual-luciferase assay system (Promega Korea, Seoul, Korea) according to the manufacturer’s instructions. All the individual in vitro assays to determine the average efficiency were performed twice and in triplicate for each treatment.

HEK293 cells were cultured in Eagle’s MEM containing 10% FBS with 1% P/S and seeded in 96-well plates at a density of 1.0 × 10⁴ cells/well. Following incubation at 37°C for 24 h, Lipofectamine^® 3000 Reagent was used to cotransfect the three vectors (expression, reporter, and Renilla control vector). Renilla control vector was used as an internal normalization standard to correct the transfection efficiency. For transfection, 1 μg of the expression vector plasmids (WT, p.Gly23Val, or p.Gly24Glu) or pcDNA 3.1 (+)were used as controls. The reporter vector used 100 ng of pGL4.33 [luc2P/SRE/Hygro] (Promega, Madison, WI), and the Renilla control vector used 10 ng of pRL Renilla Luciferase Control Reporter Vector (pRL-TK, Promega). Following incubation for 24 h, the culture medium was replaced with Eagle’s MEM containing 0.5% FBS and incubated for another 24 h. Luciferase activity was measured using the Dual-Glo^® Luciferase Assay System (Promega). We conducted the experiments according to the standard protocol and calculated the relative luciferase activity normalized to the Renilla luminescence. Each assay was performed in three separate wells and assayed six times independently.

CXCL12 was purchased from Peprotech (Rocky Hill, NJ, USA). NanoBiT® PPI starter system and Nano-Glo® live cell assay system; pBiT3.1 plasmid and Nano-Glo® HiBiT extracellular detection system; and pGL4.33 [luc2P SRE Hygro] and ONE-Glo luciferase assay system were purchased from Promega (Madison, WI, USA). Restriction enzymes were obtained from New England Bio Labs (Ipswich, MA, USA). Anti-CXCR4 antibody (Cat. No. ab124824) was purchased from Abcam (Toronto, ON, Canada). Anti-ERK antibody (Cat. No. 4695) and anti-pERK antibody (Thr202/Tyr204) (Cat. No. 4370) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-HA antibody (Cat. No. H3663) and anti-FLAG M2 affinity gel (Cat. No. A2220) for co-IP were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Green fluorescence protein (GFP) antibody (Cat. No. sc-8334), anti-β-actin antibody (Cat. No. sc-9996), and all secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All chemicals were obtained from Sigma-Aldrich unless otherwise stated.