Pgl3 luciferase reporter vector

FOSL2- or Wnt5a-knockdown cells were produced by lentivirus-mediated transduction using synthetic short hairpin RNA (shRNA) oligonucleotides (GenePharama, Shanghai, China) according to the manufacturer's protocols. The small interfering RNAs (siRNA) were used to transiently knock down CYP19A1 or FZD5 (GenePharama, Shanghai, China). The sequences of shRNA and small interfering RNA used are listed in Table S1. FOSL2 overexpressing lentivirus were purchased from GeneCopoeia (Guangzhou, China), and viral supernatant was used to infect NFs in the presence of 8 µg/ml polybrene according to the manufacturer's instructions.
The pcDNA3.1(+)-FOSL2 plasmid was created by amplifying FOSL2 from CAFs and was cloned into pcDNA3.1(+) vector. The synthesized nucleotide wild type (WT), truncated or mutant (Mut) Wnt5a promoter was inserted into pGL3 luciferase reporter vector (GenePharma, China), respectively. The reagents used in this study are as follows: the neutralizing antibody against VEGF (Bevacizumab; Roche/Genentech, Switzerland), 0.2 mg/mL; axitinib (Selleck, USA), 5 nM; CCK8 (Beyotime, China), 5 mg/mL; SQ22536 (Selleck, China), 100 µM; H89 (Selleck, China), 5 µM; rWnt5a (R&D Systems, USA), 5 µg/mL; BOX5 (Merck Millipore, USA), 5 µM. The usage of rWnt5a and BOX5 in xenografts was described in animal experiments in detail.

BT549 and Hs578T were cultured in RPMI 1640 medium (Gibco-BRL, Australia) containing 10% fetal bovine serum (FBS) (Gibco-BRL, Australia) at 37 °C in humidified atmosphere containing 5% CO₂ with 1% O₂ or 21% O₂. ATM, PFKP, or CS stable knocked down cell lines were produced by lentiviral-mediated transduction using synthetic short hairpin RNA (shRNA) oligonucleotides (GenePharama, Shanghai, China) according to the manufacturer’s protocols. The sequences of small interfering RNA and shRNA used were listed in Supplementary Table 1.
The PFKP promoter containing HIF1A-binding sites (wild type (WT): –GTACGTGGAG–) and its mutant sites (Mut: –GTAgaaaGAG–) were cloned into pGL3 luciferase reporter vector (GenePharma China) to construct the WT or Mut PFKP reporter plasmid. The reagent cisplatin was a gift from the First Affiliated Hospital of Chongqing Medical University; sodium citrate (10 mM, 12 h) was purchased from Sigma-Aldrich (St. Louis, MO, USA); KU60019 (10 μM, 12 h), MG132(40 μM, 12 h), BMS303141 (20 μM, 12 h), and SCH772984 (2 μM, 12 h) were from Selleck (USA). All experiments were performed at least three times.

Human optic atrophy 1 (OPA1) promoter (−2000 bp) were cloned into pGL3-luciferase reporter vector (GenePharma Corporation). HTR8 cells were transfected with the pGL3-luciferase reporter vector and pRL-TK-Renilla-luciferase plasmid (Promega Corp) using Lipo8000™ for 12 h. Then cells were treated with cortisol (10⁻⁶ M) with or without CBX (10⁻⁶ M) for 24 h. Luciferase assays were measured by Dual-Luciferase^® Reporter Assay System (Promega).

BT549 and Hs578T were cultured in RPMI 1640 medium (Gibco-BRL, Australia) containing 10% fetal bovine serum (FBS) (Gibco-BRL, Australia) at 37 °C in humidified atmosphere containing 5% CO₂ with 1% O₂ or 21% O₂. ATM, PFKP, or CS stable knocked down cell lines were produced by lentiviral-mediated transduction using synthetic short hairpin RNA (shRNA) oligonucleotides (GenePharama, Shanghai, China) according to the manufacturer’s protocols. The sequences of small interfering RNA and shRNA used were listed in Supplementary Table 1.
The PFKP promoter containing HIF1A-binding sites (wild type (WT): –GTACGTGGAG–) and its mutant sites (Mut: –GTAgaaaGAG–) were cloned into pGL3 luciferase reporter vector (GenePharma China) to construct the WT or Mut PFKP reporter plasmid. The reagent cisplatin was a gift from the First Affiliated Hospital of Chongqing Medical University; sodium citrate (10 mM, 12 h) was purchased from Sigma-Aldrich (St. Louis, MO, USA); KU60019 (10 μM, 12 h), MG132(40 μM, 12 h), BMS303141 (20 μM, 12 h), and SCH772984 (2 μM, 12 h) were from Selleck (USA). All experiments were performed at least three times.

The fragment of circVRK1 and the 3'-UTR of ZNF652 mRNA containing miR-337-3p binding sites were cloned into the PGL3 luciferase reporter vector (GenePharma, Shanghai, China) to construct the wild-type (WT) luciferase reporter vectors circVRK1-WT and ZNF652 3'-UTR-WT, respectively. The mutant (Mut) vectors circVRK1-Mut and ZNF652 3'-UTR-Mut were generated by mutating the binding sites of miR-337-3p in the circVRK1 sequence and the 3'-UTR of ZNF652 mRNA. According to the method of previous study [38 (link)], osteosarcoma cells were planted in 48-well plates and co-transfected with the above vectors and miR-337-3p mimics. After 48 hours, the activity of luciferase was detected by the dual-luciferase assay system (Promega, WI, USA).

Short hairpin RNA (shRNA) oligonucleotides targeting Slc6a8, p65/NF-κB, HIF1A and HIF2A, and control shRNA-NC were all purchased from GenePharma (Shanghai, China). The breast cancer cells (MDA-MB-231 and BT-549) that stably expressed above shRNAs were established by lentivirus transduction according to the manufacturer’s instructions. The sequences of shRNA used were listed in Supplementary Table 1. The promoter containing TP53/FOS/ETV4/p65/NF-κB-wild type binding sites (WT) or mutated binding sites (MUT) was cloned into pGL3 luciferase reporter vector to obtain the pGL3/Slc6a8 WT reporter and pGL3/Slc6a8 MUT reporter (GenePharma, China). The AKT inhibitor Capivasertib (AZD5363) was purchased from Selleckchem (USA).