Pcmv luciferase

The ARPE-19 cells (3 × 10⁴/well) were plated and maintained in the DMEM/F-12 medium with 10% FBS in 24-well dishes for 24 h. To measure the NF-κB activity, the ARPE-19 cells were cotransfected with pCMV-luciferase (Promega, Milan, Italy) and either pTAL-secreted alkaline phosphatase (SEAP) or pNF-κB-SEAP (Clontech, San Jose, CA, USA) at a ratio of 1 : 4 for 4 h using the polycationic detergent Lipofectamine Plus (Invitrogen-Gibco) according to the manufacturer's instructions. The ARPE-19 cells were maintained for 20 h and subsequently preincubated with or without 100 μM PUGNAc for 1 h. The cells were further incubated with or without 50 μM silibinin for 24 h before stimulation with TNF-α (20 ng/mL) for 24 h at 37°C. For each treatment, the experiments were performed in triplicate. The SEAP activity was determined in the culture supernatants, and the luciferase activity was measured in the cell lysates to normalize the transfection efficiency. The luciferase activity was assessed with the Promega Dual-Luciferase Reporter 1000 Assay System.

All of the male Balb/c nude mice used in this experiment were purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences (Shanghai, PR China) and received humane care. To figure out the effects of lncRNA‐HEIH on cancer cell dynamics in vivo, ESCC cells were transfected with pCMV‐luciferase (Promega) and selected with neomycin (800 µg/ml). ESCC cells stably expressing luciferase were infected with lncRNA‐HEIH overexpression virus as described above. Five million lncRNA‐HEIH‐up‐regulated cells (firefly luciferase‐labelled ESCC cells) and control cells were injected into the double flank of nude mice. For firefly luciferase‐labelled ESCC cells, tumour growth was monitored using the IVIS@ Lumina II system (Caliper Life Sciences, USA) 10 min after intraperitoneal injection of 4.0 mg luciferin (Gold Biotech) in 50 μl saline. The experimental protocols were approved by the Ethical Committee of the Second Military Medical University.