Prl tk reporter vector

The promoter region of wild-type MALAT1 was PCR amplified and cloned into the pRL-TK reporter vector (Promega Corporation). Chondrocytes were seeded into 48-well plates at a density of 1×10⁵ cells/ml and transfected with 300 ng luciferase reporter vector containing the promoter region of MALAT1 using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Subsequently, chondrocytes were treated with 15 or 30 µM Res for 48 h. Following 48-h treatment with Res, chondrocytes were lysed and cell lysates were collected. The relative Renilla luciferase activity was detected using a Luciferase Reporter Assay system (Promega Corporation). Each experiment was performed in triplicate.

The 3′ UTRs of RHEB, RICTOR, and RPS6KB2 were amplified by PCR from CNE genomic DNA and cloned into pRL-TK reporter vector (Promega, E2241). The predicted target site was mutated by site-directed mutagenesis. For luciferase reporter assays, the WT or MUT luciferase plasmids and miRNAs were cotransfected into HeLa cells and each experiment was repeated in triplicate. Transfected cells were lysed 30 h after transfection and luciferase activities were assayed by a Dual-Luciferase Reporter System (Promega, E1960). Total protein concentration of cell lysate was determined using Bradford assay (Bio-Rad, 500-0006) at 595 nm on a spectrophotometer (Thermo Scientific). The luciferase activity was normalized by total protein content.

The mutant 3′UTR of MALAT1/NF-κB1 was generated by mutating the miR-9 binding site sequence in the wild-type 3′UTR of MALAT1/NF-κB1. The wild-type or mutant 3′UTR of MALAT1/NF-κB1 were PCR amplified and cloned into the pRL-TK reporter vector (Promega Corporation, Madison, WI, USA). Chondrocytes were seeded into 48-well plates at a density of 1×10⁵ cells/ml and co-transfected with 300 ng luciferase reporter vector containing the wild-type or mutant 3′UTR of MALAT1/NF-κB1 and 20 pmol miR-9 mimic or scramble control using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Following 48-h transfection, chondrocytes were lysed and cell lysates were collected. Relative luciferase activities were detected using a Dual-Luciferase Reporter Assay system (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity. Each test was performed in triplicate.