β gal plasmid

To create the GLP1R 3′-UTR luciferase reporter plasmid, a pMIR-REPORT vector (Ambion, USA) was used, and nucleotides 262–461 of the GLP1R 3′-UTR were cloned into the plasmid. A similar plasmid with a mutated miR-192 seed sequence (from AGGTCAA to CATGTGC) was also constructed using a site-directed mutagenesis kit (TianGen, China).
HK-2 cells were seeded in a 24-well plate and transfected with a mixture of 1 µg of pMIR-REPORT plasmid, 0.2 µg of β-gal plasmid (Ambion, USA), and 50 nmol miR-192 or negative control (NC) mimic and inhibitor with Lipofectamine 3000 (Invitrogen, CA, USA). The cells were lysed 48 h after transfection, and luciferase activity was measured with a luciferase assay kit (Beyotime, China). All experiments were repeated three times.

A luciferase reporter assay was performed to detect YAP transcriptional activity in A101D and A375 cells exposed to pcDNA3.1-LTBP4 transfection, or shRNA-1-LTBP4 transfection. Briefly, the 8xGTIIC-luciferase plasmid, which contains a YAP -responsive synthetic promoter driving luciferase expression, was co-transfected into cells with a β-gal plasmid (Ambion, United States) using LipofectamineTM 2000. After 72 h, luciferase activity was examined. β-Gal activity was used as a normalization control for luciferase activity.

The 3′-UTRs of human or mouse Yy1 containing binding sequences of the miR-192-5p targets or the mutants (GenScript, Nanjing, China) were synthetically inserted into the 3′-UTR region of the pMIR-report luciferase plasmid (Ambion, Waltham, MA, USA). For transfection, reporter constructs (0.1 μg/well), β-gal plasmid (0.1 μg/well, Ambion, Waltham, MA, USA) to normalize experiments for transfection efficiency, and either miR-192-5p mimics (20 pmol/well) or inhibitors (100 pmol/well) were co-transfected into HEK293T cells in 24-well plates. After 24 h, the cells were harvested and lysed using Luciferase Cell Culture Lysis Reagent (Cat#E1531, Promega, Madison, WI, USA). The cell lysates were subjected to three cycles of freezing and thawing in liquid nitrogen, followed by centrifugation at 10,000× g for 10 min. Then, 10 μL of the supernatant was utilized to quantify the luciferase activity using a luciferase assay kit (Cat# E1501, Promega, Madison, WI, USA).