Dual luciferase reporter reagent

The promoter regions of STAT1 and PD-L1 were amplified from human DNA and then were cloned into a PGL4 vector (Promega). 293T cells were seeded in 24-well plates 24 h before transfection. The STAT1 or PD-L1 promoter reporter constructs were co-transfected along with PRMT5-overexpressing plasmid or a control vector using Lipofectamine 2000 (Invitrogen). After 48 h, luciferase activity was assessed using the Dual-Luciferase Reporter reagent (Promega) according to the manufacturer's protocol. Renilla luciferase was used for normalization.

The WT (wide type) or MT (mutant) 3’UTR of HES1 was amplified using PCR and cloned to the pGL3-luciferase reporter plasmid (Promega, Madison, WI, USA). Cell was transfected with miR–381 or scramble and luciferase reporter plasmid and the Renilla luciferase (Promega, Madison, WI, USA) using Lipofectamine 2000 (Invitrogen). Luciferase activity was performed using the Dual-Luciferase Reporter reagent (Promega) following to the manufacturer’s information.

Reporter plasmids were constructed by Guangzhou RiboBio (RiboBio, Guangzhou, China). 293T cells were seeded in 24-well plates. After reaching a confluence of 30%, cells were transfected with 100 ng of LIF-WT or LIF-MUT, along with 50 nM of miR-29c mimics using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Cells were harvested 24 h after transfection and lysed for luciferase assays. Luciferase activity was determined using the dual-luciferase reporter reagent (Promega, E1910, Madison, WI, USA) according to the manufacturer's instructions. Each sample was assayed in triplicate.

Sub-confluent ECs plated on 6-well-plate were incubated in Opti-MEM medium (Invitrogen, Carlsbad, CA, USA) containing Plus-Lipofectamine transfection reagents, the pG5 luciferase (pG5-Luc) and pBIND-ERK5 plasmids with pcDNA3.1-CA-MEK5α or control pcDNA3.1 vector, as we performed and described previously (41 (link)), for up to 4 h. Then, the transfection mixture was removed, ECs were washed, and ECM was added. Next, cells were treated with ponatinib at the concentrations indicated in the figures, for 24 h. Finally, cells were harvested, lysed, and luciferase activity was measured by a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA, USA), using dual-luciferase reporter reagents (Promega, Madison, WI, USA). The pG5-Luc plasmid has five Gal4 binding sites upstream of a minimal TATA box, which in turn, is upstream of the firefly luciferase gene. The pBIND-ERK5 plasmid has Gal4 fused with ERK5. Because pBIND vector also contains the Renilla luciferase gene, the expression and transfection efficiency were normalized to the Renilla luciferase activity. Therefore, relative ERK5 transcriptional activity was calculated by normalizing firefly luciferase activity according to Renilla luciferase activity (firefly:renilla luciferase activity ratio).

HEK293T cells were transfected with a human QKI promoter-luciferase reporter construct (SwitchGear Genomics), human β-catenin 3′UTR luciferase-reporter plasmid (SwitchGear Genomics), human VE-cadherin 3′UTR luciferase-reporter plasmid (last 545 bp of 3′UTR of human VE-cadherin cloned into pMIR-REPORT™ miRNA Expression Reporter Vector System; Applied Biosystems), control 3′UTR luciferase-reporter plasmid (60 bp of murine SDF-1 3′UTR cloned into pMIR-REPORT™ miRNA Expression Reporter Vector System; Applied Biosystems) or empty 3′UTR luciferase-reporter construct (SwitchGear Genomics) through the use of polyethylenimine (3 μg/1 μg DNA; Polysciences Inc). When indicated shRNA against QKI or non-targeting shRNA were co-transfected. Twenty-four hours after transfection luciferase activity was assessed with the Dual Luciferase Reporter reagents (Promega) and a GloMax^® 96 microplate luminometer (Promega) and was normalized to a constitutively expressed renilla reporter.