Pgl3 basic plasmid

The truncated sequences of ADAM9 promoter were amplified by PCR (primers listed in Supplementary Table S2) and cloned into pGL3 basic plasmids (Promega) with SacI and XhoI restriction enzyme (Takara Biotechnology). The constructed plasmids were transfected into 293T cell with pRL-TK encoding Renilla luciferase vector. After 48 h, cells were lysed, and the luciferase activity was detected using SpectroMax M5 (Molecular Devices, Silicon Valley, CA, USA). When indicated, the siRNA sequence (GGGUCUUCCUAGAGAAUAUTT) targeting human Caspr1 corresponding to the coding region was transfected into 293T cell using Lipofectamine 2000, and after 48 h, the activity of luciferase was determined. The nonsilencing siRNA sequence (GGGUCUUCCUAGAGAAUAUTT) was used as a control.

CD147 promoters were amplified via polymerase chain reaction (PCR) using HeLa cell genomic DNA. The PCR products were purified using a Cycle Pure Kit (Sangon Inc. Shanghai, China) and sub-cloned into pGL3-basic plasmids (Promega, WI, USA) using KpnI and Hindlll enzymes.

The human RanBP9 promoter or TSSC3 promoter (upstream 2000 to 0) in pGL3 basic plasmids (Promega, Madison, WI, USA) were obtained with the assistance of Invitrogen Company (Shanghai, China). The open reading frame (ORF) of TSSC3 was amplified by PCR as previously described ^{14 (link)} and ORF of RanBP9 was obtained from Invitrogen Company (Karlsruhe, Germany). Reporter assays were performed using 250 ng of human RanBP9 or TSSC3 promoter. SaOS2 cells were co-transfected with 1 ng of plamids as indicated (including overTSSC3, siTSSC3, overRanBP9 or siRanBP9). SV-40-Renilla luciferase plasmid (1 ng) was co-transfected to control the efficiency of transfection. Expression of Firefly and Renilla luciferases were analyzed 48 h post-transfection by using a luciferase assay kit (Promega), according to the manufacturer's instructions.

To verify the combination between miR-194 and TCF7L2, the sequence of TCF7L2 3’UTR was amplified and inserted into a pmirGLO vector (Promega, USA). 0.5 μg plasmid containing TCF7L2 3′UTR and 20 nM miR-194 mimic/miRNC were cotransfected into well-grown STC-1 cells by using lipofectamine 2000 (ThermoFisher, USA). Forty-eight hours after transfection, cells were lysed and the luciferase activity was measured by dual-luciferase reporter assay system (Promega, USA). To verify the combination between miR-194 and Foxa1, the luciferase activity of Foxa1 3′UTR was measured in the same way. The primer sequences for amplifying TCF7L2/ Foxa1 3′UTR were shown in Table 2.
To measured the prompter activity of gcg, the sequence of gcg promoter was amplified and inserted into pGL3-basic plasmids (Promega, USA). Cells were then cotransfected with pGL3-basic plasmid containing gcg promoter + si-TCF7L2 + pcDNA-β-catenin/ pcDNA empty vector using lipofectamine 2000 (ThermoFisher, USA). Forty-eight hours later, the luciferase activity was measured by the dual-luciferase reporter assay system (Promega, USA). To verify the promoter activity of pcsk1, the luciferase activity of the pcsk1 promoter was measured in the same way. The primer sequences for amplifying gcg/pcsk1 promoters were shown in Table 2.

Pgl3-basic plasmids (Promega, E1751) containing the Mfn1 and Dnm1l promoters were constructed separately, named as pgl3-Mfn1 and pgl3-Dnm1. As negative controls, TEAD1-binding motifs were truncated in mutant pgl3-Mfn1 and pgl3-Dnm1 plasmids. phRL-TK was co-transfected (promega) as an internal control for normalization. To overexpress YAP1, we generated plasmid of singly overexpressed wildtype YAP1 (Yap1-wt) and phosphorylation site mutant YAP1 (Yap1-S112A). For luciferase reporter assays, 293T cells were seeded in 24-well plates overnight and then co-transfected with wild or mutant luciferase reporter vectors and other indicated plasmids according to Lipofectamine 3000 Transfection Reagent (thermofisher, L3000015) manual. Dual Luciferase Reporter Assay kit (Vazyme, DL101) was used for luciferase activity assay in each group after 48 h of post-transfection.

The MIF promoter was cloned into the pGL3-basic plasmids (Promega, Madison, WI, USA) for promoter study and named pGL3-MIF. The wild-type (Wt) and mutant (Mut) PCR products containing the E2F1 binding motif in the MIF promoter were synthesized and cloned into the pGL3-basic plasmids (Generay Biotech Ltd, Shanghai, China), which were named E2F1-Wt and E2F1-Mut respectively. To explore the mechanism by which IGF2BP1 stabilized E2F1 mRNA via the m6A site in the 3'-UTR region, adenosine (A) in the m6A motif was replaced by cytosine (C) and then cloned into pmir-GLO reporter plasmids using Tsingke (Nanjing, China). Transfections were carried out using Lipofectamine-3000 transfection reagent (Invitrogen, Carlsbad, CA, USA). Each luciferase-containing plasmid (400 ng) and internal control plasmid (pRL-TK, Promega, Madison, WI, USA; 4 ng) were co-transfected. The dual-luciferase reporter gene assay kit (Promega, Madison, WI, USA) was applied to detect luciferase activities. The activities of firefly luciferase were normalized to those of Renilla luciferase.