Dual glow luciferase assay system

We searched the miRNA-binding sites to circ_MACF1 and human 3’untranslated region (3’UTR) by circInteractome web (https://circinteractome.nia.nih.gov/index.html) and ENCORI prediction software (http://starbase.sysu.edu.cn/), respectively. The fragments of circ_MACF1 and TGFBR2 3’UTR harboring the miR-942-5p pairing sites or miss-matched target sequence, provided by BGI, were individually subcloned into the psiCHECK-2 vector (Promega, Leiden, The Netherlands). The appropriate reporter construct (200 ng) was transfected into PC9/GR and A549/GR cells (1 × 10⁵) using Lipofectamine 3000 together with miRNA mimic at 30 nM. After a 48-h transfection period, we analyzed the luciferase activity with Dual-Glow Luciferase Assay System from Promega.

For detecting Nrf2 activity, 0.2 μg p-ARE/ptkRL or the control vector/ptk-RL (all vectors from Qiagen/SABioscience) were transfected into cells grown in 12-well plates using Effectene (Qiagen). For detection of Smad activity, 0.3 μg p6SRE (containing six Smad binding elements [50 (link)] or 0.3 μg pGL3 (Promega) as control were transfected together with 0.1 μg ptkRL. For analysing E-cadherin promoter activity, 0.3 μg of Ecad[–1189]-pGL3, Ecad[–1153]-pGL3 or 0.3 μg pGL3 were transfected together with 0.1 μg ptkRL. After 8 hours, medium was changed and cells were cultured overnight followed by treatments as indicated. Afterwards, cells were washed with PBS and lysed in 150 μl/well passive lysis buffer (Promega, Mannheim, Germany). Lysates were centrifuged for 2 min at 4°C, 13000 rpm. Twenty μl of the supernatant were used for the dual luciferase assay procedure, using Dual-Glow luciferase assay system from Promega. Measurements were performed with a Berthold luminometer and firefly luciferase expression was normalized to constitutive renilla luciferase expression. All measurements were carried out in duplicates.

Reporter constructs pVFir and pERuc used to quantitate EMT were constructed by fusing the human VIM promoter region (nt −1629 to −47 relative to the translation start site) and the human CDH1 promoter region (nt −1115 to −65) to the coding region of the firefly luciferase and the Renilla luciferase, respectively 17. These chimeric genes were then cloned into the SIV‐based lentivector pSIV‐gaMES4SA. Viral particles pseudotyped with the VSVG envelope were produced as described previously. Forty‐eight hours after infection with pVFir and pERuc, expression of the firefly and Renilla luciferase expression was measured with a microplate luminometer (Luminoskan Ascent, Labsystems) using the Dual‐Glow luciferase assay system (Promega). The EMT index (EMTi) was calculated as the ratio of firefly to Renilla luminescence.

Mouse Rac1 (NM_009007), Ctbp2 (NM_009980) and Slc6a9 (NM_008135) 3′UTR sequences in dual firefly luciferase/Renilla luciferase expression vectors (MmiT028031, MmiT028960 and MmiT027208) were obtained from GeneCopoeia Inc. (Rockville, MD, USA). Synthetic pre-miRNAs for mmu-miR-1a-3p (PM10617), mmu-miR-96-5p (PM10422), mmu-miR-182-5p (PM13088) and negative control were procured from Ambion (Thermo Fisher Scientific). 1.4 × 10⁵ HeLa cells/well were seeded in 24-well plates; 24 h later, cells were co-transfected with 400 ng 3′UTR plasmids and 16 pmoles of each pre-miRNAs using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific). 24 h post-transfection, cells were analysed using a Dual-Glow luciferase assay system (Promega, Madison, WI, USA). ANOVA and post-hoc Tukey’s multiple comparison test were performed and p < 0.05 values were accepted as statistically significant.

For miR-221-promoter luciferase assay, U937 cells grown in a 24-well plate were transfected by using Lipofectamine LTX (Life Technologies) with 10 ng of pRL-TK (Promega, Madison, WI), 300 ng of (-1600) MIR-222/221-Luc (kindly provided by Carlo Croce, Ohio State University), in combination with 300 ng of either pCMV5-RUNX1B (Addgene, Cambridge, MA), pcDNA3.1-AML1-MTG8-V5 [28 (link)], pGEM-CMV-CBFB, or pGEM-CMV-CBFB-MYH11 (both kindly provided by Paul Liu, NIH, Bethesda, MD). For RUNX1-3′UTR luciferase assay, cells seeded into 24-well plates were co-transfected with 10 ng of the pRL-TK and either 500 ng of pGL4.13-RUNX1-3′UTR plasmid, containing the RUNX1-3′UTR downstream of the firefly luciferase coding sequence (kindly provided by Yoram Groner, Weizmann Institute, Israel), or 500 ng of the control pGL4.13 plasmid. The concentration of transfected DNA was kept constant by adding an appropriate amount of empty pcDNA3.1. After 24 hours, cells were lysed in passive lysis buffer and measured for luciferase activity using the Dual Glow Luciferase Assay System (Promega) and a Veritas Luminometer (Tuner Biosystems, Sunnyvale, CA) as per manufacturer′s instructions. Statistical significance was calculated by using the Student’s t-test.