Pgl3 luciferase reporter plasmid

The vector expressing Myc-TEAD4 (pReceiver-M43) and short hairpin RNA (shRNA) targeting YAP (psi-nH1) were ordered from Genecopoeia (Rockville, MD). PCR-amplified human14-3-3ζ was subcloned into pFlag-CMV^TM-2 vector (Sigma, St Louis, MO) for transient transfection and c-Flag pcDNA 3 vector (Addgene, Cambridge, MA) for stable transfection. 14-3-3ζ L98/100 A, S37A, and S37E were generated using a QuickChange^® Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). The PKM2 promoter luciferase reporter gene was constructed by cloning the wild-type or mutant HRE region of PKM2 into SacI/XmaI sites of pGL3 luciferase reporter plasmid (Promega, Madison, WI) with the following primers: wt-HRE: 5′-CGAGCTCGAGTTGAGACCATCCTGGCCAA-3′(forward) and 5′-CCCGGGGAAGACGGGGTTTCGCCACGT-3′ (reverse). mut-HRE: 5′-CGAGCTCGGTTGAGACCATCCTGGCCAGT-3′ (forward) and: 5′-CCCG
GGGA GCCAAGACGGGGTTCTCAAC-3′(reverse). Small interfering RNA (siRNA) targeting 14-3-3ζ and HIF-1α were purchased from GenePharma (Shanghai, China). ERK2 siRNA (#6578) was obtained from Cell Signaling Technology.

ETS2 binding motif sequences on CXCL1 promoter were cloned into pGL3 luciferase reporter plasmid (Promega). Empty vector or ETS2 reporter were transfected into cells using FuGENE® HD (Promega). pRLTK Renilla Luciferase vector was co-transfected as internal control. After 24 h, cells were harvested, and Firefly and Renilla luciferase activities were measured with Dual-Luciferase Reporter Assay System (Promega).

The wild type circ_0020123 sequences (circ_0020123-WT), mutant circ_0020123 sequences (circ_0020123-MUT), wild type THBS2 3′UTR sequences (THBS2-WT), mutant THBS2 3′UTR sequences (THBS2-MUT) were cloned into pGL-3 luciferase reporter plasmid (Promega, Madison, WI, USA). Then, the plasmid and miR-590-5p or miR-NC were co-transfected into A549 and H1299 cells by Lipofectamine 2000 (Thermo Fisher Scientific). After transfection for 36 h, the Dual-Luciferase Reporter Assay System (Promega) was performed to detect the luciferase activity.

The pGL3 luciferase reporter plasmid (Promega, Madison, WI, USA) was used to construct luciferase reporter plasmids of E2F1. The Lipofectamine 3000 reagent (Invitrogen) was used for transfection of the luciferase reporter plasmid according to the manufacturer's instruction. After amplification, human ANP32E or E2F1 cDNA was cloned into the pSin‐puro‐retro vector (Clontech, Mountain View, CA, USA). Short‐hairpin RNA (shRNA) targeting ANP32E or E2F1 in a vector with pLKO‐puro were used (Sigma‐Aldrich). Next, 2 × 10⁴ cells were added and infected by a retrovirus that was produced by pLKO‐puro‐ANP32E‐shRNA transfection into 293FT cells for 72 h. Subsequently, these cells were transfected with the pMSCV‐neo‐luci plasmid. Cells expressing ANP32E‐shRNA‐luci were cultured with 250 μg·mL⁻¹ G418 and 0.5 μg·mL⁻¹ puromycin for 10 days to produce stable cell lines.