Pgl3 basic vector

The circDCAF8-WT, circDCAF8-MUT, NAP1L1-WT, NAP1L1-MUT were cloned into pGL3-basic vector (GenePharma, Shanghai, China). The WT or MUT vector was co-transfected into HEK-293T cells with miRNA mimics using Lipofectamine 3000. The dual luciferase reporting system (Promega) was used to measure the luciferase activity after 48 h of incubation.

The recombinant vector or pGL3-Basic vector (GenePharma, Shanghai, China) were cotransfected with the pRL-CMV vector (GenePharma) containing a Renilla luciferase reporter gene (as a normalizing control) into either the miR-19b knockdown or control stable P19 cells. The Dual Luciferase Reporter Assay system (Promega) was used to analyze the firefly and Renilla luciferase activities 36 h later.

The wild type (WT) or mutant type (MUT) sequences of LEMD1 promoter region were inserted into the pGL3 Basic vector (GenePharma, Shanghai, China) to construct the reporter plasmids of LEMD1 promoter; after that, they were co-transfected with Ov-NC and Ov-SOX4 into cells via Lipofectamine 2000 (Invitrogen, CA, USA). After 48 h, the Dual-Luciferase reporter assay system (Promega) was employed to estimate the luciferase activity.

The promoter region of SYNCRIP (from −1391 to 138) was chosen to design primer and then amplified by PCR and inserted into the pGL3‐Basic vector (GenePharma Company). In this study, luciferase reporter plasmids included SYNCRIP promoter‐pGL3‐Basic (referred to as pGL3‐promoter) and pGL3‐Basic (negative control). 293T cells (1 × 10⁴ per well) were inoculated in 48‐well plates with 200 µL culture medium and then cotransfected with the target siRNA and plasmid (si‐ctrl or si‐LncNT5E and pGL3‐Basic or pGL3‐promoter) and pRL‐TK plasmid expressing Renilla luciferase (Promega). After incubation for 48 hours, cells were harvested, and luciferase activity was measured with a Dual‐Luciferase Reporter Assay System (Promega). Renilla luciferase activity was used for normalization. The luciferase activity was expressed in terms of the ratio of firefly luciferase activity to Renilla luciferase activity.

Wild-type (WT) and mutant human ACSL4 promoters were synthesized and subcloned into the pGL3-Basic vector by GenePharma (Shanghai, China). An empty vector (control) or the WT, or deleted (Del) ACSL4 promoter plasmid (1.5 μg/well) was transfected into Caco-2 cells. Then, cells were lysed and analyzed with the TransDetect Double-Luciferase Reporter Assay Kit (FR201, TransGen). Luciferase activities were measured using a Varioskan LUX multimode microplate reader (Thermo Scientific, USA).

To investigate whether C/EBPα regulates Munc18b expression via binding to the promoter of Munc18b, the upstream regions (-2000/200) of Munc18b gene with WT or mutation of the binding motifs 1 or 2 with C/EBPα were amplified and subcloned into the pGL3-Basic vector by GenePharma (Shanghai, China) to generate Munc18b promoter constructs. The cells were co-transfected with Munc18b promoter construct or empty pGL3-Basic vector and C/EBPα plasmid or its corresponding empty vector. The cells were then lysed and analyzed with Dual luciferase reporter assay system (Promega, WI, USA).

The pGL3‐basic vectors (GenePharma) with either circ_0007331‐WT or circ_0007331‐MUT were co‐transfected with miR‐200c‐3p mimic or mimic NC into HEK 293T cells utilizing Lipofectamine 2000 reagent (Thermo Fisher). Correspondingly, the pGL3‐basic vectors with either HIF‐1α‐WT or HIF‐1α‐MUT were co‐transfected with miR‐200c‐3p mimic or mimic NC into HEK 293T cells utilizing Lipofectamine 2000 reagent. The luciferase activities were measured by the dual‐luciferase assay system after 48 hours.