Prl tk internal control vector

The SIAH1 promoter fragment was amplified using PCR and ligated into the PGL3-basic vector (Promega). The empty PGL3-basic vector served as the negative control. A total of 1.5–3 × 10⁵ cells per well were placed in 24-well plates for cell transfection. A total of 1 mg pGL3 plasmid, 100 ng pRL-TK internal control vector (Promega), and shPHF19/Flag-PHF19 vector were co-transfected into 293FT cells in serum-free Opti-MEM Reduced Serum Medium (Life Technologies). After 4–6 h, the culture medium was added to each well to a final volume of 1 ml. After a further incubation of 48 h, a luciferase reporter assay was performed according to the manufacturer’s instructions (Promega). Luciferase activity was normalized to pRL-TK activity. Each experiment was performed in triplicate.

The promoter region of FBXW7 was ligated into a pGL3 vector via polymerase chain reaction. The empty pGL3-basic vector and pRL-TK internal control vector were instantaneously transfected into 293FT cells as a negative control. Then, the pGL3 plasmid, the pRL-TK internal control vector (Promega) and a shTRIP13/TRIP13 vector were co-transfected into 293FT cells by transfection with Lipofectamine 2000. After 48 h of cell culture, luciferase reporter assays were performed according to the manufacturer’s instructions (Promega). There were three replicate experiments in each group.

UMNSAH/DF-1 cell (Cell Bank of the Chinese Academy of Science, China) was a spontaneously immortalized chicken cell line derived from 10-day-old East Lansing Line (ELL-0) eggs. We cultured the cells as described previously in complete growth medium—Dulbecco's modified eagle medium (GIBCO, USA) and 10% fetal bovine serum (GIBCO, USA)—at 39°C in a humidified 5% CO₂/95% air incubator. These cells are adherent with a fibroblast-like morphology.
For western blotting analysis and ELISA, DF-1 cells seeded in 24-well plates were grown overnight to 80–90% confluence prior to transfection with 1 μg/well of plasmid (pcDNA3.1⁺, control) using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's protocol. After 24 h, cells were stimulated by 10 μg/mL poly[I:C]. Then, the culture supernatant and cells were collected 12 h later.
For the dual-luciferase reporter assay, DF-1 cells seeded in 96-well plates were grown overnight to 80–90% confluence prior to transfection with 0.2 μg recombinant plasmid, 0.05 μg pNF-κB-Luc (no eukaryotic selection, Agilent, USA), and 0.01 μg pRL-TK internal control vector (Promega, UK)/well (pcDNA3.1⁺, control) using Lipofectamine 2000 according to the manufacturer's protocol. After 24 h, cells were stimulated by 10 μg/mL poly[I:C], and luciferase levels were measured 12 h later.

HEK293 T cells were cultured in 24-well plates until 50–60% confluency and then transfected with an NF-κB-responsive reporter vector (Beyotime), pRL-TK internal control vector (Promega), and target plasmid. The cells were washed with PBS and collected at 24 h after transfection. Transcriptional activity was measured using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA).

The wild type and mutant 3′-UTR regions of PHB containing predicted miR-27a target site were synthesized (Tsingke Biotechnology Co., Ltd.) and were cloned into the pGL3 vector (Promega, E1741) immediately downstream of the stop codon of the luciferase gene. Either the wild-type or mutant PHB 3′-UTR-luciferase was co-transfected with miR-27a mimic/inhibitor in GSCs using Lipofectamine. PRL-TK internal control vector (Promega, E2241) was co-transfected as the endogenous control for luciferase activity. After 48 h, luciferase activity was measured using Dual-Luciferase Reporter Assay (Promega, E1910). The luciferase activity was normalized to PRL-TK activity.