Usinglipofectamine 3000

BGC seeded into 24-well plates were transfected with 150 ng of NF-κB-Luc using
Lipofectamine™ 3000 (Invitrogen, CA, USA). The reporter plasmid pGL3Luc-5XNF-kappaB was
constructed with double-strand oligonucleotides that contain five repeats of NF-κB binding
consensus sequence (5′-GGGGAATTTCC-3′) (Wong et al., 2011 (link)), cloned in the pGL3Luc-promoter
vector (Promega, Madison, USA). Briefly, the plasmids were mixed with Optimem, p300
reagent, and Lipofectamine 3000. The mix was incubated for 10 min at room temperature, and
the cells were incubated with this mixture for 24 h. Then, the cells were treated with
caffeic acid with or without D-Asp for a further 24 h. Cells were subsequently harvested
and lysed with 150 μl of 1X reporter lysis buffer (Promega), and protein lysates were
obtained by two freeze-thawing cycles. Luciferase assays were performed with the
Luciferase Assay System (Promega) with equal amounts of the protein lysates. Detection was
performed with an Infinite M200 PRO plate reader (TECAN, Männedorf, Switzerland).

HEK293T cells (ATCC, Manassas, VA) and MIA PaCa-2 cells (human
pancreatic cancer; ATCC, Manassas, VA) were maintained in Dulbecco’s
modified Eagle’s medium (Corning, Lowell, MA) supplemented
with 10% fetal bovine serum (Corning, Lowell, MA) and penicillin–streptomycin
(Corning, Lowell, MA). All cells were grown at 37 °C and 5% CO₂ in a humidified incubator and tested negative for mycoplasma
using the MycoAlert Mycoplasma Detection Kit (LT07-318, Lonza, Morristown,
NJ).
Lentivirus was produced by transfection of HEK293T packaging
cells with three packaging plasmids^{30 (link)} (pMDL,
pRSV-REV, and pCMV-VSVG) and the lentiviral reporter plasmid using
Lipofectamine 3000 (Invitrogen, Waltham, MA) according to the manufacturer’s
recommended protocol. The supernatant containing lentivirus was then
collected 72 h later, aliquoted and stored at −80 °C.
MIA PaCa-2 cells were seeded on a 6-well plate (Corning, Glendale,
AZ) at a density of 3 × 10⁵ cells/well and transduced
with 500 μL lentiviral supernatant plus polybrene (MilliporeSigma,
Burlington, MA) to a final concentration of 8 μg/mL. Transduced
cells were then expanded further in culture and FACS sorted for mStrawberry
expression (BD-FACS Aria, BD Bioscience, Franklin Lakes, NJ).

MiR-127 inhibitor and negative control (NC) were designed and synthesized by
GenePharma (Shanghai, China). Cell transfection was performed using
Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions.
After transfection for 48 h, the efficiency of transfection was monitored by
quantitative real-time polymerase chain reaction (qRT-PCR).