12 well cell culture plate

ACC-LC-73, HS24, PC1, PC10, and SK-MES-1 cells were grown to 40–60% confluency in 12-well cell culture plates (Falcon, Tokyo, Japan), and then the medium was changed to Opti-MEM (Thermo Fisher Scientific, Tokyo, Japan). A total of 40 pmol of miRNA mimic/inhibitor (Thermo Fisher Scientific, Tokyo, Japan), miRNA inhibitor (Thermo Fisher Scientific, Tokyo, Japan), or FAM46C siRNA (Sigma, Tokyo, Japan) was transfected into cells using Lipofectamine 2000 (Thermo Fisher Scientific, Tokyo, Japan) according to the manufacturer’s instructions. After incubating for 24 hours, the cell medium was changed to RPMI1640 supplemented with 10% FBS. Transfected cells were collected at 48 hours after transfection. Total RNA was isolated from cells using TRIzol reagent (Thermo Fisher Scientific, Tokyo, Japan), and 1 μg was reverse-transcribed into cDNA using ReverTra Ace (TOYOBO, Osaka, Japan). Expression of FAM46C or GAPDH mRNA was examined using Step One (Thermo Fisher Scientific, Rockford, IL, USA) and the TaqMan gene expression assay (FAM46C: hs00214530, GAPDH: 4326317E). Expression of MYC mRNA was examined using Step One and KOD SYBR qPCR Mix (TOYOBO, Osaka, Japan) with the following primer sequences: 5′-GGTCTCCACACATCAGCACAA-3′, 5′-TCTTGGCAGCAGGATAGTCCTT-3′. Relative expression was evaluated with the ∆∆Ct method. All RT-qPCRs were performed in triplicate.

MiaPaCa-2 cells were plated in 12-well cell culture plates with at a density of 20,000 cells per well. Twenty-four hours later, apelin was added to the wells at a final concentration of 1 µM and then added every 24 h. Cells were detached and counted every 24 h.
Migration was conducted using 8 µm pore size membrane transwell inserts for 12-well cell culture plates (BD Falcon, Le Pont de Claix, France). After 4 h of serum starvation, medium in the upper chamber was replaced, whereas DMEM containing 5% fetal calf serum, with or without 1 µM apelin, was added to the lower chamber for 18 h. Cells that migrated through the filter membrane were dyed with 0.2% crystal violet.

Transport experiments were performed as described previously [24 (link)] with some modifications. All studies were conducted with HBSS supplemented with 10 mM HEPES (pH 7.4). Cell monolayers were preincubated for 30 min at 37 °C with the study buffer in both the apical (AP) and basolateral (BL) sides, and TEER value was measured. The inserts were then transferred to a 12-well cell culture plate (BD, Franklin Lakes, NJ, USA) containing 1.5 mL of prewarmed study buffer (pH 7.4, 1% v/v DMSO) in each well. Transport experiments were initiated by replacing the AP side of the cell monolayers with 0.5 mL of test substance solution (5 µM) in the study buffer (pH 7.4, 1% v/v DMSO). The cell monolayers were then incubated at 37 °C on a rocker operated at 30 rpm. Experiments were terminated at 120 min by removing the inserts. Aliquots (100 µL) of both the AP and BL solutions were transferred to a 96-well plate followed by dilution with an equal volume of acetonitrile containing the internal standard for sample analysis. The analytical methods are described below. TEER value was measured thereafter to determine if the integrity of the cell monolayers were maintained during the experiments.