Inverted phase contrast microscopy

The Lv-shRARβ A549 parental, Lv-shRARβ CD166⁺EpCAM⁺ and Lv-shRARβ A549 CD166^-EpCAM^- and non-infected cells (control). Briefly, the cells were re-suspended in a ratio of 1:1 (v/v) of growth factor-reduced Matrigel (BD Biosciences) attachment plate (Corning, Inc., Oneonta, NY, USA). The sphere was grown in serum-free DMEM-F12 (Gibco) supplemented with 20 ng/ml of epidermal growth factor (EGF), 10 ng/ml of basic fibroblast growth factor (bFGF), 1% B27 supplement (Invitrogen), and 1 % antibiotic-antimycotic (Gibco). The size and number of the formed spheroids were assessed after 21 days of culture using inverted phase-contrast microscopy (Olympus).

Cells were seeded at a density of 10⁶/wells in six-well ultralow attachment plates (Corning Inc, Corning, NY). Sphere culture system was constitute of MacCoy's 5A or DMEM, N2 supplement(Sigma Biochemicals, St. Louis, MO), 1% methylcellulose(Sigma Biochemicals, St. Louis, MO), 20 ng/ml EGF(PeproTech, Rocky Hill, NJ) and 20 ng/ml bFGF(PeproTech, Rocky Hill, NJ). Fresh aliquots of EGF and bFGF were added every two days. At 8–10 days, spheres containing at least 50 cells each were observed by inverted phase contrast microscopy (Olympus, Melville, NY).

In brief, miR330-5p inhibitor or miR-330-5p mimic transfected PC cell migrations were detected using a wound-healing assay. The wound was generated in the cells with 90%–95% confluence by scratching the surface of the plates with a sterile pipette tip. The cells were then incubated in the absence or presence of 3 μmol/L ATO for 20 h and then photographed with an inverted phase-contrast microscopy (Olympus, Japan).

Human NPCs were purchased from American Science Cell Research Laboratories and cultured in NPC Medium (NPCM) containing 500 ml basal medium, 10 ml Foetal Bovine Serum (FBS), 5 ml NPC Growth Supplement, and 5 ml penicillin/streptomycin solution (P/S) (HyClone, Logan, UT, U.S.A.) at 37°C, 5% CO₂ in a humidified incubator. The culture medium was refreshed the next day to remove unattached cells and residual DMSO (Solarbio, Beijing, China), and then it was changed every 2–3 days. When NPCs reached approximately 80–90% confluence, they were split and subcultured at a 1:3 ratio with a 0.25% (w/v) trypsin (Sigma, St.Louis, MO, U.S.A.) solution. The third-generation NPCs were randomly divided as follows: (1) Control (CON) group: cultured in NPCM; (2) HG group: cultured in 0.2 M HG concentration; (3) HG+LIR group: cultured in HG medium containing various concentrations of liraglutide (10, 100, or 1000 nM); (4) HG+LIR+LY group: cultured in HG medium containing liraglutide and LY294002 (LY, 20 μM). The cellular morphology was observed by inverted phase-contrast microscopy (Olympus, Tokyo, Japan). The cell vitality, apoptosis rate, reactive oxygen species (ROS) level, and the expression of GLP-1R, Akt, p-Akt, and caspase-3 of the groups were detected under the same experimental conditions after incubation for 48 h.