Matrigel chamber

Matrigel chambers (Corning) were used to determine the effect of D3 depletion on invasiveness as per the manufacturer’s protocol. In brief, SCC CTR, D2KO, and D3KO cells treated with T3 (30 nM) were harvested, re-suspended in serum-free medium, and then transferred to the hydrated Matrigel chambers (200,000 cells per well). The chambers were then incubated for 5 days in culture medium. The cells on the upper surface were scraped off and washed away, whereas the invaded cells on the lower surface were fixed and stained with 1% crystal violet in 20% ethanol for 10 min at room temperature. Finally, invaded cells and migrated cells were counted under a microscope and the relative number was calculated.

RASSF1A expression was induced in H1299 with 1 μg/μl doxycycline 24 h invasion. After 24 h, cells were trypsinized and (1 × 10⁵) were cultured in serum‐free medium in the upper wells (in triplicate) of transwell Matrigel chambers (Growth factor reduced, Corning) or transwell inserts, coated with thin layer of collagen (2 mg/ml) and allowed to invade towards bottom wells supplemented with 10% FBS conditional media. After 24 h of incubation, invading cells were fixed and stained with Richard‐Allan Scientific™ Three‐Step Stain (Thermo Fisher Scientific), photographed, and counted manually using Adobe Photoshop software.

Cell invasion and migration abilities were evaluated using Transwell assays. For the invasion assay, Matrigel chambers (Corning, NY, USA) were used according to the manufacturer's instructions. A total of 2.5 × 10⁴ cells/well were resuspended in 100 μl fetal bovine serum-free medium in the upper chamber (8 μm pore size, CoStar, Corning, NY, USA) of a Transwell system. The lower chamber was filled with 0.6 ml medium supplemented with 10% FBS. After incubation for 48 h at 37°C, the invasive cells were fixed with 100% methanol and stained with 0.5% crystal violet before counting under an inverted microscope. For the migration assay, 2.5 × 10⁴ cells were plated in uncoated Transwell upper chambers. The number of cells that migrated across the membrane was estimated under an inverted microscope (Nikon, Tokyo, Japan) at 20× magnification.

Cells (4 × 10⁴) in RPMI medium without FBS were seeded in matrigel chambers (Corning) and deposited into 24-well plates with 500 μl of RPMI/10% FBS per well. Cells were incubated during 22 h at 37°C and 5% CO₂. Then, chambers were stained and fixed with 0.05% crystal violet in 20% methanol for 1 h. Invasive cells were counted under a Nikon Eclipse TS100 inverted microscope.

Trypsin (0.25%) was employed to disperse HCC cell lines (HCC-LM3, Huh-7), which were then centrifuged, resuspended, and spread into the wells of a 24-well culture plate. Matrigel Chambers (8 µm pore size; Corning, Beijing, China) were harnessed in the invasion test instead of the migration assay. The upper chamber, coated with Matrigel beforehand, accommodated 5 × 10⁴ transfected cells. A medium with 10% FBS supplemented with 400 μL of RPMI-1640 was placed in the lower chamber. Following 24 h of incubation at 37 °C, the cells that failed to migrate were removed from the upper compartment. The Transwell membrane was immobilized with 4% paraformaldehyde for 10 min, dyed with 0.5% crystal violet, and then washed in double distilled water. An inverted microscope was adopted for counting. All trials were implemented in triplicate and repeated three times.

HGF migration was assessed using Matrigel chambers (two wells separated by Matrigel on a 6.5-mm diameter polycarbonate membrane with 8-μm pores; Corning Costar, MA) as described previously [31 (link)] with modifications. Cells were starved overnight in serum-free medium containing 0.1% BSA and then pre-incubated with SB203580 (a p38 inhibitor), SP600125 (a JNK inhibitor) or U0126 (a p42/p44 inhibitor) for 1 h before stimulation with K6F. The lower compartment of the well received 500 μl of control medium supplemented with K6F (1 μg/mL), ScK6F (1 μg/mL), plain medium as a negative control, or platelet-derived growth factor (10 ng/ml) as a positive control. The upper compartments received 200 μl of cells (25,000) in serum-free medium containing 0.1% BSA. The cells were incubated for 12 or 24 h at 37°C. Cells on the upper surface of the membrane were completely removed by sweeping with cotton swabs and cells on the lower surface of the membrane were fixed in methanol and stained with hematoxylin. Five digitized images (×40 magnification) from the migrated cell area were captured at random and cells were counted. Migratory activity was expressed as the relative invasion capacity compared with that measured in control medium.