Methanol ar

The 24-well transwell units with 8 mm I.D. polyester membrane with 8 μm pore size polycarbonate filters (Corning, USA) were employed to investigate the influences of miR-4521 upregulation and FAM129A downregulation on the in vitro migration and invasion properties of 786-O and ACHN cells. For migration assays, 600 μL of RPMI-1640 with 15% FBS was loaded into each lower chamber. 10,000 and 2000 cells from each of the 786-O and the ACHN groups were separately loaded into one upper chamber in 200 μL of RPMI-1640 medium and incubated at 37 °C with 5% CO₂ for 24 h. The cells on the upper surface of the insert that did not migrate were carefully wiped off using cotton swabs. The cells that migrated to the lower surface of the filter were fixed in methanol (AR, Sigma, USA) for 30 min, dried for 5 min at RT, stained in 0.1% crystal violet for 40 min, washed with PBS (200 μL), and counted by randomly selecting five fields per well using an upright light microscope (Olympus, Japan) at a magnification of ×200.
For invasion assays, the filter surface of an insert transwell unit was first coated with 50 μL ice-cold ECM gel (1:5 dilution with RPMI 1640, Sigma, USA) by incubating at 37 °C for 8 h. The loading numbers for the 786-O and the ACHN group cells were 7500 and 15,000, respectively, in 200 μL of RPMI-1640. The remaining steps were the same as for the migration assays.

The effect of CRKL and ETV6 deregulations on the migration and invasion abilities of HepG2, HCCLM3 and HuH7 cells were examined using the Boyden transwell chamber assay. Briefly, 1 × 10⁴ cells in 200 μl serum-free DMEM were seeded onto the upper compartment of transwell with 8 μm pore size polycarbonate filters (Corning, USA). The chambers were then placed into 24-well plates containing 600 μl DMEM with 20% FBS and incubated for 24 h at 37 °C with 5% CO₂. For invasion assay, the inserts were first coated with 50 μl 2.5% ECM gel (Sigma, USA) in DMEM, and incubated at 37 °C for 1 h. 1 × 10⁴ cells in 200 μl serum-free DMEM were seeded onto the upper compartment of the transwell. The chambers were then placed into 24-well plates containing 600 μl DMEM with 20% FBS and incubated for 24 h at 37 °C with 5% CO₂. The non-migrated and non-invaded cells on the upper surface of the insert were removed by swabbing, the migrated and invaded cells onto the lower surface were fixed with methanol (AR, Sigma, US) for 30 min, stained with 0.1% crystal violet for 40 min, washed with phosphate buffered solution (PBS), counted using an upright light microscope (Olympus, Japan) with 100× magnification. Five random field views were counted and averaged.