Matrigel invasion chamber

Matrigel invasion chambers (Corning 354480) were placed in 24-well plates and rehydrated with FBS-free DMEM medium at 37 °C in a CO₂ incubator for 2 h. After removing the medium, 4 × 10⁴ cells of MDA-MB-231 were seeded into Matrigel invasion chambers and control chambers (Corning, 354578) with 125 µL of Opti-MEM medium. Then another 125 µL of each compound (YL2, adamantane, ND1-YL2, or YL2-HyT6) in Opti-MEM medium was added to each chamber. The bottom of the well was filled with 500 µL of DMEM medium containing 10% FBS 1% PS. The chambers were then incubated at 37 °C with 5% CO₂ for 24 h. After removing the medium, the chambers were washed with cold PBS. Then noninvading cells on the upper surface of the membrane were removed by scrubbing with a cotton swab. Cells on the bottom side of the membrane were fixed with 500 µL of 4% formaldehyde for 10 min at rt. After washing with PBS, the bottom side of the chamber was stained with 500 µL of Hoechst 33342 (5 µg/mL) for 5 min at 37 °C in the CO₂ incubator. After washing with PBS, cells were observed with a fluorescence microscope (Nikon, Tokyo, Japan, Eclipse TE 2000). The percent invasion (% invasion) of each sample was determined with the following equation: $\%\mathrm{Invasion}=\frac{Mean \# of cells invading through Matrigel chamber membrane}{Mean \# of cells migrating through control chamber membrane}\times 100$

Cell invasion was assessed using 24-well Matrigel invasion chambers (Costar, Corning, USA) with 8-μm-inserts. Cells were transfected with 30 nM of the miR-1 precursor or the negative control. Forty-eight hours later, the cells were trypsinized. Subsequently, the cells were resuspended in 100 μl of serum-free media and added to the inserts, which were placed into wells with 600 μl of 10% serum-containing media. After 24 h of incubation, the membranes of the inserts were carefully washed with cold PBS, fixed with 4% paraformaldehyde (Sigma, USA) for 15 min, and stained with crystal violet (0.005%, Beyotime, China) for 15 min. The cells on the upper side of the membrane were removed with a cotton swab. Images of invaded cells were collected using a Nikon Ti-S microscope (Nikon, USA).

Cell invasion assays were performed using Matrigel® Invasion Chambers (8-μm pore size, Corning Costar Corporation) according to the manufacturer’s protocol. Cells (2 × 10⁴) were suspended in 200 μl DMEM containing 1% FBS and added to the upper chamber. DMEM (700 μl) containing 10% FBS was added to the lower chamber. Cells were incubated for 20 h at 37 °C with 5% CO². After incubation, cells on the upper surface of the membrane were removed with a cotton swab. Cells on the lower surface of the membrane were fixed with 4% paraformaldehyde for 15 min and stained with 0.2% crystal violet for 10 min. The numbers of invasive cells were calculated on the entire lower surface. The experiment was repeated three times in triplicate.