Cell proliferation was performed as described.87 For analysis of cell proliferation, 2 × 104 cells/well were seeded in 12-well dishes. For 4 days, the cells (in triplicates) were trypsinized and enumerated every 24 h using a hemocytometer.
Invasion assay was conducted using CytoSelect 96-Wells Cell Invasion Assay Kit (Cell Biolabs Inc., CA, USA) as described.88 Cells (1 × 105) were seeded in 96-well culture plates, and the assay was conducted according to the manufacturer's protocol.
Wound-healing/migration assay was performed as described.89 In brief, 1 × 106 cells were seeded in 60-mm plates and grown until 90% confluent. Prior to the experiment, the cells were incubated with mitomycin C (10 μg/ml), which inhibits cell division. After 2–3 h of incubation at 5% CO2 and 37 °C, the cell monolayer was wounded with a 200 μl pipette tip. Cells were then washed thrice with phosphate-buffered saline (PBS), and images of this wound were acquired by Nikon eclipse TE2000-U microscopy (Nikon Instruments Inc., Melville, NY, USA) at various time points (0, 12 and 24 h).
Drug-resistance analysis was performed as described previously.90 In brief, 1 × 105 cells were seeded in 12-well dishes. Following overnight incubation at 5% CO2 and 37 °C, they were treated with 0.5 μm doxorubicin and incubated for another 48 h. They were then enumerated and represented as percent (%) of surviving cells. Full paper
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