Giemsa s solution

We planted the non-targeting control and BIRC5 siRNA transfected cells separately on a Transwell upper chamber (Falcon) with a pore size of 8.0 μM, coated with Matrigel® Matrix (Corning) and serum-free medium, then put the upper chamber onto 12-well plates with 10% fetal bovine serum medium for 24 h of further cultivation. We fixed the Transwell membrane with 4% paraformaldehyde for 15 min, then stained with Giemsa’s solution (Merck) and took photographs and counted cells under a light microscope (Olympus IX700).

Cells were tranfected with the expression plasmid for TAp63α or with the empty plasmid pcDNA3 (Invitrogen, Carlsbad, CA, USA) using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's suggestions. Forty-eight hours after transfection, cells were transferred to the fresh medium containing G418 (at a final concentration of 800 mg/mL). Two weeks after the selection, G418-resistant colonies were fixed with methanol and stained with Giemsa's solution (Merck, Darmstadt, Germany).

Cells were seeded at 2 × 10⁵ cells/six-well plate and allowed to attach overnight. Cells were transfected with the empty plasmid or with the expression plasmid for TAp73α. Forty-eight hours after transfection, cells were transferred to fresh medium containing 400 μg/ml of G418 (Sigma–Aldrich). Two weeks after the selection, G418-resistant colonies were fixed, stained with Giemsa's solution (Merck Millipore), air-dried and photographed.

HK1 cells stably expressing RERG or empty vector and HK1_EBV cells transfected with RERG or empty vector after 48 h were counted and plated at 500 cells/dish into 60-mm dishes. Then cells were incubated at 37 °C and at an atmosphere of 5% CO₂ for 2 weeks. Once the cells formed visible colonies, the colonies were washed twice with phosphate-buffered saline and fixed with 70% ethanol for 20 min. The cells were appropriately stained with Giemsa’s solution (Merck, Darmstadt, Germany) and allowed to air dry at room temperature. The experiments were triplicated and the numbers of colonies were microscopically counted.

For invasion assay, Boyden chambers (Neuro Probe, Inc., USA) were used according to the manufacturer’s protocol. Briefly, polycarbonate membrane (8 μm pore size, 25 × 80 mm, Neuro Probe, Inc., USA) was pre-coated with 10 μg of human fibronectin (Sigma, MO, USA) on the lower side and matrigel on the upper side. Cells (1.5 × 10⁴) were plated in the top chamber in 50 μl of starvation medium (0.1% FBS) containing drugs or DMSO. After 16 h, stationary cells were removed from the top side of the membrane, whereas migrated cells in the bottom side of the membrane was fixed in 100% methanol and stained with 10% Giemsa’s solution (Merck, Germany) for 1 hr. Invaded cells were counted under the light microscope (400x, ten random fields from each well). All experiments were performed in triplicates.

Cellular migration ability was determined by the trans-well cultivation using Boyden chambers (Neuro Probe, Inc., Gaithersburg, MD, USA). A polycarbonate membrane (8 μm pore size, 25 mm × 80 mm, Neuro Probe, Inc., USA) was precoated with 10 μg of human fibronectin (Sigma, MO, USA) on the side immersed at the lower chamber fulfilled with the conditioned medium (10% FBS). Cells (1.5 × 10⁴) were seeded in the top chamber containing 50 μL of starvation medium (0.1% FBS). For TNF-α effects, cells were preincubated with TNF-α (R&D Systems) at 10 ng/mL for 24 h prior to the trans-well cultivation. After the incubation for 4 h, the remaining cells on the top side of the membrane were removed prior to fixing the migrated cells on the bottom side of the membrane with 100% methanol followed by staining with 10% Giemsa’s solution (Merck, Germany) for 1 h. The migrated cells were finally counted under a microscope. All experiments were performed in triplicates and repeated three times.

Cells were plated on glass coverslips in 24-well-plates (1 × 10⁵ cells per well) and infected with bacteria at an MOI of 10 CFU per cell as described above. After 1, 4, or 24 h post-infection, cells were fixed in situ with 3.7% formaldehyde in PBS for 10 min at room temperature and stained with Giemsa [12 drops of Giemsa's solution (Merck, Germany) in 10 ml distilled water] for 20 min at room temperature. Samples were then washed with water and observed under a light microscope at x1,000.