, Brea, CA) The assay was performed in a six-well plate Fifty H

, Brea, CA). The assay was performed in a six-well plate. Fifty HCC cells were resuspended in 2× culture medium and mixed with 1% liquid agar in the ratio of 1:1. The mixture was added on top of the base agar layer (0.5% agar in culture medium). Culture medium was added on top of the solidified agar and was incubated in a CO2 incubator at 37°C for 2 weeks. The total number of colonies formed was counted, and images of colonies were taken under a microscope. Transwell migration assay was performed as previously described.19 Briefly, Selleckchem p38 MAPK inhibitor 1 × 105 cells in serum-free culture medium were added to the upper chamber of the transwell insert (Corning Inc., Corning, NY), whereas the lower

chamber was filled with

medium containing 10% fetal bovine serum. Cells were allowed to migrate at 37°C for 12 hours. Cells that migrated through the membrane to the lower surface of the transwell were fixed with methanol and stained with crystal violet. HCC cells (1 × 106) were resuspended in 100 µL of phosphate-buffered saline (PBS) and injected subcutaneously (SC) to the left or right side of 8-week-old male BLB/cAnN nude mice. Tumor size was measured weekly, and tumor volume was calculated with the following formula: 1/2 length × width2. Mice were sacrificed on Z-IETD-FMK mouse week 5, and tumors were harvested. Firefly-luciferase–labeled MHCC97L cells (MHCC97L-Luc) were established as previously described20 and infected with shSUV39H1 viral particles. MHCC97L-Luc cells (1 × 106) were resuspended in 20 µL of Dulbecco’s modified Eagle’s medium with high glucose/Matrigel (1:1) and orthotopically injected into the left hepatic lobe of 8-week-old male BLB/cAnN nude mice. For bioluminescent analysis, 100 mg/kg of D-luciferin were injected intraperitoneally

into mice 5 minutes before imaging. Mice were sacrificed on week 5, then images of in vivo tumor growth and ex vivo lung and lymph node metastasis were taken using an IVIS 100 Imaging System (Xenogen, Hopkinton, MA). All animal experiments were performed according to the Animals (Control of Experiments) Ordinance (Hong Kong) and the Institute’s guidance on animal experimentation. Senescence-associated beta-galactosidase (β-Gal) activity assay was performed according to the previously see more described protocol.21 Briefly, HCC cells were seeded at 2 × 104 cells per well into a 24-well plate and incubated for 5 days. Cells were washed with PBS twice and fixed in 2% formaldehyde in PBS for 5 minutes. After washing with PBS twice, cells were incubated with staining solution (0.2 M of citric acid/sodium phosphate [pH 6], 0.5 M of K4[Fe(CN)6], 0.5 M of K3[Fe(CN)6], 5 M of NaCl, 1 M of MgCl2, and 1 mg/mL of X-gal) at 37°C for 12-16 hours and washed with PBS twice. Three microscopic images of each well and three wells were counted for each treatment.

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