tetrazolium (MTS) assay
5 × 103/wells MHCC97H and Hep3B cells were seeded in a 96- well plate. Cells were transfected with indicated plasmids after being cultured for 24 hours. 20 μL MTS buffer (K300-500, Amyjet Scientificlnc, China) was added to each well and incubated for 2-4 hours after cells were transfected for 24 hours. The absorbance
value was measured at 450 nm. Five duplicated wells were set for each group. Cell survival rates = (optical density (OD)EXP − ODEmpty/ ODControl − ODEmpty)%.
The experiment was repeated 3 times.
2.4 | Immunohistochemistry (IHC)
Formalin-fixed paraffin-embedded 5-μm tissue sections were incu- bated at 60°C for 30 minutes and then deparaffinized in xylenes, rehydrated through a graded series of alcohol concentrations for 5 minutes and rinsed by water for 2 minutes. After antigen retrieval by 1mMTris-EDTA (pH = 8.0) was performed, all sections were rinsed with phosphate buffer saline (PBS) for three times and left to block at room temperature in 3% H2O2-methanol for 10 minutes, followed by
2.7 | Transwell assay
Cells were resuspended using the DMEM supplemented with 10 g/L bovine serum albumin (BSA) and adjusted to the final density of 1 × 104 cells/mL. The Transwell chamber was put into a 24-well plate, and the upper chamber surface of the bottom membrane of the Transwell chamber was coated with Matrigel (40111ES08, Yeasen BioTechnologies co., Ltd., Shanghai, China) at the ratio of 1:8 and air-dried overnight at 4°C. After routine digestion, cells
MA et Al.
were resuspended in a culture medium, adjusted to a cell density of 1 × 105 cells/mL, and 200 μL of the cell suspension was added to the top of the Transwell chamber covered with Matrigel (BD, San Jose, CA, USA). The lower chambers were supplemented with 600 μL medium containing 20% FBS. The Transwell chamber was collected following 24 hours of incubation at 37°C, where the Matrigel and cells were removed with a cotton swab, and subjected to fixation and then stained by crystal violet dissolved in methanol. Stained cells were counted in five random fields per well using an inverted microscope (XDS-800D; Caikon, Shanghai, China). The invasive cells were counted. Triplicates were set in each group. This experiment was repeated for at least 3 times.
2.8 | MatrigelTM pseudo-tube formation
Matrigel (Shanghai Shanran Biological Technology Co., Ltd., Shanghai, China) was placed in a refrigerator at 4℃ overnight to be melted into yellow gel-like fluid. A total of 70 μL yellow gel-like fluid was added to pre-cooled 96-well plate by pre-cooled micropipette, followed by incubation at 37°C for solidification. After cells were transfected for 48 hours, they were starved for 1 hour and resuspended in DMEM to prepare for cell suspension. Cell suspension (1 × 105 cells/mL)
was seeded into pre-coated 96-well plate and added with medium of different groups. The cell plate was then incubated at 37°C for 18 hours. Each group was set up with independent triplicates. Three contiguous fields of pictures were taken by an upright microscope (×100) (Leica, Weztlar, Germany), and the number of intact capillary lumens surrounded by cells was analysed by Image J software. This experiment was repeated for at least 3 times.