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 3-(4,5-dimethylthiazol-2-yl)-5(3-

carboxymethonyphenol)-2-(4-sulfophenyl)-2H-

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

 

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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¡æ 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.