Fig. 2
The gain- and loss-of-function experiments indicated the involvement of EGFL8 in migration, invasion and apoptosis of HCC cells. a The expression of EGFL8 in HCCLM3, Hep2B and Hep3B liver cancer cell lines was significantly lower than that in HL-7702 normal liver cell line. EGFL8 expression level was lower in high-metastatic liver cancer cell line HCCLM3 than low-metastatic liver cancer cell lines HepG2. EGFL8 expression in the latter was also lower than almost non-metastatic liver cancer cell line Hep3B. Data were showed as mean ± SEM. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. b The overexpression or inhibition efficiency of EGFL8 protein in HCCLM3 or Hep3B cell lines after lentivirus infection was determined by Western blot. GAPDH was also measured as loading control. Data were showed as mean ± SEM. ***, P < 0.001. Full-length blots are presented in Supplementary Fig. 1. c Wound healing assay showed that 24 h and 72 h wound closure rates were all remarkably reduced in HCCLM3EGFL8 cells but notedly elevated in Hep3BshEGFL8 cells. DAPT (N-[N-(3,5-Difluorophenacetyl-L- alanyl)]-(S)-phenylglycine t-butyl ester) treatment could significantly decrease wound closure rate of Hep3BshEGFL8 cells, whatever 24 h or 72 h. Magnification, × 200. Data were showed as mean ± SEM. *, P < 0.05, **, P < 0.01, versus HCCLM3Vector cells; #, P < 0.05, versus Hep3BshCtrl cells; ###, P < 0.001, versus Hep3BshCtrl cells; N.S., no significance, versus Hep3BshCtrl cells; §§§, P < 0.001, versus Hep3BshEGFL8 cells. d The results of Transwell invasion assay showed the number of HCCLM3EGFL8 cells invaded through the matrigel was significant decreased than that of HCCLM3Vecter cells. However, the number of Hep3BshEGFL8 cells permeated through the matrigel was obviously increased than that of Hep3BshCtrl cells and DAPT treatment could significantly reduce this number. Magnification, × 200. Data were showed as mean ± SEM. **, P < 0.01; ***, P < 0.001. e The MTT assay showed there was no significant difference between the growth curves of HCCLM3EGFL8 and HCCLM3Vector cells. Although a very subtle increase of proliferation could be found in Hep3BshEGFL8 cells compared with Hep3BshCtrl cells, which did not lead to a significant difference between the proliferation of these two groups of cells. DAPT treatment did not exhibit a significant influence on the proliferation of Hep3BshEGFL8 cells. N.S., no significance. f The apoptosis levels of HCC cells were determined by flow cytometric analysis and the cell percentage in each quadrant was shown. The percentage of apoptotic cells in every group of HCC cell was shown by histogram. Data were showed as mean ± SEM. **, P < 0.01; ***, P < 0.001. N.S., no significance