Fig. 3.

TNFAIP1 regulates HCC cell migration, invasion and metastasis in vitro and in vivo. Cell migration and invasion assay for MHCC97H cells infected with TNFAIP1 or the control lentivirus using transwell membranes. a. The migrated and invaded MHCC97H cells were stained with crystal violet. Scale bars, 25 μm. b. The number of migration and invasion of MHCC97H cells was counted and analyzed in five random fields of each filter (**P < 0.01, ***P < 0.001, Student's t-test). Cell migration and invasion assay for SMMC7721 cells infected with shTNFAIP1 or shControl lentivirus using transwell membranes. c. The migrated and invaded SMMC7721 cells were stained with crystal violet. Scale bars, 25 μm. d. The number of migration and invasion of SMMC7721 cells was counted and analyzed in five random fields of each filter (**P < 0.01, ***P < 0.001, Student's t-test). e. Photomicrographs of metastatic lung nodules in nude mice by tail-vein injection of MHCC97H-TNFAIP1 and Control stable cells. The arrows indicate the metastatic nodes on the surface of the lung (n = 7/group). f. Lung nodules were stained by hematoxylin and eosin (H&E) staining kit and the number of lung metastatic foci in each group was calculated (n = 7) (**P<0.01, Student's t-test). Scale bar, 200 μm. g. Photomicrographs of metastatic lung nodules in nude mice by tail-vein injection of SMMC7721-shTNFAIP1 and shControl stable cells. The arrows indicate the metastatic nodes on the surface of the lung (n = 7/group). h. Lung nodules were stained by H&E staining kit and the number of lung metastatic foci in each group was calculated (n = 7) (**P < 0.01, Student's t-test). Scale bar, 200 μm. i. RT-qPCR analysis of the mRNA level of TNFAIP1 in lungs of nude mice (***P < 0.001, Student's t-test). j. RT-qPCR analysis of the mRNA level of CCND1, MMP2, MMP9 and TNFAIP1 in MHCC97H cells infected with TNFAIP1 or the control lentivirus and in SMMC7721 cells infected with shTNFAIP1 or shControl lentivirus (**P < 0.01, ***P < 0.001, Student's t-test). k. Western blot analysis of the protein expression of CCND1, MMP2, MMP9 and TNFAIP1 in MHCC97H infected with TNFAIP1 or the control lentivirus and in SMMC7721 infected with shTNFAIP1 or shControl lentivirus. l. Immunostaining of xenograft tumors from MHCC97H-TNFAIP1, MHCC97H—Control, SMMC7721-shTNFAIP1 and SMMC7721-shControl groups were conducted using a specific anti-CCND1, MMP2, MMP9 or TNFAIP1 antibodies (DAB staining, brown) and counterstained with haematoxylin (blue) (n = 5/group). Scale bar, 25 μm. m. Western blot was performed on the same tumor samples for detection of CCND1, MMP2, MMP9 and TNFAIP1. Data are presented as means ± SEM from triplicate independent experiments. P-values were determined by two-tailed Student's t-test (**P < 0.01, ***P < 0.001).