Fig. 4

The overexpression or knockdown of ANGPT2 in HCC cells and their exosomes in vitro. a, b Recombinant lentiviruses that carried the pLV-hANGPT2-mCherry vector or control lentiviruses (only carried pLV-mCherry) were used to infect Hep3B and MHCC97H cells to obtain stable cell lines that overexpressed ANGPT2 fused with mCherry (named Hep3B-ANGPT2 and MHCC97H-ANGPT2, respectively) and matched control stable cell lines (named Hep3B-CT and MHCC97H-CT, respectively). Real-time qPCR detected the relative mRNA levels of ANGPT2 in the stable cell lines (a). Immunoblotting showed the expression levels of ANGPT2 in the stable cell lines and their exosomes, which showed that the levels of exosomal ANGPT2 were increased correspondingly (b). n = 4 for each group, ***P < 0.001, Student’s t-tests. c, d Recombinant lentiviruses that carried lentiCRISPRv2-ANGPT2gRNA or control lentiviruses (only carried lentiCRISPRv2) were used to infect Hep3B and MHCC97H cells to obtain stable cell lines that knocked down ANGPT2 (named Hep3B-ANGPT2crispr and MHCC97H-ANGPT2crispr, respectively) and matched control stable cell lines (named Hep3B-V2 and MHCC97H-V2, respectively). Genomic DNA sequencing detected mutations in the ANGPT2 gene in stable cell lines (c). Immunoblotting showed the expression levels of ANGPT2 in the stable cell lines and their exosomes, which showed that the levels of exosomal ANGPT2 were decreased correspondingly (d)