Fig. 8: DRAM1 promotes metastasis of HCC cells via enhanced autolysosomes.

A Levels of p62 and LC3 protein were examined by WB. B The WB shows the protein stability of VAMP8 in the CHX chase assay at the indicated time points. C Levels of p62 and LC3 protein were examined by WB in HepG2 cells. D Levels of p62 and LC3 protein were examined by WB in ATG7 KO and Control cells. E, F Representative images of cell colonies in transwell assays with the indicated group. Transwell migration assay (Upper) and transwell invasion assay (Down) in HepG2 cells. Representative Figures from three independent experiments are shown. The quantification of migrated cells per field is shown in the bar graphs. Data are shown as means ± SD from three independent experiments. Scale bar: 100 μm. G The representative image shows the immigration of Control and sh-ATG5 HepG2-GFP cells, with or without stable overexpression DRAM1, the images were acquired at 3 dpi at the tail region in WT (AB) zebrafish embryos. The cells were infected with lentivirus encoding GFP and approximately 300 HepG2-GFP cells were injected into the Duct of Cuvier (DoC) at 2 dpf (top image). Micro-metastases developed at 3 dpi (5 dpf) in the tail region (GFP, HepG2 cells). Scale bar: 100 μm. H Quantification of the number of tumor clumps in the tail region at 3 dpi. The number of tumor clumps was calculated in tail region by Image J, n = 9 larvae. I Representative images showing the migration of Control and ATG7 KO 97H-GFP cells, with or without stable overexpression DRAM1, in the hindbrain region at 2 dpi. The white cycle indicates the hindbrain region. Scale bar: 100 μm. (J) Quantification of the number of migrated tumor cells out of the hindbrain region at 2 dpi, n = 6 larvae. Data are shown as means ± SD. Data in (A–E, G–J) are representative of three independent experiments, P-values were calculated by two-tailed Student’s t tests (F, H, J). P-values are indicated in the figure.