Fig 2: High expression of CFL1 is associated with poor survival in HCC patients. (A) Protein levels of CFL1 in 124 paired HCC and adjacent tissues were detected by immunohistochemistry. Paired samples t-test was used. (B) The mRNA expression level of CFL1 in TCGA and GTEx’s liver hepatocellular carcinoma (LIHC) dataset including 160 tumor free tissues and 369 tumor tissues. The box plot is generated by GEPIA2 with jitter (size = 0.4). Red cluster: tumor samples; blue cluster: normal samples. Genes with higher | log2FC| values (>1) and lower Q–values (<0.01) were considered differentially expressed genes and marked the symbol *. Mean OS (C) and DFS (D) between patients with high and low CFL1 expression in our cohort. Mean OS (E) and DFS (F) between patients with high and low CFL1 expression from TCGA. Kaplan Meier analysis and log rank test were used.

Fig 3: Silencing CFL1 inhibited migration and invasion of HCC cells. (A) HepG2 or (B) Hep3B cells were transfected with individual CFL1-specific siRNAs or control siRNA for 24–72 h, and cell lysates were collected and subjected to western blotting. After transfection, a scratch was made in monolayers of (C) HepG2 or (D) Hep3B cells, and the migration ability of the cells was detected with an inverted microscope.

Fig 4: NJXA suppressed pulmonary tumor metastasis in mice. Six week-old male nude mice were injected with 1 × 106 HepG2 cells via tail veins. After injection the mice were randomly divided into three groups (n = 8), and treated with vehicle control (1% DMSO in normal saline), NJXA (20 mg/kg), or 5-FU (2 mg/kg every 2 days) via intraperitoneal injection. After treatment for 16 days, the mice were sacrificed. (A) Body weight analysis every 2 days during the whole experiments. (B) Representative lung images and HE staining of lungs from each group. (C) Quantitative analysis of metastatic nodes in lung. (D) Lung weight analysis after treatment. (E) Immunohistochemistry staining of CFL1, F-actin and G-actin in lung tissues. All the data are presented as means ± SD. **P < 0.01 and ***P < 0.001 compared to the control (n = 3).

Fig 5: Biological function of HBx on cell viability, cytoskeleton remodeling and lipid metabolism in vitro(A) The HBx promotes the Huh-7 cells proliferation compared with the control group (*p < 0.05). (B) The total and free intercellular cholesterol concentration of Huh-7/myc-HBx cell were significantly increased compared with control (*p < 0.05). (C) The lipid droplet was accumulated in Huh-7/myc-HBx cells using the oil red O staining. (D) The ADFP mRNA level of Huh-7/myc-HBx cells was significantly increased compared with control group (*p < 0.05). (E) The cytoskeleton and lipid metabolism related proteins, including CDC42, CFL1, PPAR? and ADFP in Huh-7/myc-HBx cells were significantly increased compared with control group. (F) The lipid droplet was obviously accumulated in 12M and 24M p21HBx/+ mice using the oil red O staining.