Figure 3.

The expression of ASNS suppressed the proliferation of and colony formation by HCC cell lines. (A) Establishment of transient and stably transfected HCC cell lines. The SMMC7721 cells were transfected with the GV142/ASNS (ASNS) or GV142/GFP (con) plasmid. The MHCCLM3 and MHCC97H cells were infected with lentivirus/siASNS (siASNS) or lentivirus/scrambled control (Con) and evaluated by an immunoblotting assay. (B) The HCC cells (2 × 10³ cells per well) were seeded in 96-well plates and cultured overnight at 37 °C. Cell proliferation was detected using the CCK-8 assay at various time points according to the manufacturer's instructions. (C) Images of cells stained with 4,6-diamidino-2-phenylindole (DAPI) were captured using an sCMOS camera with an ImageXpress Micro XL high-content screening system (Molecular Devices, Sunnyvale, CA, USA) and were analysed using MeteXpress 4.0 software (Molecular Devices). The cell cycle distribution was calculated. (D) A panel clonogenic assay of two HCC cell lines after ASNS knockdown. The HCC cells (2 × 10³ cells per well) were seeded in 6-well plates and incubated for 10 days, and then the number of cell colonies was counted. (E) Knockdown of ASNS promoted xenograft tumour growth in nude mice. The MHCCLM3-con and MHCCLM3-siASNS cells (5 × 10⁶ cells per mouse) were inoculated into the flanks of nude mice, and tumour growth was observed. Error bars represent the s.d. *P<0.05, **P<0.01.