Transfect HCC cells with ISX fusion GFP expression plasmid or ISX shRNAi, select stable clones. 2. Grow clones in MEM culture medium with 10% FBS and 1x NEAA. 3. Prepare 0.6% and 1.2% agar in ddH2O, keep warm at 65°C. If the agar solidifies, repeat the preparation steps. 4. Form bottom gel layer with 1 ml of 2x MEM culture medium with 20% FBS and 1 ml 1.2% agar. 5. Harvest and count cells, dilute to 1 x 10^4 cells/ml in 2x MEM with 20% FBS. 6. Mix 1 ml diluted cells with 1 ml 0.6% agar, solidify at 37°C for 30 min. 7. Add 2 ml 10% FBS MEM culture medium to each well. Repeat the medium addition every 3 days. 8. Culture for 2 weeks, changing medium every 3 days. 9. Stain colonies with 0.05% crystal violet-75% ethanol or 40% methanol, count colonies larger than 0.5 mm. If the staining is not clear, repeat the staining process.