The mice were kept in a pathogen-free environment with a temperature range of 22-26°C, humidity (40-60%) and subjected to a 12-hour light and 12-hour dark cycle. To establish stable cell lines, HepG2 cells were transfected with luciferase-IRES-GFP-pcDNA, and Hep3B cells were transfected with luciferase-IRES-GFP-pcDNA, luciferase-IRES-GFP-WT-PHF2 or luciferase-IRES-GFP-PHF2-C23A. Cells stably expressing the indicated plasmids were selected with G418. For gene silencing, PHF2-targeting siRNAs were transfected directly into HepG2 cells using in vivo-jetPEI on the first day of transplantation. To prevent gene recovery, siRNAs/jetPEI were administered via the tail vein 5 days after following Polyplus' recommendation. HepG2 cells (2 × 10^6) and Hep3B cells (5 × 10^5) were orthotopically injected into the livers of NSG mice. After the injection of HCC cells, each group was randomly divided into two subgroups; one was fed a normal diet, and the other was fed a custom-made 45% PA-enriched diet. At the end of the experiment, the mice were sacrificed, and their tumor tissues were stored at -80°C for western blotting and RT-qPCR analysis.

Palmitic acid (PA) is the most common fatty acid in humans and mediates palmitoylation through its conversion into palmitoyl coenzyme A. Although palmitoylation affects many proteins, its pathophysiological functions are only partially understood. Here we demonstrate that PA acts as a molecular checkpoint of lipid reprogramming in HepG2 and Hep3B cells. The zinc finger DHHC-type palmitoyltransferase 23 (ZDHHC23) mediates the palmitoylation of plant homeodomain finger protein 2 (PHF2), subsequently enhancing ubiquitin-dependent degradation of PHF2. This study also reveals that PHF2 functions as a tumor suppressor by acting as an E3 ubiquitin ligase of sterol regulatory element-binding protein 1c (SREBP1c), a master transcription factor of lipogenesis. PHF2 directly destabilizes SREBP1c and reduces SREBP1c-dependent lipogenesis. Notably, SREBP1c increases free fatty acids in hepatocellular carcinoma (HCC) cells, and the consequent PA induction triggers the PHF2/SREBP1c axis. Since PA seems central to activating this axis, we suggest that levels of dietary PA should be carefully monitored in patients with HCC.