Type 2 collagenase

Mouse liver cells purification protocol was adapted from refs. ^{31 (link),32 (link)}. Briefly, liver cells were isolated following a two-step protocol of Pronase/Collagenase digestion. Firstly, the liver was perfused with HBSS (Sigma #H6648) containing 0.2 mg/mL EDTA. Secondly, a perfusion with 0.4 mg/mL Pronase (Sigma #P5147) and 2 mg/mL Collagenase Type II (Worthington #LS004196) in HBSS was performed. Finally, the liver was perfused with HBSS containing 0.4 mg/mL Pronase, 2 mg/mL Collagenase Type II and 0.1 mg/mL DNase I (Roche #R104159001). The liver was minced and further digested with HBSS containing 0.4 mg/mL Pronase, 2 mg/mL Collagenase Type II and 0.1 mg/mL Dnase I for 25 min with shaking at 37 °C. To stop digestion, DMEM (Thermo Fisher #31966047) was added. The resulting liver cell suspension was filtered and washed three times through centrifugation at 300 g for 4 min in 2% FBS-PBS. The suspension was then subjected to density gradient centrifugation using 20% Percoll (GE Healthcare #17-0891-01) to remove dead cells. Resuspension of the cells in ACK lysis buffer (Thermo Fisher #A1049201) allowed the lysis of red blood cells. Cell suspension was then centrifuged and resuspended in 2% FBS-PBS to proceed with scRNA-seq analysis using 10X Genomics Chromium Single-Cell 3’ according to the manufacturer’s instructions.

NOD/SCID mice (Sankyo Laboratory Co. Ltd., Tsukuba, Japan) were bred and maintained in accordance with our institutional guidelines for the use of laboratory animals. A total of 2 × 10⁶ HCC cells stably expressing shRNA against G9a or luciferase were implanted into the subcutaneous space on the right and left sides of the backs of recipient NOD/SCID mice, respectively. In the BIX-01294 treatment model, a total of 2 × 10⁶ Huh1 or Huh7 cells were implanted into the subcutaneous space on the backs of NOD/SCID mice. BIX-01294 (10 mg/Kg) was administered intraperitoneally 3 times a week. Tumor formation and growth were observed weekly. Subcutaneous tumors were also subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry with anti-H3K9me2, anti-CASP3, and anti-Ki67 (DAKO, Carpinteria, CA) antibodies. For the analyses of xenograft tumors, small pieces of tumor were digested in DMEM containing 5 mg/mL collagenase type II (Roche). The cell suspension was centrifuged on Ficoll (IBL, Gunma, Japan) to remove dead cells and debris. The harvested cells were subjected to sphere formation assays. All experiments were performed in accordance with the institutional guidelines for the use of laboratory animals.

Collagenase type II was from Roche (Lisbon, Portugal). D-[[¹⁴C(U)]-glucose (250 mCi/mmol/L) was from Scopus Research BV (Wageningen, The Netherlands). Human insulin, Actrapid, was kindly supplied by Novo Nordisk A/S (Paço de Arcos, Portugal). N-heptane was from Merck-&-Co., Inc. (Whitehouse Station, NJ, USA). Optiphase Hisafe was from PerkinElmer, Inc. (Waltham, MA, USA). RNeasy^® MiniKits were from QIAGEN Sciences (Germantown, MD, USA). High Capacity cDNA Reverse Transcriptase kits were from Applied Biosystems (Forest City, CA, USA). PCR primers were designed using Beacon Designer software and synthesized by IDT-Integrated DNA Technologies, Inc. (BVBA, Leuven, Belgium). SYBRGreen Supermix was from Quanta Biosciences (Gaithersburg, MA, USA). All other reagents were from Sigma (St. Louis, MO, USA). ECF reagent was from GE Healthcare (Little Chalfont, UK).