Hek293e

LuloHya coding DNA sequence (AF132515) was codon-optimized for mammalian expression and synthesized by BioBasic Inc. VR2001-TOPO vector containing LuloHya sequence (Vical Incorporated) was transformed in One Shot TOP10 Chemically Competent E. coli (Invitrogen). Plasmid DNA was prepared using the EndoFree plasmid MEGA prep kit (Qiagen, Valencia, CA). Recombinant protein expression was carried out at the SAIC Advance Research Facility (Frederick, MD). Briefly, human embryonic kidney cells (HEK293E; American Type Culture Collection, Manassas, Virginia) were transfected with 1 mg of plasmid DNA and supernatants were collected 72 h after transfection and shipped frozen to our laboratory for protein purification. Recombinant LuloHya was purified by affinity chromatography followed by size-exclusion chromatography, using Nickel-charged HiTrap Chelating HP and Superdex 200 10/300 GL columns, respectively (GE Healthcare Life Science, Piscataway, NJ). All protein purification experiments were carried out using the AKTA purifier system (GE Healthcare Life Science, Piscataway, NJ). Purified protein was separated in a NuPAGE Novex 4–12% Bis-Tris Protein Gels (Life Technologies) and visualized by Coomassie stain. Protein identity was verified by Edman degradation at the Research Technologies Branch, NIAID, NIH.

3T3-L1 fibroblasts obtained from the Howard Green Laboratory (Harvard Medical School) were cultured in high glucose Dulbecco’s modified eagle medium (DMEM) (Gibco by Life Technologies) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco by Life Technologies), and 1× GlutaMAX (Gibco by Life Technologies) at 37°C and 10% CO₂. Cells were differentiated into adipocytes as described previously (Fazakerley et al., 2015 (link); Norris et al., 2018 (link)) and used for experiments 7–12 days after initiation of differentiation. 3T3-L1 adipocytes stably expressing FLAG-W80A or FLAG-W80A-T309A Akt2 were generated using retrovirus as previously described and characterised previously (Kearney et al., 2019 (link)). HEK293E, HeLa, and HCC1937 cell lines were obtained from the American Type Culture Collection and grown in the medium described above to culture 3T3-L1 cells. MCF7 cells were a gift from Associate Prof. Jeff Holst (Centenary Institute) and were validated by STR. Cells were maintained in Minimum Essential Media (Gibco by Life Technologies Cat#10370–021), with the addition of 10% (v/v) FBS, 1× GlutaMAX, and 1 mM sodium pyruvate (Gibco by Life Technologies) at 37°C and 5% CO₂. Cells were routinely tested for mycoplasma contamination and found to be contamination-free.

Human embryonic kidney 293E (HEK293E), HCT-116, HT-29 (colon cancer), HepG2 (liver cancer), PC3 (prostate cancer), and CT26 (mouse colon cancer) cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). SNU-398 (liver cancer) and SNU-638 (gastric cancer) cell lines were purchased from the Korean Cell Line Bank (KCLB; Seoul, Korea). HEK293E and HepG2 cells were maintained in DMEM with 10% fetal bovine serum (FBS) at 37°C in 5% CO₂. HCT-116, HT-29, PC3, SNU-398, SNU-638, and CT26 cells were maintained in RPMI1640 with 10% FBS at 37°C in 5% CO₂. The stable SNU-449Cp (TM4SF5-low), SNU-449Tp, and SNU-449T₇ (both highly TM4SF5⁺) liver cancer transfectant cell lines were maintained as previously described.⁷ (link) Luciferase-expressing SNU-449T₇ cells (SNU-449T₇-luc) were previously described.¹⁰ (link) C8161 cells (melanoma) were a kind gift from Dr. C.-R. Jung (Korea Research Institute of Bioscience and Biotechnology [KRIBB], Daejeon, Korea).³¹ (link)

Human embryonic kidney 293E (HEK293E) cells (American Type Culture Collection (ATCC), Manassas, VA, USA) were maintained in DMEM containing 10% fetal bovine serum (FBS) at 37°C in 5% CO₂. SW480 (colon cancer; ATCC), SNU-398 (liver cancer), and SNU-638 (gastric cancer; Korean Cell Line Bank, Seoul, Korea) cells were maintained in RPMI1640 containing 10% FBS. HepG2 (liver cancer; ATCC) cells were maintained in Eagle’s Minimum Essential Medium containing 10% FBS. Human umbilical vein endothelial cells (HUVECs) were maintained using an EGM-2 BulletKit (Cambrex BioScience, Walkersville, MD, USA).

All of the DNA sequences were codon-optimized for mammalian expression systems and synthesized by BioBasic Inc. VR2001-TOPO (Vical Incorporated, San Diego, CA, USA). The vector harboring the protein sequences were transformed in One Shot TOP10 Chemically Competent E. coli (Invitrogen, Waltham, MA, USA). Recombinant protein expression was carried out at the SAIC Advanced Research Facility (Frederick, MD, USA). Briefly, human embryonic kidney cells HEK293E (American Type Culture Collection, Manassas, VA, USA) were transfected with 1 mg of plasmid DNA, and supernatants were collected after 72 h of transfection. Recombinant proteins were purified by affinity chromatography followed by size-exclusion chromatography, using Nickel-charged HiTrap Chelating HP and Superdex 200 10/300 GL columns, respectively (GE Healthcare Life Science, Chicago, IL, USA). All protein purification experiments were carried out using the AKTA purifier system (GE Healthcare Life Sciences, Chicago, IL, USA). Purified protein was separated in a NuPAGE Novex 4–12% Bis-Tris protein gels (Thermo Fisher Scientific, Waltham, MA, USA) and visualized by Coomassie stain using the eStain protein stain system (GenScript, Piscataway, NJ, USA). Protein identity was verified by Edman degradation at the Research Technologies Branch, NIAID, NIH.