Penicillin streptomycin

Cells were collected and washed twice with phosphate-buffered saline (PBS) in order to remove serum and then suspended in serum-free DMEM/F12 supplemented with 1% penicillin/streptomycin (GenDEPOT Inc.) and 2% B27 supplement (Invitrogen, Waltham, MA, USA). A total of 25 ng/ml of hEGF and hbFGF (Peprotech, Rocky Hill, NJ, USA) were added every other day. The cells were subsequently cultured for 7 days in Ultra-low-attachment 6-well plates (Corning Inc. Corning, NY, USA) at a density of 5 × 10³ cells/well.

MV4-11 cells were purchased from American Type Culture Collection (ATCC, VA, United States). The cells were passaged for less than 1 month, and mycoplasma infection was checked by PCR once a week. The growth medium was Iscove’s Modified Dulbecco’s Medium (IMDM; ThermoFisher, United States) supplemented with 10% fetal bovine serum (FBS; Corning, United States) and 1% penicillin/streptomycin (GenDEPOT, United States). The cells were maintained in a humidified atmosphere with 5% CO₂ at 37°C. Cell viability was determined using the WST-8 assay (Cyto XTM cell viability assay kit; LPS solution, Daejeon, South Korea) in accordance with the manufacturer’s protocol.

Parental SNU449, SNU761 hepatocellular carcinoma, or HCC827 lung cancer cells lacking TM4SF5, or Hep3B or Huh7 hepatocellular carcinoma endogenously expressing TM4SF5 (Korean Cell Bank) were confirmed for their identities by the provider, and either infected with control retrovirus (SNU449_Cp) or TM4SF5-retrovirus (SNU449_Tp) as previously described [12 (link)], or transiently or stably transfected with a mock control or a FLAG-tagged TM4SF5 plasmid [13 (link)]. Stable cells were selected with 500 μg/ml G418 (A.G. Scientifics). The cells were maintained in RPMI-1640 (WelGene Inc., Korea) containing 10% fetal bovine serum (FBS, GenDEPOT Inc., Barker, TX) and 1% penicillin/streptomycin (GenDEPOT Inc.). Cancer-associated fibroblasts (CAFs) were a kind gift from Prof. Young-Joon Surh (Seoul National University). In certain experiments, the cells were transfected with the indicated expression control vector, TM4SF5 shRNA (shTM4SF5) [26 (link)], or TM4SF5 N-glycosylation mutants [N138A/N155Q (NANQ)] [15 (link)]. Alternatively, the cells were infected with control adenovirus or adenovirus expressing siRNA against p27^Kip1 [27 (link)], or they were infected with retrovirus containing a constitutively active mutant RhoA (Q63L). In cases, different cells were co-cultured in 3D gel conditions at the indicated mixture ratio.

The 3T3-L1 preadipocyte and stromal vascular fraction (SVF) cell lines were obtained from Dr. Hyun Ho Choi and Dr. Kai Sun at UTHealth Houston, respectively. Dulbecco’s Modified Eagle’s Medium (DMEM, GenDEPOT, Baker, TX, USA) supplemented with 10% fetal bovine serum (FBS, GenDEPOT, TX, USA) and 100 mg/mL of penicillin/streptomycin (GenDEPOT) was used to culture 3T3-L1 and SVF cells at 37 °C in a 5% CO₂ incubator until 90% confluence. Confluent cells were maintained in differentiation induction medium consisting of 10 mg/mL of insulin (Sigma, St. Louis, MO, USA), 0.25 mM dexamethasone (Sigma), and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma) in 10% FBS-contained DMEM for two days, followed by maturation medium containing 10% FBS and 10 mg/mL of insulin for six days. Nobiletin (NOB) (Selleckchem, Houston, TX, USA) was first added at 10 or 20 μM concentrations to differentiation induction media and maintained in subsequent media.
To generate Rora/Rorc double knockdown cell lines (Ror DKD), we carried out CRISPR as previously described [36 (link)]. The gRNAs, sense and antisense, were designed using the https://crispr.dbcls.jp/ program (accessed on 12 January 2020) and cloned into the BsmB1 site of the GeCKO vector [37 (link)] for 3T3-L1 transfection followed by puromycin selection. Clones with Ror DKD were selected based on expression analyses.

All cell lines were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). The cell lines were passaged for less than 2 months as ATCC protocols, and mycoplasma infection was checked by PCR once a week. Growth medium for BT549, MDA-MB-453, MCF7, and T47D cells were Dulbecco’s modified Eagle medium (DMEM; Corning, NY, USA), and MDA-MB-231 cells were maintained in RPMI 1640 (Welgene, Daegu, South Korea) supplemented with 10% fetal bovine serum (Corning) and 1% penicillin/streptomycin (GenDEPOT, USA) or 1% ZellShield™ (Minerva Biolabs, Berlin, Germany). The cells maintained under standard conditions, at humidified atmosphere of 5% CO2 at 37°C.