Hek293 cells

Human hepatoma Huh-7, Hep3B cells, human embryonic kidney Hek293 cells, breast carcinoma MCF-7, and MDA-MB-231 cells were all purchased from Bioresource Collection and Research Center (BCRC; Hsinchu, Taiwan) with authentication. They were maintained in DMEM medium supplemented with 10% (v/v) fetal bovine serum (FBS) at 37° C under 5% CO₂ and 100 % humidity. Plasmid transfection was done using transit-LT1 transfection reagent (Mirus Bio LLC., Medison, WI, USA) according to the manufacturer's instruction. Transfected cells were selected and enriched with culture medium containing G418, hygromycin B, or puromycin. For the treatment of exosome at MCF-7 cells, 2 × 10⁵ cells were plated overnight and incubated at serum-free medium for 4 hr. Exosome solution with desired protein equivalent was added and pipetted gently into the medium and kept at incubator for additional 24 hr. The cells were then harvested for PCR, or western blot.

ESCs were established from mouse (C57BL/6 J or C57BL/6J x 129X1/svJJmsSlc) blastocysts. ESCs were cultured at 37 °C under 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM)-high glucose (D6429-500ML, Sigma) supplemented with 1% fetal bovine serum (FBS), 17.5% KSR (10828028, Gibco), 0.2% 2-mercaptoethanol (21985-023, Gibco), and 1 × 10³ units/mL ESGRO mouse LIF (ESG1107, Millipore). HEK293 cells (RIKEN BRC) were cultured in DMEM supplemented with 10% FBS. Cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocols, harvested 48 h later, and sorted using FACSAriaII (BD Biosciences). The molar ratio of the dCas9-peptide array fusion vector, scFv-GFP-TET1CD vector, and gRNA vector in the transfection was 1:2:4, respectively. The ESCs were used for the tetraploid complementation experiment after 12 days in culture.

HEK293 cells (human embryonic kidney cell line) and Vero cells (African green monkey kidney cell line) were obtained from the RIKEN BioResource Center Cell Bank. Vero/TMPRSS2^{43 (link)} were obtained from the Japanese Collection of Research Bioresources Cell Bank. 293T cells were maintained in Dulbecco’s modified Eagle’s Medium (DMEM) containing 10% fetal calf serum (FCS). Vero cells and Vero/TMPRSS2 cells were maintained in DMEM containing 5% FCS and 1 mg/ml G418 (Invivogen) was added to the growth medium for Vero/TMPRSS2 cells.

Mouse embryonic fibroblast26 (link) (MEF, gift from Dr. Akira Kitamura, Hokkaido University) and HEK293 cells (Riken BRC, Ibaraki, Japan) were maintained in a 5% CO₂ humidified atmosphere at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Hyclone Lab., Logan, UT, USA), 1 × 10⁵ U l⁻¹ penicillin G and 100 mg l⁻¹ streptomycin sulfate (Wako, Osaka, Japan). The day before the experiment, cells (early passage) were plated on a 35-mm glass-base dish (AGC Techno Glass, Ltd., Shizuoka, Japan) to 60–70% confluence for live-cell imaging or a 6-well plate (Thermo Fisher Scientific, Waltham, MA, USA) for expression assay. The medium was replaced by phenol red-free medium (Opti-MEM, Life Technologies, Gaithersburg, MD, USA) before confocal imaging. Microinjection of the DNA probe was conducted at the confocal microscope stage by combining Femtojet (Eppendorf, Hamburg, Germany) and Injectman NI2 (Eppendorf) with Femtotip (Eppendorf) as an injection needle. Injection pressure was adjusted to deliver the desired amount of DNA.

HEK293 cells (RCB1637: Riken Bioresource Center, Tsukuba, Japan) were cultured in a minimum essential medium (Thermo Fisher Scientific, Tokyo, Japan) supplemented with 10% foetal bovine serum under a 5% CO₂ atmosphere at 37 °C. The culture medium was changed every 3 days, and cells were passaged at 80% confluence using a 0.02% ethylenediaminetetraacetic acid (Sigma-Aldrich, St. Louis, MO)/DPBS solution.

All in vitro experiments were performed by using HEK293 cells (RIKEN BRC, Ibaraki, Japan) stably expressing hTRPA1, which were prepared by a T-Rex^TM system (Invitrogen). The cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 mg/mL), Zeocin™ (0.1 mg/mL), and blasticidin S (0.05 mg/mL) at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. The cells were treated with tetracycline (1 µg/mL) the day before experiment.