Amphotericin

Thirty fertilized eggs of Single Comb White Leghorn chickens were purchased from a local hatchery (Koiwai Farm Co., Ltd, Shizukuishi, Iwate, Japan). Embryonic cells from various tissues were prepared as described previously (Kita and Makino, 2014 ). Briefly, fertilized eggs were incubated for 19 days, and embryos were taken from eggs. After decapitated, breast muscles, liver, spleen and kidney were removed and minced finely with scissors. Minced tissues were gently digested using 0.25% (w/v) trypsin, pipetted several times and passed though the gauze to remove the crumble of tissues. Cells were seeded in a Type-1 collagen-coated 48-well plate (Corning, Bedford, MA, USA) with Medium 199 including 2.5 µg/ml amphotericin, 100 units-100 µg/ml penicillin-streptomycin, 50 µg/ml gentamycin and 10% fetal calf serum (FCS) and incubated at 37°C in 5% CO₂/95% air (v/v). All reagents except penicillin-streptomycin for preparation of various tissue cells were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Penicillinstreptomycin was purchased from Biological Industries Ltd. (Kibbutz Beit Haemek, Israel).

Bovine earlobe fibroblasts have been previously reported [19 (link)]. Pieces of swine earlobes were obtained from the Rakuno Gakuen Field Education Center
(Ebetsu, Japan) when pigs (aged 3 days) were ear-notched. The swine earlobe tissue blocks were cut into fragments, and vigorously washed with phosphate-buffered saline (PBS) containing 200
U/ml penicillin, 200 µg/ml streptomycin, and 500 ng/ml amphotericin B (Nacalai Tesque, Kyoto, Japan).
Subsequently, the tissues were treated with 2 mg/ml collagenase type I (Wako Pure Chemical, Osaka, Japan) in Tyrode’s solution and cut into small pieces using scissors. The
tissue slices were further digested for 30 min at 37°C with strong agitation. After extensive washing with PBS and centrifugation, the pellet was resuspended in Dulbecco’s modified Eagle
medium (DMEM) containing 10% fetal calf serum (FCS), 200 U/ml penicillin, 200 µg/ml streptomycin, and 500
ng/ml amphotericin, and filtrated with a cell strainer (70 µm; Corning, New York, NY, USA) to remove debris. The cells were cultured in
DMEM supplemented with 10% FCS at 37°C, 5% carbon dioxide, and 95% humidity.

HEK293T, MDA-MB-231, LM2, and SCP28 cell lines were maintained in DMEM medium (Sigma) supplemented with fetal bovine serum (10%; Gemini Bio Products), penicillin/streptomycin (1%; Corning), and amphotericin (0.2%; Corning). SUM159-M1a cells were grown in F12 medium supplemented with 10% FBS, 10 μg/mL insulin, and 20 ng/mL EGF. Plasmids or siRNAs were transfected into cells using Lipofectamine 2000 or RNAiMax following the manufacturer's manual (Life Technology).
To generate lentiviruses for USP20 overexpression, pLEX empty vector, pLEX-USP20, or pLEX-USP20-C154S was cotransfected with VSV-G and p8.9 into HEK293T cells. Media containing viruses were collected 2 and 3 d after transfection. Recipient LM2 cells were incubated with virus-containing media supplemented with 2 μg/mL polybrene for 24 h. To generate stable cell lines, puromycin was used to select the infected cells.