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3 protocols using Collagen-coated dish

1

Acquisition and Characterization of Diverse Tumor Cell Lines

The melanoma cell line B16F10 was purchased from the Cell Resource Center for Biomedical Research (Sendai, Japan). The MC-38 mouse colon adenocarcinoma cell line was provided by Takeshi Setoyama and Tsutomu Chiba at Kyoto University. The BrafV600E melanoma cell line (Dhomen et al., 2009 (link)) was provided by Caetano Reis e Sousa at the Francis Crick Institute. 4T1 mammary tumor cells were purchased from ATCC (Manassas, VA) and maintained on a collagen-coated dish (AGC Techno Glass, Tokyo, Japan).
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2

Establishment of Engineered 4T1 Breast Cancer Cell Lines

4T1 mammary tumor cells were purchased from ATCC (Manassas, VA, USA) and maintained on a collagen‐coated dish (AGC Techno Glass, Tokyo, Japan) in RPMI‐1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS (Sigma‐Aldrich, St. Louis, MO, USA) and 1% penicillin–streptomycin (Nacalai Tesque, Kyoto, Japan) at 37°C in a humidified atmosphere containing 5% CO2. A BindShut II dish (AGC Techno Glass) was used for suspension culture. For establishment of 4T1 cell lines expressing iRFP, 4T1 cells were infected with the lentivirus produced from pCX4bsr‐iRFP. After blasticidin S selection at 10 μg/mL for 1 week, the bright cell population was collected by FACS (FACSAria II; BD Biosciences, Franklin Lakes, NJ, USA) and subjected to single cell cloning. For the expression of tdTomato, piggyBac transposon plasmids encoding the tdTomato gene (pPBbsr2‐tdTomato) and pCMV‐mPBase were cotransfected into 4T1 cells by using Lipofectamine 3000 (Thermo Fisher Scientific). After blasticidin S selection (10 μg/mL) for 1 week, the bright cell population was collected by FACS. For knockdown experiments, 4T1 cells were infected with lentiviruses encoding shRNAs (Table S1) or scramble control. After puromycin selection for 1 week, knockdown efficiency was confirmed by immunoblotting.
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The melanoma cell line B16F10 was purchased from the Cell Resource Center for Biomedical Research (Sendai, Japan). The MC-38 mouse colon adenocarcinoma cell line was provided by Takeshi Setoyama and Tsutomu Chiba at Kyoto University. The BRAF V600E melanoma cell line (Dhomen et al., 2009) (link) was provided by Caetano Reis e Sousa at the Francis Crick Institute. 4T1 mammary tumor cells were purchased from ATCC (Manassas, VA) and maintained on a collagen-coated dish (AGC Techno Glass, Tokyo, Japan). All cell lines were cultured in complete RPMI medium (Thermo Fisher Scientific) containing 10% FBS (Sigma-Aldrich, St. Louis, MO), 1 mM sodium pyruvate (Thermo Fisher Scientific), 50 μM 2-mercaptoethanol (Nacalai Tesque), 1%
GlutaMAX solution (Thermo Fisher Scientific), 1% MEM Non-Essential Amino Acids (Thermo Fisher Scientific), 10 mM HEPES solution (Thermo Fisher Scientific), 50 μM 2-mercaptoethanol (Nacalai Tesque), 100 units/ml penicillin, and 100 μg/ml streptomycin (Nacalai Tesque, Kyoto, Japan). Mycoplasma contamination is regularly checked using PlasmoTest mycoplasma detection kit (InvivoGen, San Diego, CA).
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