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Seno et al. Cancer Drug Resist 2019;2:335-50 I http://dx.doi.org/10.20517/cdr.2019.01 Page 337

containing 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin. All cells were cultured at 37 °C
with 5% CO under humidified conditions. The mouse leukemia cell line L1210 (JCRB cell bank, Osaka, Japan)
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was maintained in RPMI1640 containing 10% FBS, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL
streptomycin.

To prepare the CM from miPS-CSCs, cells were cultured until reaching 80% confluence, after which the
culture medium was replaced with serum-free culture medium containing Insulin-transferrin-delenium-x
(ITS-X, Life Technologies). CM was collected at 20 h after medium replacement, centrifuged, and then
filtered using a 0.45-µm filter (Millipore). For selection of a stem cell-like population in miPS-CSCs, cells
were cultured in the medium containing CM of each miPS-CSC (1:1 to DMEM), 0.3 µg/mL for miPS-B16cm
cells, or 1 µg/mL for the rest of miPS-CSCs of puromycin for 7 days with one or two passages. The medium
was replaced every 24 h during the selection.

After verifying tumorigenicity by injection into the mice biaxillary, the miPS-LLCcm primary cells were
obtained from the primary tumors, and the miPS-LLCcm LMT cells were obtained from the resulting lung
metastatic tumors.

Drug screening and evaluation of concentration-dependent cytotoxicity of daunorubicin
All test compounds were provided as Screening Committee of Anticancer Drugs(SCADs) Inhibitor Kit 1
(ver. 2.4) and 2 (ver. 2.0) from SCADs supported by Grant-in-Aid for Scientific Research on Innovative Areas,
Scientific Support Programs for Cancer Research from the Ministry of Education, Culture, Sports, Science
and Technology, Japan.

Stem cell populations of miPS-CSCs selected in the presence of puromycin were seeded in gelatin-coated
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96-well plates at a density of 1 × 10 cells/well in medium containing CMs and DMEM at a 1:1 ratio. After
24 h, test compounds were added at a concentration of 1 μmol/L. Cell viabilities at 48 h were determined as
follows. After incubation, the wells were added with 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide, yellow tetrazole (MTT, Sigma-Aldrich) to a final concentration of 0.6 mg/mL, and the plate
was incubated for 4 h. Formed formazan crystals were dissolved in 10% (w/v) SDS with 0.02 N HCl and
incubated overnight. Finally, the absorbance of each well was measured at 570 nm using an MTP-800AFC
microplate reader (Corona, Japan).

For the analysis of dose-dependent cytotoxicity of daunorubicin against various cells, cells were seeded in
96-well plates. Daunorubicin was added at various concentrations after incubation for 24 h. Viable cells
were evaluated by MTT assay as described above. The experiment was independently repeated three times.
The concentrations at which cell growth was inhibited by 50% (IC ) were determined based on the survival
50
curve obtained by MTT assay.

For the preparation of DNA, RNA, and proteins and for FACS analysis, cells were seeded in a 60-mm dish at
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a density of 7 × 10 cells/dish. After 24 h, cells were treated with daunorubicin at varying incubation periods.

Analysis of apoptotic features
To analyze the production of the apoptotic DNA ladder, cells were lysed with a lysis buffer (10 mmol/L Tris-
HCl, pH 7.4, 5 mmol/L EDTA, and 0.2% Triton X-100), vortexed, and incubated on ice for 30 min. The cells
were then centrifuged (17,400 × g, 30 min) to remove precipitated intact genomic DNAs. Supernatants were
collected, and fragmented DNA was precipitated with ethanol. Precipitants were re-dissolved in extraction
buffer containing 10 mmol/L Tris-HCl (pH 7.4) and 5 mmol/L EDTA and subsequently treated with RNase
A for 5 h at 37 °C. Afterwards, cells were added with 1/10 volume of buffer containing 100 mmol/L Tris-HCl