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Page 338 Seno et al. Cancer Drug Resist 2019;2:335-50 I http://dx.doi.org/10.20517/cdr.2019.01

(pH 8.0), 100 mmol/L EDTA, and 250 mmol/L NaCl, after which the samples were treated with Proteinase
K at 65 °C overnight. DNA fragments were extracted with phenol/chloroform/isoamyl alcohol (25:24:1),
precipitated with ethanol, and analyzed by 1.5% or 2% agarose gel electrophoresis.

Annexin V Apoptosis detection kit APC (Affymetrix) was used for the detection of phosphatidylserine as
an apoptosis marker. Cells were treated with 100 nmol/L daunorubicin for 3 h, harvested, and stained with
annexin V-APC according to the manufacturer’s protocol. Stained cells were analyzed by flow cytometry
using BD Accuri C6 (BD Biosciences) equipped with the FlowJo software (Tree Star, Inc., CA).

RNA preparation, cDNA synthesis, and quantitative PCR
Total RNA was extracted from the cells treated with 100 nmol/L daunorubicin for varying incubations
periods using Trizol reagent (Ambion). After DNase I (Takara, Japan) treatment, RNA was re-purified
using Trizol reagent. cDNA was synthesized from 3 μg of RNA using SuperScript III First Strand
Synthesis Kit (Invitrogen) using oligo-dT primers. Quantitative real-time PCR was performed on a Light
Cycler 480 (Roche) instrument and SYBR Green I Master Mix (Roche) according to the manufacturer’s
instructions. The following primers were used for PCR. Noxa, 5’-GAGTGCACCGGACATAACTG-3’
and 5’-CTCGTCCTTCAAGTCTGCTG-3’; Bax, 5’-TAGCAAACTGGTGCTCAAGG-3’ and
5’-TCTTGGATCCAGACAAGCAG-3’; Puma, 5’-GTACGAGCGGCGGAGACAAG-3’ and
5’-GCACCTAGTTGGGCTCCATTTCTG-3’; p21, 5’-GCCCGAGAACGGTGGAACTT-3’ and
5’-GACAAGGCCACGTGGTCCTC-3’; Mdm2, 5’-CTAGCTTCTCCCTGAATGCC-3’ and
5’-TTGCACACGTGAAACATGAC-3’; Nanog, 5’-AGGGTCTGCTACTGAGATGCTCTG-3’ and
5’-CAACCACTGGTTTTTCTGCCACCG; Gapdh, 5’-AACGGCACAGTCAAGGCCGA-3’ and
5’-ACCCTTTTGGCTCCACCCTT-3’. The expression levels of Noxa, Bax, Puma, p21, Mdm2, and Nanog
genes were normalized against Gapdh expression levels.

Protein preparation and western blotting
Whole cell lysates were prepared in 50 mmol/L Tris-HCl, pH 7.5 containing 150 mmol/L NaCl, 5 mmol/L
EDTA, and 0.5% w/v Triton X-100. Protein concentrations were determined by Bio-Rad Protein Assay (Bio
Rad, VA) using normal IgG (Bio Rad) as a standard. Cells that detached from dishes after daunorubicin
treatments were collected by centrifugation at 5000 × g at 4 °C for 5 min, washed twice with ice cold PBS,
and lysed as described above. Cell Fractionation Kit-Standard (ab109719, Abcam, UK) was used to obtain
the mitochondrial, nuclear, and cytoplasmic protein fractions according to the manufacturer’s protocol.
Afterwards, 15 or 30 μg of protein of whole cell lysate, and the equivalent volume of the cell fractionation
were analyzed by SDS-PAGE and electrically transferred on PVDF membranes. The primary and secondary
antibodies were used to detect the proteins on the membrane. The following primary antibodies were used
for western blotting: anti-p53 antibody in 1:1000 dilution (#2524, Cell Signaling Technology, MA), anti-β-
tubulin antibody in 1:600 dilution (#2146, Cell Signaling Technology, MA), anti-Histone H3 antibody in
1:2000 dilution (ab1791, Abcam, UK), anti-Nanog antibody in 1:1000 dilution (ab80892, Abcam, UK), anti-β-
actin antibody in 1:1000 dilution (#4970, Cell Signaling Technology, MA), anti-caspase-3 antibody in 1:1000
dilution (#9665, Cell Signaling Technology, MA), anti-cleaved caspase-3 antibody in 1:500 dilution (#9664,
Cell signaling Technology, MA), anti-caspase-7 in 1:1000 dilution (#9492, Cell Signaling Technology, MA),
anti-caspase-9 antibody in 1:1000 (#9504, Cell Signaling Technology, MA), anti-ICAD antibody in 1:1000
dilution (ab108521, Abcam, UK), anti-cleaved PARP-1 antibody in 1:1000 dilution (#9544, Cell Signaling
Technology, MA) and anti-EndoG antibody in 1:1000 (Cell Signaling Technology, MA) were employed.
As the secondary antibodies, HRP-conjugated anti-rabbit IgG antibody in 1:10000 dilution (#7074, Cell
Signaling Technology, MA), and HRP-conjugated anti-mouse IgG antibody in 1:5000 dilution (sc-2005, Santa
Cruz Biotechnology, CA). Immunoreactivity signals were developed using an ECL kit (GE Healthcare, CA)
and detected using Light Capture II (ATTO, Japan).