healthmedicinet

Mice

K19 ^CreERT
mice were kindly provided by Guoqiang Gu (Vanderbilt University, Nashville, TN, USA)
24]. ROSA26-lox-stop-lox-YFP reporter (ROSA-YFP) mice, obtained from the Jackson Laboratory, were crossed with K19 ^CreERT
mice to generate K19 ^CreERT
/ROSA-YFP mice. Atg5 ^flox/flox
mice have been described previously 25] and were kindly provided by Dr. Noboru Mizushima (Tokyo University, Tokyo, Japan).
Atg5 ^flox/flox
mice were crossed with K19 ^CreERT
mice to generate Atg5 ^flox/flox /K19 ^CreERT+
mice. C57BL/6 J (B6) mice were from CLEA Japan (Tokyo, Japan). All mice used were
of the B6 background. For tamoxifen (TAM) treatment, mice were injected with 10 mg/kg
TAM (Cayman Chemical, Ann Arbor, MI, USA) intraperitoneally (i.p.) three times (on
days 1, 3, and 5). For CQ treatment, mice were injected with 50 mg/kg CQ (Sigma-Aldrich,
St. Louis, MO, USA) i.p. at the times indicated. All animal studies were approved
by the Animal Care and Use Ethics Committee at the Institute for Adult Diseases, Asahi
Life Foundation.

Tumor induction

Atg5 ^flox/flox /K19 ^CreERT+
(Atg5-deficient mice) and Atg5 ^flox/flox
mice (Cre-negative littermates, used as control mice) were injected i.p. with 12.5 mg/kg
AOM (Sigma-Aldrich) on day 1. After 5 days, mice received water supplemented with
2.5 % DSS (MP Biomedicals, Irvine, CA, USA) for 5 days, after which the mice were
maintained on regular water for 14 days and subjected to two further DSS treatment
cycles. On days 60, 62, and 64, the mice were injected i.p. with 10 mg/kg TAM. On
day 67, the mice were sacrificed to analyze colon tumors. Macroscopic colon tumors
were counted, and the longest diameter of each tumor was measured using a caliper
in a blinded fashion.

Cell lines

Four established colon cancer cell lines, HCT116, SW48, DLD1, and SW837, were used
26], 27]. HCT116 and SW48 cells harbor the wild type p53 gene, while DLD1 and SW837 cells
are mutated in the p53 gene 26], 27]. HCT116 cells were maintained in McCoy’s 5A medium containing 10 % fetal bovine serum
(FBS). SW48 and SW837 cells were maintained in Leibovitz’s L-15 medium containing
10 % FBS. DLD1 cells were maintained in RPMI 1640 medium containing 10 % FBS. Hank’s
Buffered Salt Solution (HBSS) was used to induce amino acid starvation conditions.
The cell lines were obtained from the American Type Culture Collection (Baltimore,
MD, USA), and all media formulations were obtained from Sigma-Aldrich.

Antibodies and reagents

The following primary antibodies were used for immunoblotting and immunohistochemistry:
anti-Atg5, anti-Atg7, anti-LC3, anti-p62, anti-PARP, anti-cleaved caspase 3, anti-BiP,
anti-p53, anti-phospho-eIF2?, anti-phospho-JNK, anti-phospho-Chk1, anti-phospho-p53,
anti-actin (all from Cell Signaling, Beverly, MA, USA), anti-CK19, anti-proliferating
cell nuclear antigen (PCNA) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA),
anti-Ki67 (Dako, Carpinteria, CA, USA), anti-p53 (Vector Laboratories, Birmingham,
CA, USA), anti-CHOP (Thermo Fisher Scientific, Waltham, MA, USA), and anti-yellow
fluorescent protein (YFP) (MBL, Tokyo, Japan). CQ diphosphate salt (Sigma-Aldrich)
was dissolved in PBS at the indicated concentrations.

RNA interference

Small interfering RNAs (siRNAs) targeting Atg5 (MISSION siRNA, Sigma-Aldrich) and
BiP (Dharmacon siGENOME SMART pool siRNA, GE Healthcare, Pittsburg, PA, USA) or the
non-silencing control (5’-AATTCTCCGAACGTGTCACGT-3’) were transfected into cells using
Lipofectamine RNAimax (Invitrogen, Waltham, MA, USA) for 72 h. Immunoblotting was
used to verify that the siRNAs reduced cellular protein expression by more than 80 %.

Immunoblotting

Cells or mouse tissues were disrupted in lysis buffer (20 mM Tris, pH 7.5, 150 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 1 % Triton X [Sigma-Aldrich], 2.5 mM sodium pyrophosphate,
1 mM glycerophosphate, 1 mM Na
₃
VO
₄
, 1 ?g/ml leupeptin). The lysates were electrophoresed by SDS-PAGE, transferred to
a polyvinylidene difluoride membrane (GE Healthcare), and blocked for 1 h in Tris-buffered
saline-Tween 20 with 5 % dry milk. The membrane was incubated overnight at 4 °C with
the primary antibody and subsequently washed and incubated with a secondary horseradish
peroxidase (HRP)-conjugated antibody. The immunocomplexes were detected using a chemiluminescence
detection kit (Luminata Classico Western HRP; Merck Millipore, Darmstadt, Germany).
Images were obtained using the LAS 4000 image analyzer (Fujifilm, Tokyo, Japan).

Immunohistochemistry

Formalin-fixed and paraffin-embedded mouse tissues were cut at a thickness of 3 ?m,
deparaffinized, and incubated in citrate buffer at 95 °C for 40 min for antigen retrieval.
Endogenous peroxidase activity was blocked using 3 % H
₂
O
₂
. The tissue sections were incubated overnight with rabbit primary antibody, followed
by a polyclonal goat anti-rabbit immunoglobulins/biotinylated secondary antibody (DAKO)
for 30 min, and then exposed to Streptavidin/HRP (DAKO) for 10 min. The Mouse-on-Mouse
Immunodetection kit (Vector Laboratories) was used as the mouse primary antibody for
mouse tissues, according to the manufacturer’s instructions. The chromogenic reaction
was performed using the Liquid DAB Substrate Chromogen System (Dako). YFP expression
in the colons of mice was examined by immunofluorescence staining. The tissues were
incubated with anti-YFP antibody followed by Alexa Flour 594-conjugated goat anti-rabbit
secondary antibody (Molecular Probes, Eugene, OR, USA) for 30 min, and the nuclei
were visualized by DAPI staining (Takara, Tokyo, Japan) for 1 min. The proportion
of Ki67-positive cells was determined by counting more than 500 cells in three Ki67-concentrated
lesions, and the numbers of cleaved caspase 3-positive cells per field were counted.

Real-time RT-PCR

Total cellular RNA samples were isolated from mouse colon tissues and from HCT116
cells using NucleoSpin RNA II (Takara). The cDNAs were generated from 1-?g total RNA
by reverse transcription using Transcriptor Universal cDNA Master (Roche, Branchburg,
NJ, USA). The mRNA expression levels of mouse Atg5, interleukin (IL) 1-?, chemokine
(C-X-C motif) ligand 1 (CXCL1), p53 upregulated modulator of apoptosis (PUMA), Noxa,
Bax, CHOP, BiP, spliced X-box binding protein 1 (sXBP1) and of human Atg5, Ulk1, Atg7,
C-X-C chemokine receptor type 4 (CXCR4), SOX9, CD44, CXCL1, IL8, cellular inhibitor
of apoptosis protein 1 (cIAP1), PUMA, Noxa, Bax, CHOP, BiP, and spliced XBP1 were
determined by quantitative real-time RT-PCR using the LightCycler 480 instrument II
real-time PCR System (Roche). GAPDH mRNA was used as an internal control. The primer
sequences used are available on request.

Flow cytometric analysis of apoptosis

Colon cancer cells (1.0?×?10
⁴
/ml) were seeded into 24-well plates and 24 h later were treated with siRNAs for 72 h
or with CQ for 24 h. Cells were detached by trypsinization and exposed to early and
late apoptotic detection reagents (GFP Certified Apoptosis Detection Kit, Enzo life
Sciences, Farmingdale, NY, USA) for 15 min. Samples were analyzed by flow cytometry
using the FL2 channel for Annexin V detection (early apoptosis) and the FL3 channel
for PI detection (late apoptosis) using a fluorescence-activated cell sorter (FACS)
(accuriC6, BD Biosciences, Ann Arbor, MI, USA).

Cell growth assay

The extent of cell growth was assessed using the Cell Counting Kit-8 (CCK-8) from
Dojindo Laboratories (Kumamoto, Japan). Cells (1.0?×?10
⁴
/ml) were seeded into 96-well plates (day 0) and 24 h later were transfected with
siRNAs (day 1) for 48 h. CCK-8 solution was added to each well for 2 h. The absorbance
at 450 nm was determined using a multi-mode reader (SpectraMax, Molecular Devices,
Sunnyvale, CA, USA).

Statistical analysis

Statistical analysis was performed using Welch’s t-test, Mann–Whitney U-test, and one-way analysis of variance with Dunnett’s multiple comparison test, where
appropriate. Differences were considered statistically significant at p??0.05.