SUPPLEMENTAL FIGURE LEGENDS
Supplemental Figure S1. 4-hydroxytamoxifen excision of Atg3 is specific, and Cremediated excision is not inherently toxic. A. A7F cells (which do not express Cre-ER) and
A3C cells were cultured with 4OHT for the indicated times, and Atg3 protein was analyzed by
immunoblot. A non-specific band is indicated by <. B. A7F cells were cultured with 4OHT for
the indicated times, with media being changed, cells being split, and new 4OHT being added on
day 2, and survival was assessed by propidium iodide exclusion flow cytometry. C,D. CrePos
cells (which express Cre-ER and are Atg3+/+) were cultured in the presence or absence of 4OHT.
(C) Cell accumulation over time was measured by quantitation on a Coulter Z2 Particle Counter,
and (D) survival was measured by flow cytometry. E. Bone marrow from IL7RF/F Cre-ER or
Cre-ER negative mice was cultured in either 4OHT for two days or 4 μM etoposide for 12 hours,
and γH2A.X (phospho histone H2A.X serine 139), and Cre protein levels were analyzed by
immunoblot. Asterisks denote p < 0.05 by Student’s t-Test of experimental samples compared to
control samples. Data shown are representative of three or more experiments.
Supplemental Figure S2. Establishment and verification of A3C Glut1 and Bcl-2
overexpression models. A. The expression of Glut1 and Bcl-2 in A3C cells was established by
immunoblot, with NGF being the vector control. B. NGF, Glut1, and Bcl-2 A3C cells were
cultured with ethanol or 4OHT, and Atg3 protein levels were analyzed by immunoblot. C. Live
NGF and Myc-Glut1-expressing cells were stained with anti-myc and FITC anti-mouse IgG1,
and Myc-Glut1 surface expression was assessed by flow cytometry.
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Supplemental Figure S3. Atg3 excision leads to apoptosis. A,B. NGF cells were treated with
4OHT for the indicated times, and stained with anti-Bax and propidium iodide. (A) Active Bax
and (B) sub-diploid DNA content were assessed by flow cytometry. Asterisks denote p < 0.05
by Student’s t-Test of treated samples compared to control samples. Data shown are
representative of three or more experiments.
Supplemental Figure S4. Atg3 is critical for survival in metabolic stress. A3C NGF cells
were cultured with ethanol or 4OHT to delete Atg3, then cultured in media with or without
addition of 2 mM 2-deoxyglucose (2DG), and viability was assessed. Asterisks denote p < 0.05
by Student’s t-Test of treated samples compared to control sample.
Supplemental Figure S5. Disruption of autophagy leads to minor changes in organic acid
and amino acid levels. A-D. Bcl-2-expressing A3C cells were cultured for two days with either
ethanol or 4OHT to inhibit autophagy, then cultured in the presence or absence of both 4OHT
and IL3 for twenty four hours, and (A) organic acids, (B) high-abundance amino acids, (C)
medium-abundance amino acids, and (D) low-abundance amino acids were measured by tandem
mass-spectrometry. Means and standard errors of triplicate samples are shown. Data shown are
representative of three or more experiments. Asterisks denote p < 0.05 by Student’s t-Test of
IL3-withdrawn samples compared to control samples.
Supplemental Figure S6. Atg3 deletion does not cause overt metabolic stress. A. Bcl-2expressing cells were treated ± 4OHT for two days, then cultured with the nutrients indicated and
without IL3 for 24 hours, and ATP levels were measured from cell lysates. Asterisks denote p <
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0.05 by Student’s t-Test of 4OHT treated samples compared to control samples. B. Bcl-2expressing cells were cultured for two days with either ethanol or 4OHT to inhibit autophagy,
then cultured in the presence or absence of both 4OHT and IL3 for twenty four hours, and
expression of components of the AMP-kinase pathway were measured by immunoblot. C. NGF
and p185 BCR-Abl-expressing cells were cultured with either ethanol or 4OHT for the indicated
times to inhibit autophagy, and phosphorylation of the AMPK downstream target acetyl-CoA
carboxylase was measured by immunoblot. Data shown are representative of three or more
experiments.
Supplemental Figure S7. Excision of Atg3 in Bcr-Abl-expressing A3C cells disrupts
autophagy. A. The expression of p185 BCR-Abl and BCR-Abl / Glut1 (B/G) was established
by immunoblot. B. NGF and BCR-Abl-expressing cells were run on a low percentage
polyacrylamide gel to resolve BCR-Abl, c-Abl, and phospho-BCR-Abl (pBCR Tyr 177)
expression by immunoblot. HSP90 serves as a loading control. C. Control NGF, BCR-Abl,
and B/G-expressing cells were cultured with ethanol or 4OHT for two days, and Atg3 loss and
p62 accumulation were observed by immunoblot. D. NGF, Bcl-2, and BCR-Abl-expressing
cells were cultured with ethanol or 4OHT for 2 days, then cultured without IL3 and with 40 μM
chloroquine (CQ) for 10 hours, and LC3-II protein accumulation was assessed by immunoblot.
E. BCR-Abl-expressing cells were cultured in the presence or absence of 0.1 μM imatinib for 12
hours, and phospho-BCR-Abl and downstream targets of BCR-Abl signaling were assessed by
immunoblot. Data shown are representative of three or more experiments.
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Supplemental Figure S8. Glut1 expression does not rescue BCR-Abl dependence on
autophagy. A,B. (A) BCR-Abl or (B) BCR-Abl/Glut1-expressing cells were cultured for two
days with either ethanol or 4OHT to delete Atg3, then cultured in the presence or absence of both
4OHT and IL3, and viability was assessed. Means and standard deviations of triplicate samples
are shown. Data shown are representative of three or more experiments. Asterisks denote p <
0.05 by Student’s t-Test of 4OHT-treated samples compared to control-treated samples.
Supplemental Figure S9. BCR-Abl-expressing cells growing out of long-term 4OHTtreated cultures express Atg3 and are autophagy-competent. BCR-Abl-expressing AC3 cells
were cultured in the presence or absence of both IL3 and 4OHT, with media being changed and
new 4OHT added on day two (Figure 6B, C). After fifteen days, cells from each triplicate
sample that grew out of the cultures 4OHT ± IL3 4OHT were analyzed by immunoblot for Atg3
and LC3-II. Samples were compared to untreated BCR-Abl-expressing A3C cells and cells
treated with 4OHT for three days (left panel).
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