Yuan et al.
Supplementary Information
Gossypol and an HMT G9a Inhibitor Act in
Synergy to Induce Cell Death in Pancreatic
Cancer Cells
Yuan Yuan1,2, Alicia J. Tang1, Adam B. Castoreno1, Szu-Yu Kuo3, Qiu Wang1, Petric Kuballa4,
Ramnik Xavier4, Alykhan F. Shamji1, Stuart L. Schreiber1,2* and Bridget K. Wagner1*
Affiliations:
1
Chemical Biology Program, Broad Institute, Cambridge, MA
2
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA
3
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA
4
Center for Computational & Integrative Biology, Massachusetts General Hospital, Boston, MA
* Corresponding authors: bwagner@broadinstitute.org; stuart_schreiber@harvard.edu
Broad Institute, 7 Cambridge Center, Cambridge, MA 02142; Tel: (617) 714-7363, Fax (617)
714-8943
1
Yuan et al.
Supplementary Figure Legends
Figure S1. Lack of functional p53 renders cancer cells more resistant to the G9a inhibitor
BRD4770. The relative survival rate after 24-hour treatment of five cancer cell lines with
BRD4770.
The
cellular
metabolism
levels
were
detected
by
formazan
reduction
spectrophotometrically for 24 hours (reference 13).
Figure S2. Cellular p53 activity upon BRD4770 treatment in p53 wild-type and mutant cell
lines. (A) Western blot analysis of p53, phospho-p53 (Ser15) and acetyl-p53 (Lys382) levels in
MCF7, HPAC, PC3 and HeLa cells after three-day treatment with BRD4770. Tubulin was used
as an internal loading control. Cellular p53 activity was measured using a luciferase reportergene assay in (B) MCF7 cells and (C) PANC-1 cells upon treatments of BRD4770 and Nutlin-3
at indicated concentrations for 2 days. Activity of the firefly luciferase p53-reporter was
normalized to constitutively active Renilla luciferase. Data represent the mean and standard
error of three independent experiments for each condition.
Figure S3. Compounds that enhance BRD4770 toxicity in pancreatic cancer cells were
identified by measuring cellular ATP levels. PANC-1 and hHPNE cells were treated with
2.5µM BRD4770 and a collection of 198 bioactive compounds for 72 hr. Cellular ATP levels
were then measured and normalized to the DMSO negative-control wells on each plate. Four
compounds enhanced BRD4770-induced cell death in tumorigenic PANC-1 cells, but not in nontumorigenic hHPNE cells
Figure S4. Compounds that enhance BRD4770-induced pancreatic cancer cell death were
identified by measuring live-cell metabolism. PANC-1 cells were seeded in 96-well plates
that were pre-coated with 92 cytotoxic drugs in four doses.(13) 2.5µM BRD4770 was added to a
2
Yuan et al.
set of plates while DMSO was added to another set of plates as negative controls for 48hr. The
cellular metabolism levels were monitored by formazan reduction spectrophotometrically for
24hr. Four compounds enhanced BRD4770-induced pancreatic cell death in a dose-dependent
manner.
Figure S5. Western blot analysis of G9a protein levels in tumorigenic PANC-1 cells and the
immortalized human pancreatic ductal cell line hHPNE. Tubulin was used as an internal loading
control.
Figure S6. UNC0638, a G9a inhibitor structurally unrelated to BRD4770, also synergizes
with gossypol to induce cell death in pancreatic cancer cells. (A) ATP levels in PANC-1
cells after three-day treatment with the indicated concentrations of gossypol and UNC0638.
Data represent the mean and standard error of four independent replicates for each
combination. (B) Heatmap of the combination index (CI) for each combination of gossypol and
UNC0638 in PANC-1 cells. The strongest synergistic effect was observed at a combination of 2
µM gossypol and 2.5 µM UNC0638, with CI=0.43. (C) ATP levels of hHPNE after three-day
treatment with the same combination of gossypol and UNC0638. (D) Heatmap of the CI for the
same compound combination in hHPNE cells.
Figure S7. Addition of gossypol does not make the p53 wt cancer cell line MCF7 more
sensitive to BRD4770. (A) Cellular ATP levels after 24- and 72-hour treatment of MCF7 cells
with BRD4770 in the absence or presence of gossypol. Data represent the mean and standard
error of six biological replicates. (B) Caspase-3/7 activities in MCF7 cells were measured after
24-hour and 72-hour treatment with BRD4770 in the absence or presence of gossypol. Results
3
Yuan et al.
were normalized by cellular ATP levels, and data represent the mean and standard error of six
independent replicates.
Figure S8. Gossypol and BRD4770 do not induce cell apoptosis in PANC-1 cells.
Caspase3/7 activation in PANC-1 cells after one-day (A) and three-day treatment (B) with the
indicated concentrations of gossypol and BRD4770. Data represent the mean and standard
error of four independent replicates for each combination.
Figure S9. Gossypol and BRD4770 synergize to induce cell death in HeLa cells stably
transfected with mCherry-eGFP-LC3. (A) Cellular ATP levels were measured after 72hr
treatment with the indicated concentrations of gossypol and BRD4770. Data represent the mean
and standard error of four independent experiments for each combination. (B) Heatmap plot
representing the combination index (CI) for each compound combination. The strongest
synergistic effect was observed at a combination of 1µM of gossypol and 1.25µM of BRD4770,
(CI=0.28).
Figure S10. Effects of BRD4770 and gossypol on LC3-II levels in PANC-1 and HeLa cells.
Quantification of Western blots for LC3-I and LC3-II protein levels in response to (A) 48-hour
treatment with the indicated concentrations of gossypol, BRD4770, Torin 1 and Bafilomycin A1
in PANC-1 cells and (B) 24-hour treatment with the indicated concentrations of gossypol and
BRD4770 in HeLa cells. The lysosomal inhibitors e64D and pepstatin A were added to indicate
treatments to determine whether LC3-II levels were further increased by blocking degradation of
autophagosomes.
4
Yuan et al.
Figure S11. Diverse autophagy inducers do not synergize with BRD4770 to induce cell
death in PANC-1 cells. PANC-1 cells were treated with (A) rapamycin (mTOR inhibitor), (B)
tamoxifen (PI3K inhibitor), (C) clonidine (imidazoline-1 receptor agonist), (D) carbamazepine
2+
2+
(inositol inhibitor), (E) verapamil (Ca channel blocker), (F) loperamide (Ca channel blocker),
and (G) PI3k inhibitor in combination with BRD4770. The cellular ATP levels were measured
72hr after treatment and the CI was calculated at each combination.
Figure S12. ABT-737 synergizes BRD4770 to induce cell death in PANC-1 cells with a
much lower efficiency compared to gossypol. (A) ATP levels in PANC-1 cells after three-day
treatment with the indicated concentrations of ABT-737 and BRD4770. Data represent the mean
and standard error of four independent replicates for each combination. (B) Heatmap of the
combination index (CI) for each combination of ABT-737 and BRD4770 in PANC-1 cells. The
strongest synergistic effect was observed at a combination of 32 µM ABT-737and 10 µM
BRD4770, with CI=0.34. (C) ATP levels of PANC-1 cells after three-day treatment with the same
combination of HA14-1 and BRD4770. (D) Heatmap of the CI for HA14-1 and BRD4770
compound combination.
Figure S13. High concentrations of BRD4770 or UNC0638 induce LC3 expression in HeLa
cells. Cells were treated for 24 hours with 10M BRD4770 or 5mM UNC0638, followed by
imaging for LC3 fluorescence. Scale bar = 20m.
Figure S14. Quantification of western blot analysis by imageJ. The protein levels were
normalized by tubulin control on each blot.
5
Yuan et al.
Figure S1
6
Yuan et al.
Figure S2
7
Yuan et al.
Figure S3
8
Yuan et al.
Figure S4
9
Yuan et al.
Figure S5
10
Yuan et al.
Figure S6
11
Yuan et al.
Figure S7
12
Yuan et al.
Figure S8
13
Yuan et al.
Figure S9
14
Yuan et al.
Figure S10
15
Yuan et al.
Figure S11
16
Yuan et al.
Figure S12
17
Yuan et al.
Figure S13
18
Yuan et al.
Figure S14
19