SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1. Hippuristanol Alters Resistance to DNA Damaging Agents. A.
Kaplan-Meier plot demonstrating response of Tsc2+/-E-Myc tumor-bearing mice to rapamycin
(Rap), doxorubicin (Dxr), and Rap+Dxr treatment. p < 0.02 for Rap+Dxr versus Rap; p < 0.001
for Rap+Dxr versus Dxr. Note that this experiment was performed at the same time as the one
presented in Fig. 1B but the data is presented separately here for clarity (the DXR cohort
response in this study is the same as the one shown in Fig. 1B) B. Polysome fractionation of
cultured Tsc2+/–Eμ-Myc lymphoma cells exposed to 50 nM hippuristanol for 1 hr. C. Viability of
cultured Tsc2+/–Eμ-Myc lymphoma cells exposed to 50 nM hippuristanol for 1hr and 4 hrs, as
determined by FACS analyses using propidium iodine (PI) staining. D. Representative
micrographs of Tsc2+/–Eμ-Myc lymphoma sections stained by TUNEL. Bars represent 50 μm.
C57BL/6 mice bearing well-palpable tumors were injected with Rap. Twenty-four hours later,
mice were injected again with Rap alone or in combination with Dxr. Six hours after treatment,
tumors were extracted and stained. Note that this experiment was performed at the same time as
the one presented in Fig. 1E. E. Representative micrographs of Tsc2+/–Eμ-Myc lymphoma
sections stained for Ki-67 expression. Bars represent 50 μm. C57BL/6 mice bearing wellpalpable tumors were injected with vehicle, Hipp, or Rap. Twenty-four hours later, mice were
injected again with Hipp or Rap alone or in combination with Dxr. Six hours after treatment,
tumors were extracted, fixed, and stained.
Supplementary Figure 2. Long-term Administration of Hippuristanol is Well Tolerated. A.
Hippuristanol does not affect weight gain in mice. Six to 8 week old mice were injected daily for
5 days with 10 mg/kg hippuristanol and body weight monitored before beginning of treatments
(day 0) as well as one day after the last treatment (day 6); n=10. B. Hippuristanol does not alter
liver aminotransferase activity. Five C57Bl/6 mice were administered daily injections of Hipp
for 5 days. Alanine (ALT) and aspartate (AST) aminotransferase levels were determined one day
after the last injection. The bar represents the mean of the individual measurements (represented
as circles). C. Quantitation of individual cell populations from mice administered daily with 10
mg/kg Hipp or vehicle for 5 consecutive days. Flow cytometry was used to identify and
quantitate the cell populations staining for B220+ (B cells), Ly-6G+ (granulocytes), CD11b+
(monocytes/macrophages, granulocytes), or CD4+ (T cells). D. Western blot analysis of Pgp-1 in
HeLa and HeLa/ Pgp-1 cell lines. E and F. Titration of silvestrol (Sil), doxorubicin (Dxr) and
hippuristanol (Hipp) on HeLa and HeLa/ Pgp-1 cell lines. Cells were treated for 48 hrs with
increasing concentrations of Sil or Dxr (E), or Hipp or Dxr (F) followed by assessment of cell
proliferation using the MTS assay according to the instructions of the manufacturer (Promega).
Supplemental Figure 3. Dose-Response Curves to Hippuristanol and Doxorubicin in EμMyc lymphomas expressing Mcl-1 cDNA or shBim or shNoxa hairpins. Cells of the indicated
genotype were exposed to different concentrations of Hippuristanol (A, C, E) or Doxorubicin (B,
D, F) for 18 hrs after which time the fraction of viable cell population was determined by flow
cytometry. n = 3, Results are expressed as the mean ± SD.
Supplemental Figure 4. Dose-Response Curves to Hippuristanol and ABT-737 in Arf-/-EMyc and p53-/-E-Myc lymphomas. Cells of the indicated genotype were exposed to different
concentrations of Hippuristanol (A, B) or ABT-737 (C, D) for 18 hrs after which time the
fraction of viable cell population was determined by flow cytometry. n = 3, Results are expressed
as the mean ± SD.
Supplemental Figure 5. Relationship between Hippuristanol and ABT-737 in Arf-/-EMyc/Mcl-1, Arf-/-E-Myc/Bcl-2, p53-/-E-Myc/Mcl-1 and p53-/-E-Myc/Bcl-2 lymphomas. AD. Cells of the indicated genotype were exposed to the indicated drug concentrations for 18 hrs
after which time the fraction of viable cell population was determined by flow cytometry. The
data is representative of 3 different experiments with the SD presented. E and F. Combination
index (CI) plot for Hipp and ABT-737 in Arf−/−Eμ-Myc/Bcl-2 (E) and p53−/−Eμ-Myc/Bcl-2 (F)
cells. The primary data was taken from Panels B and D.
Supplemental Figure 6. Relationship between Hippuristanol and ABT-737 in Arf-/-E-Myc,
Arf-/-E-Myc/Mcl-1/Bcl-2,
p53-/-E-Myc
and
p53-/-E-Myc/Mcl-1/Bcl-2
lymphomas.
Arf−/−Eμ-Myc cells (A), Arf−/−Eμ-Myc/Mcl-1/Bcl-2 (B), p53−/−Eμ-Myc (C), and p53−/−EμMyc/Mcl-1/Bcl-2 (D) were exposed to the indicated drug concentrations for 18 hrs after which
time the fraction of viable cell population was determined by flow cytometry. The data is
representative of 3 different experiments with the SD presented.
Supplemental Figure 7. Synergy between ABT-737 and eIF4AI Suppression is p53dependent. A. Flow cytometry analysis of GFP-negative Arf-/-E-Myc/Bcl-2 cells used to
determine the viable population from the experiment presented in Fig. 5B at the indicated days.
Error bars indicate SEM; n = 3. B. Competition assay of p53−/−Eμ-Myc/Bcl-2 lymphomas
infected with retroviruses expressing the indicated shRNAs. Flow cytometry was used to
determine the %GFP+ population over a 6-day time course following the last infection. Data
from cells infected with shRNAs to FLuc, Mcl1, and eIF4E are grouped in the left panel, while
those targeting eIF4AI/II are grouped in the right panel for clarity. Error bars indicate SEM; n =
3. C. Synergy between eIF4AI suppression and ABT-737 in p53−/−Eμ-Myc/Bcl-2 lymphomas.
Following the last infection, cells were exposed to vehicle or the indicated concentrations of
ABT-737 for 18 hrs at which time flow cytometry was used to determine the %GFP+ cells. Data
from cells infected with shRNAs to FLuc, Mcl1, and eIF4E are grouped in the left panel, while
those targeting eIF4AI/II are grouped in the right panel for clarity. Error bars indicate SEM; n =
3.
Supplemental Figure 8. Sensitivity of Human Lymphoma and Leukemia Cells to
Hippuristanol. A. Dose response curves assessing sensitivity of human lymphoma and leukemia
cells to hippuristanol. Cells were exposed to the indicated drug concentrations for 18 hrs after
which time the fraction of viable cell population was determined by flow cytometry. The data is
representative of 2 different experiments with the SD presented. EoL-1, human eosinophilic
leukemia; Sc-1, B cell follicular lymphoma; Jeko-1, Mantle cell lymphoma; MOLT-3, T cell
acute lymphoblastic leukemia; Mino, Mantle cell lymphoma; Ri-1, Non-Hodgkin’s lymphoma;
Mv411, biphenotypic B myelomonocytic leukemia; Grant 519, Mantle cell lymphoma; hMB,
human CD133+ cells overexpressing Myc and Bcl-2 and modeled after “double hit” lymphomas.
B. Heat maps of combination indices (CI) plot displaying the synergistic effect for the
combination of Hipp and ABT-737 in human lymphoma and leukemia cells. Synergy is defined
as 0.25 < CI < 0.75 whereas strong synergy is CI < 0.25. Concentrations of ABT-737 ranged
from 625 to 40 nM (green overhead dose line) or from 2.5 M to 0.156 M (orange overhead
dose line) with 2-fold serial dilutions. Hippuristanol dilutions ranged from 2.5 M to 20 nM
(blue overhead dose line) with 2-fold serial dilutions. C. Western blot analysis of extracts
prepared from the indicated tumor cell lines. The proteins detected by immunoblotting are
indicated to the right. Note that extracts from the Namalwa Burkitt’s lymphoma cell line (Fig. 6)
are analyzed on this panel set. TP53 mutational status of cell lines was determined from data at
http://p53.fr.