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Zheng et al.
Supplementary Figure Legends
Supplementary Figure S1. Related to Figure 1.
Unravelling the mechanism of C646 action. A–C, ChIP analysis was performed on MCF7 cells treated
as in Fig. 1A, PCR products for the indicated genes were labeled with SYBR-green dye. Quantification
was based on ΔCt values from specific reactions with anti-p53 antibody subtracted from non-specific
reactions with control IgG antibody, normalized to input signals. Fold enrichment of PCR products
amplified from (A) MDM2, (B) CDKN1A and (C) ALB (negative control) genes bound to p53 protein. D,
MCF7 cells were treated with or without 1 μM of C646, followed by 0.4 µM of doxorubicin for 48 h.
Immunoblot shows input levels of p53 and actin loading control. Indicated acetyl-lysines (AcK) were
detected from immunoprecipitated p53. E, MCF7 cells were treated with or without 1 μM of C646,
followed by the following agents: control (Con), 10 μM 5-fluorouracil (5-FU), 12.5 μM cisplatin (Cis), 10
nM camptothecin (Cpt), 2 µM etoposide (Eto), 25 nM actinomycin D (ActD) and 400 nM doxorubicin
(Dox). Graph shows CellTiter-Glo signal intensities expressed in relative luminescence units (RLU).
Bars, mean ± SD; n ≥ 3; t-test (ns = no sig dif; *, p < 0.05; **, p < 0.01). F, Protein levels of p53 and
K382-acetylated p53 at 48 h post treatment.
Supplementary Figure S2. Related to Figure 2.
C646 prevents specific p53 lysines from acetylation. A, Bar graph shows the quantified cell densities
from Fig. 2D. B, Quantified cell densities from Fig. 2G. C, Contour plot represents the relative proportion
of labelled cells from Fig. 2E. D, Relative proportion of labelled cells from Fig. 2H. Bars, mean ± SD; n
≥ 3; t-test (ns = no sig dif; *, p < 0.05; **, p < 0.01).
Supplementary Figure S3. Related to Figure 3.
C646 reduces the stabilization of p53 protein. A–E, HCT116, MCF7 and U2OS cells were treated with
doxorubicin alone (Dox) or a combination of C646 and doxorubicin (C+D), followed by addition of
cycloheximide, an inhibitor of translational elongation. (A) Immunoblot shows the dynamic levels of p53
at various time points after cessation of protein synthesis. Line charts expresses p53 protein levels as
a percentage relative to cycloheximide pre-treatment in (B) HCT116, (C) MCF7 and (D) U2OS cells. (E)
Bar graph displays the half-lives of p53 in the respective cell lines. Bars, mean ± SD; n ≥ 3; t-test (ns =
no sig dif; *, p < 0.05; **, p < 0.01).
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Zheng et al.
Supplementary Figure S4. Related to Figure 5.
C646 selectively protects human bone marrow over colorectal cancer cells. A, Sigmoidal plots show
doxorubicin dose-responses of primary human bone marrow cells treated as in Fig. 5B at 24 and 48 h
post treatment. B, Sigmoidal plots show doxorubicin dose-responses of human colorectal cancer cells
HCT116 p53+/+ and p53-/- treated as in Fig. 5C at 24 and 48 h post treatment. C, IC50 of doxorubicin in
human BM and HCT116 cells at 24 and 48 h as determined from supplementary Fig. S4A and S4B. D,
The therapeutic index of doxorubicin is expressed as the ratio of its IC50 in normal BM cells to colorectal
cancer cells (N/C). Bar graph shows the therapeutic indices as calculated from the data in
supplementary Fig. S4C. Bars, mean ± SD; n ≥ 3; t-test (ns = no sig dif; *, p < 0.05; **, p < 0.01).
Supplementary Figure S5. Related to Figure 6 and 7.
C646 protects bone marrow function while preserving doxorubicin efficacy. A, Mouse BM cells were
treated as in Fig. 6C. Graph shows the mRNA levels of the indicated genes as determined by RT-qPCR.
B, Mouse BM cells were treated as in Fig. 6D for 24 h and subjected to caspase 3/7 assay. Graph
shows the signal intensities of the early apoptosis marker expressed in relative luminescence units
(RLU). C, Mouse BM cells were treated as in Fig. 6E and harvested at 24, 48 and 72 h time points,
stained with propidium iodide and counted by an ADAM-MC automatic cell counter. Graph shows the
live cell counts relative to mock treatment. Bars, mean ± SD; n ≥ 3; t-test (ns = no sig dif; *, p < 0.05; **,
p < 0.01). D, HCT116 p53-/- cells were treated with or without 1 μM of C646, followed by 0, 0.4 or 2.0
µM of doxorubicin for 48 h. Immunoblot shows protein levels of p73, K321-acetylated and K327acetylated p73, with cleaved PARP as apoptosis marker and actin as loading control. E, Plots show
doxorubicin dose-responses of mouse BM cells treated as in Fig. 7A at 24 and 48 h post treatment.
F, Doxorubicin IC50 as calculated from Fig. 7A and supplementary Fig. S5E. G, Plots show C646 doseresponses of mouse BM cells treated as in Fig. 7B at 24 and 48 h post treatment. H, Mice were treated
as in Fig. 7D for 7 days and thereafter, the bone marrow and HT29 tumor cells were extracted,
homogenized and lysed. Immunoblot shows protein levels of p53, E2F1, and their transcriptional
targets, with cleaved PARP as apoptosis marker and actin as loading control.
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