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Supporting figure legends
Fig. S1 Selection of the experimental system. (A) Scheme showing the strategy for
the selection of the experimental system. (B) MCF-7 cells, SJSA cells, HCT116
TPp53+/+ cells and HCT116 TP53-/- cells were treated with five p53 activating
compounds, RITA, nutlin, actinomycin D (ActD), cisplatin (CDDP) and
5-fluorouracil (5-FU), with or without pretreatment with p53 inhibitor PFT-α.
Apoptosis was assayed by FACS of Annexin V-propidium iodide (PI) stained cells,
Rhodamine 123 (Rho123)-PI stained cells or Sub-G1 population detection using PI
staining. Cell cycle was assessed by FACS of PI-stained cells.
Fig. S2 Hierarchical clustering analysis of the shRNA screen data from present
study (MCF-7) and the previously published shRNA screen data (in HCT116
cells, Sullivan et.al., 2012). Shown is the heatmap in which the abundance of
individual shRNAs is indicated by blue color: light blue, low abundance; dark blue,
high abundance.
Fig. S3 Network analysis of SLNs and RNs revealed Sp1 as the only RN at the
center of the network with p53.
Fig. S4 Sp1 is a key determinant of p53-mediated apoptosis. The depletion of Sp1
in MCF-7 (A) and HCT116 cells (B) with two different Sp1 shRNAs rescued cells
from inhibition by RITA as assessed by a colony formation assay. (C) Ectopic
expression of Sp1 promotes apoptosis induced by nutlin in MCF-7 and HCT116 cells,
but not in HCT116 TP53-/- cells as assessed by FACS of Annexin V-PI stained cells.
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Fig. S5 Effect of Sp1 depletion on the gene expression profile induced by RITA in
MCF-7 cells. The plot presents the fold change of gene expression significantly
affected by RITA in MCF-7 cells. Data are presented as Log2(RITA/DMSO). p <
0.001.
Fig. S6 shRNA screen data for p53-induced pro-apoptotic genes. The
experimental procedure of genome-wide short hairpin RNA (shRNA) screen in MCF7
cells and data processing were performed as described in SI Materials and Methods.
The blue bars indicate the ratio of read counts between RITA-treated cells and
DMSO-treated cells (RITA versus DMSO). The red curve indicates -Lg(p value).
Fig. S7 Comparison of gene expression profiles induced by RITA and nutlin in
Sp1-depeleted and non-depleted cells. Venn diagrams show the intersection of
genes significantly changed by RITA or nutlin treatment in MCF-7 cells without (A)
or with (B) Sp1 depletion. p < 0.001. (C) Heatmap representation of expression
profiles of cell cycle arrest genes in MCF-7 cells with or without Sp1 depletion upon
RITA or nutlin treatment. Columns indicate arrays and rows indicate genes.
Fig. S8 Pathways enriched with SLNs. Pathway analysis of SLNs identified via
analysis of the genome-wide shRNA screen data was performed using DAVID tool. p
< 0.05.
Fig. S9 Pathways enriched with 227 p53 target genes co-repressed by p53 and
Sp1 upon RITA treatment. Pathway analysis of these 227 genes was performed
using DAVID tool. p < 0.05.
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Fig. S10 Network analysis of 227 p53-targets co-repressed by p53 and Sp1.
Analysis of functional connections between proteins encoded by these 227 genes was
performed using STRING software. Only nodes having at least two connections are
shown.
Fig. S11 Binding profiles of p53 on selected genes. Binding profiles of p53 on
selected genes in MCF-7 cells treated with DMSO, RITA or nutlin were plotted using
ChIP-seq data. Regions amplified by ChIP-PCR are indicated in each gene.
Fig. S12. Ectopic expression of Sp1 promotes the binding of Sp1 to p53 target
genes and p53-mediated pro-apoptotic transcriptional repression in MCF-7 cells
upon nutlin treatment. (A) Binding of Sp1 to selected genes (described in Fig. 4)
was assessed by ChIP-PCR in nutlin-treated MCF-7 cells with or without ectopic
expression of Sp1. (B) The mRNA level of selected genes was examined by qPCR. In
(A) and (B) data are presented as mean ± SD, n = 3.
Fig. S13. Sp1 protein level is important for the formation of p53/Sp1 complex. (A)
The amount of Sp1 bound to p53 upon RITA or nutlin treatment of MCF-7 cells was
detected by p53 co-immunoprecipitation followed by immunoblotting. Right panel:
equal amount of immunoprecipitated p53 was loaded to assess bound Sp1. (B) The
amount of Sp1 bound to p53 upon nutlin treatment of MCF-7 cells with or without
ectopic Sp1 expression was detected by p53 co-immunoprecipitation followed by
immunoblotting. Equal amount of immunoprecipitated p53 was loaded to assess
bound Sp1.
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Fig. S14. Sp1 is not required for the induction of MDM2 transcription by p53.
mRNA level of MDM2 in MCF-7 cells with or without Sp1 depletion upon RITA or
nutlin treatment was examined by qPCR. Data are presented as mean ± SD, n = 3.
Table S1 Raw data of the genome-wide shRNA screen
Table S2 Validation of genome-wide shRNA screen. 15 genes were selected from
screen based on three criteria (p value < 0.1, FDR < 0.3, p(wZ) < 0.1) and re-tested
for validation purpose. Blue indicates reduced viability (synthetic lethal node, SLN),
purple indicates enhanced viability (resistance node, RN). Results were compared
with statistic analysis of screen data.
Table S3 List of RNs and SLNs. These resistance nodes and synthetic lethal nodes
were selected based on a stringent criteria: p value < 0.05, FDR < 0.1, p(wZ) < 0.1.
Table S4 List of RNs that have been identified as direct transcriptional targets of
Sp1.
Table S5 Prediction of Sp1 response elements in 173 'RITA/nutlin common'
targets using oPOSSUM software.
Table S6 Prediction of Sp1 response elements in 54 ‘RITA-specific’ p53 targets
using oPOSSUM software.
Table S7 List of primers
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