Download Supplementary Figure legends Figure S1: Characterization of

Supplementary Figure legends
Figure S1: Characterization of cellular response to Myc and MycVD.
A: Immunoblot comparing levels of endogenous Myc in MCF10A, HeLa or HCT116 cells with
Myc expressed from the PGK promoter or Myc-ER (SFFV promoter) in pools of infected
MCF10A cells. Vinculin was used as loading control.
B: Cumulative growth curves of exponentially growing cells. 1.5 x 105 MCF10A cells expressing
moderate levels of Myc or MycVD and control cells were seeded and triplicate biological
samples were counted and re-plated at the indicated time points.
C: Both Myc and MycVD promote exit from G0. The indicated pools of MCF10A cells were
starved for 24 hrs. At the start of the experiment, cells were re-stimulated by addition of full
medium. The panels show FACS assays documenting the DNA content (propidium iodide) of
cell populations at the indicated times after stimulation together with control samples (n = 2).
D: High expression levels of Myc WT are counterselected more rapidly than MycVD in HMLE
cells. Cells were harvested at the indicated time points after infection. Relative Myc WT and
MycVD levels are quantified on the right. Vinculin was used as loading control (n=1).
E: Phase contrast pictures documenting the morphology of MCF10A cells infected with ctr, Myc
WT or MycVD, driven by either PGK (left panel) or SFFV (right panel) promoter. Scale bars =
100 µm.
Figure S2: Characterization of Myc-induced apoptosis.
A: Immunoblots documenting levels of Myc-ER and MycVD-ER proteins in nuclear extracts
after incubation with the indicated concentrations of 4-OHT for 1 h. Histone H2B was used as
loading control.
B: Phase contrast pictures documenting cell death upon activation of Myc-ER, but not MycVDER in HMLE cells. Scale bar = 100 µm
C: FACS assays documenting percentage of early ("AnnexinV+/PI-") and late apoptotic
("AnnexinV+/PI+") HMLE cells under the indicated experimental conditions (200 nM 4-OHT, 48
hrs). Error bars show SD (n = 3). p-values were calculated using a Student´s t-test (****: p <=
0.0001).
Figure S3: Myc-induced apoptosis requires transcription and involves Bim and p53.
A: Induction of apoptosis requires transcriptional activity of Myc. MCF10A-Myc-ER cells were
infected with viruses expressing a doxycycline-inducible shRNA targeting MAX. shMAX was
induced with 1 g/ml doxycycline. 24 hrs later, Myc-ER was activated by addition of 200 nM 4OHT. 48 hrs later, cells were harvested and the percentage of AnnexinV-positive cells was
determined by FACS analysis (upper panel). The lower panel shows control immunoblots. Error
bars represent SD (n = 3). p-values were calculated using a Student´s t-test (**: p<=0.01; ****: p
<= 0.0001).
B: Induction of apoptosis by mutant alleles of Myc. MCF10A cells were infected with
lentiviruses expressing either Myc WT, Myc∆BR, which lacks the basic region, or MycD, which
carries three mutations in the leucine zipper that preclude dimerization with Max (Huang et al,
2004). The upper panel shows FACS assays documenting the percentage of early apoptotic
("AnnexinV+/PI-") and late apoptotic ("AnnexinV+/PI+") cells. The lower panel shows
immunoblots documenting expression of Myc, cleaved PARP and Vinculin as loading control.
Error bars represent SD (n = 3). p-values were calculated using a Student´s t-test (*: p<=0.05;
****: p <= 0.0001).
C: Induction of apoptosis requires p53. The experiment was performed as before, except that
MCF10A-Myc-ER cells were superinfected with vectors expressing either an shRNA targeting
luciferase as control or depleting p53 (TP53). Upper panel: FACS analysis. p-values were
calculated using a Student´s t-test (****: p <= 0.0001). Lower panel: Immunoblots documenting
knockdown of p53 and cleavage of PARP.
D: Depletion of Bim attenuates Myc-induced apoptosis. MCF10A-cells were superinfected with
control shRNA, or an shRNA targeting Bim (BCL2L11). The analysis was performed as before.
Upper panel: FACS analysis. p-values were calculated using a Student´s t-test (***: p<=0.001).
Lower panel: Immunoblots documenting knockdown of BIM.
E: Overexpression of Bcl2 attenuates Myc-induced apoptosis. MCF10A-cells were superinfected
with control viruses, or viruses expressing BCL2. The analysis was performed as before. Left
panel:
FACS
analysis
(****:
p<=0.0001).
Right
panel:
Immunoblots
documenting
overexpression of BCL2.
F: Both Myc-ER and MycVD-ER induce expression of Bim. The panels show immunoblots
documenting expression of Bim splice variants and p53 after induction of either Myc-ER or
MycVD-ER for 96 hrs.
Figure S4: Analysis of Myc-dependent changes in gene expression.
A: Activation of Myc-ER or MycVD-ER does not significantly alter cellular RNA content.
MCF10A cells were stimulated with 100 nM 4-OHT for 24 hrs before the amount of total RNA
per cell was determined. Error bars show SD (n = 3).
B: qRT-PCR analyses validating microarray results. CCND2 is shown as an example for an
activated gene, DKK1 and ITGB4 are examples of genes repressed by Myc-ER, but not by
MycVD-ER. Error bars represent SD of technical triplicates (n=1).
C: Examples of previously identified sets of activated (upper table) or repressed (lower table)
Myc target genes, regulated in response to Myc-ER and MycVD-ER as determined by GSEA
analysis (C2: curated gene sets). NES: normalized enrichment score. FDR: False discovery rate.
D: Attenuation of MycVD-ER mediated repression. Normalized enrichment scores for all
significantly regulated gene sets (1543 sets, GSEA C2, FDR <=0.25 for Myc-ER 4-OHT versus
control) are plotted for Myc-ER and MycVD-ER. 28 sets of previously identified Myc target
genes sets are shown in red.
Figure S5: De-repression of Myc target genes in response to depletion of Miz1.
A:
GO-term analysis documenting functional annotations of genes repressed by Myc WT
(log2FC <= -0.5), but less by Myc VD (log2FC >= 0.5 for the difference between Myc WT and
Myc VD) in U2OS cells (Walz et al., 2014). The x-axis indicates significance as –log10 q value
(Benjamini); Top 10 significant GO-terms are shown.
B: Left panel: Expression of Myc-repressed genes in U2OS cells (Walz et al., 2014). x-axis
shows the difference in Myc-dependent expression in control versus Miz1-depleted cells. y-axis
shows the difference in expression between Myc and Myc VD. Data are represented in 57 bins
with 70 genes each.
Right panel: Same analysis as in left panel except that y-axis shows the difference in expression
between Myc-ER and MycVD-ER in MCF10A cells. Data are represented in 30 bins with 70
genes each. r: Spearman’s rank correlation coefficient.
Figure S6: Chromatin binding of Myc and Miz1.
A: Immunoblot documenting expression of Miz1 and Myc-ER fusion proteins. Where indicated,
100 nM 4-OHT was added for 24 hrs. Tubulin was used as loading control. All three observed
bands are specific for Miz1.
B: Venn diagram documenting overlap of Miz1 binding sites in murine neuronal progenitor cells
(NPCs) and in MCF10A cells. Data for NPCs are taken from (Wolf et al, 2013). p-value is based
on a hypergeometric distribution.
Figure S7: Repression of SRF target genes.
A: Table summarizing the GSEA C3 motif analysis. Shown are known transcription factor
families and corresponding gene sets that are significantly enriched in promoters of Myc-ER
versus MycVD-ER repressed genes. NES = normalized enrichment score; FDR = false discovery
rate.
B: Summary of microarray analyses of gene expression in MCF10A-Myc-ER and MycVD-ER
cells upon incubation with 100 nM 4-OHT for 24 hrs. The plot is taken from Figure 2D, and
genes that are contained in the SRF_C gene set (Figure 5A) are indicated by red dots.
C: Examples of ChIP-sequencing tracks for genes bound by Myc/Miz1 complexes and by SRF
(SRF data are from MCF7 cells). Fold changes above tracks are taken from the microarray
analysis.
D: qRT-PCR assays documenting expression of the indicated SRF target genes after induction of
Myc-ER (100 nM, 24 hrs). Before activation of Myc-ER, cells had been superinfected either with
control viruses or with viruses expressing a constitutively active RhoA allele (RhoAQ63L). Error
bars represent SD of technical triplicates from one experiment (n = 2).
Figure S8: Regulation of apoptosis by Myc and ∆N-Mal.
A: Caspase 3/7 activity assays documenting attenuation of Myc-dependent apoptosis in response
to addition TRAIL in response to expression of ∆N-Mal (doxycycline-inducible). Error bars show
SD of triplicate biological assays (n = 3).
B: FACS assays documenting percentage of early apoptotic (AnnexinV-positive) and late
apoptotic (AnnexinV/propidium iodide-double positive) cells under the indicated experimental
conditions. Error bars show SD of triplicate biological assays (n = 3).
C: MCF10A cells were cultured as before. Inhibitors of Ilk (Cpd 22, EMD MILLIPORE) and Fak
(FAK inhibitor 14, Santa Cruz Biotechnology) were added at the indicated concentrations. 2 hrs
later, cells were lysed and lysates probed with antibodies against AktpS473, total Akt and Gapdh
as loading control.
D: MCF10A MYC-ER cells were transfected with either 100 nM control siRNA or siRNA
targeting AKT1/2 and incubated for 72 hours before analysis (n=2).
Supplementary Table S1: Statistics of ChIP-Sequencing reads.
Supplementary Table S2: Antibodies used in this study.
Supplementary Table S3: Oligonucleotides for qRT-PCR and shRNA-mediated depletion.