Zaccara et al., Supplemental text, Figure Legends and Figures
Supplemental Figure Legends
Figure S1. Doxorubicin and Nutlin-3a treatment of MCF7 cells result in similar,
low-level toxicity.
(A) WST toxicity assay upon treatment with Doxorubicin (Doxo) and Nutlin-3a
(Nutlin). MCF7vector cells were treated for 16 hours at the indicated doses. Results
are shown as percentage reduction in the absorbance signal compared to the mock
treatment.
(B) FACS analysis on MCF7vector cells. Cells were treated for 16 hours at the chosen
doses of 1.5μM Doxo and 10μM Nutlin. Cells were treated with Camptothecin (5μM)
as positive control. Staining with Annexin-V and To-Pro3 was performed.
Percentages of cell death are shown in each square.
(C) Western Blot analysis of MCF7vector and MCF7shp53 cell extracts. p53 protein
levels after Doxo and Nutlin treatment were measured. p21, a well-known p53
target, was included as a control. GAPDH was used as reference protein for loading
control.
Figure S2. Polysomal profiling of MCF7vector and MCF7shp53 cells.
Profiles after sucrose gradient fractionation of cytoplasmic extracts prepared from
MCF7vector (A) and MCF7shp53 (B) cell lines. The conditions tested were Mock,
Doxo (1.5μM) and Nutlin (10μM) after 8 hours or 16 hours of treatment. The subpolysomal fractions (sub: free RNA, small-40S and large-60S, monosomes-80S) and
the polysomal fractions (pol) were separated. Sub and pol fractions were combined
in two separated tubes for RNA extraction. The 16-hour images are also presented
in Figure 1 of the main body, as the microarray expression experiments were
performed on those extracts.
Figure S3. Gene Ontology (GO) analysis on uncoupled Differentially Expressed
Genes (DEGs).
Plots report the output of the DAVID functional annotation cluster tool, with the
most enriched functional categories identified in our list of DEGs. For each category,
genes associated with that category are reported on the left.
(A) GO analysis of polysomal upregulated DEGs.
(B) GO analysis of subpolysomal downregulated DEGs.
(C) GO analysis of polysomal downregulated uncoupled DEGs.
Figure S4. miR-34a expression levels quantified by qPCR.
(A) miR-34a expression levels in MCF7vector and MCF7shp53 cells treated with
Doxorubicin and Nutlin-3a. Data are plotted relative to U6 levels, used as a reference
gene, and the mock condition. Means ± SD of three technical replicates are shown
(B) Ectopic overexpression of miR-34a in the MCF7vector cell line. The graph shows
the miR-34a levels quantified by qPCR after transfection of the pre-miR-34a
expression plasmid. Results are presented as fold of change relative to both U6
levels and the empty miR-expression plasmid psiUx, that was used as negative
control. A pre-miR-636 overexpressed plasmid was transfected as an additional
control of miR-34a specificity, and miR-34a levels were quantified also in this
condition. Means ± SD of three technical replicates are shown.
(C) Relative miR-34a expression levels quantified in MCF7vector cells transfected
with an LNA miR-34a inhibitor or a negative control (Control A) and treated 48
hours later with Doxo or Nutlin for 16 hours. Data are plotted relative to U6 levels
and each mock condition. Means ± SD of three technical replicates are shown.
Figure S5. Enrichment of regulatory elements in the UTRs of coupled DEGs
The enrichment analysis of post-transcriptional regulatory elements for coupled
DEGs after Doxorubicin and Nutlin-3a treatment was performed with AURA 2
(aura.science.unitn.it/). A heatmap graph based on the enrichment p-values
adjusted for multiple testing with the Benjamini-Hochberg method is shown. We
analyzed also the enrichment of regulatory elements for DEGs in common to both
treatments, referred to as “common”.
Target mRNAs for several RBPs were particularly enriched among down-regulated
coupled DEGs. Several miRNAs were also identified as putative regulators of the
coupled DEGs, although in most cases the relationships were not statistically
significant. Curiously, miR-17-19b-1, -21 and -146a targets were enriched among
the upregulated DEGs, suggesting that these miRNAs might be downregulated by
Doxo and Nutlin treatment.
Figure S6. Gene Ontology (GO) analysis on additional groups of transcriptional
/translational uncoupled DEGs.
Gene Ontology analysis on uncoupled DEGs that were upregulated (A) or
downregulated (B) only in total RNA or were upregulated in subpolysomal RNA (C).
Shown for each group are the most enriched ontology terms with the relative pvalue, the gene names in those categories, and a Venn diagram presenting the
overlap between DEGs for the two treatments (see also Table S1E). (A,
B)Transcriptionally modulated DEGs may represent direct p53 targets for which
changes in cytoplasmic mRNA levels have not occurred yet. GO analysis for these
targets indicated an enrichment for lysosome, glycerolipid metabolic process and
nuclear lumen terms.
(C) Subpolysomal upregulated genes could result from transcriptional upregulation that had already subsided at the time point chosen for analysis but did
not yet reach translation; these genes were enriched for kinase inhibitor activity and
amino-acid biosynthesis ontology terms. The figure is organized as in panels A and B.
Figure S7. Heatmap representing enriched GO terms for DEGs in the three RNA
preparations that were analyzed by expression microarrays.
(A) Doxorubicin and Nutlin-3a treatments did not markedly differ in the activation
of biological responses, based on the observation of Gene Ontology and pathways
analyses. Both drugs modulated the expression genes identified with the p53
pathway, a term that was enriched at a similar level of statistical significance among
the upregulated-coupled DEGs (panel A). That finding is consistent with the
canonical p53 function as a transcription factor and with a level of coordination
between transcription and translation enabling the execution of the p53-dependent
responses. However, apoptotic terms are more significantly enriched in the
upregulated polysomal (pol) and subpolysomal (sub) DEGs than in the upregulated
total (tot) DEGs. This finding uncovers a new layer of complexity in the modulation
of p53-dependent apoptosis.
(B) No clear differences in GO terms were identified when downregulated DEGs
were analyzed in the three different fractions.
Table S1. Lists of Differentially Expressed Genes in all the different categories.
We present in a friendly format the lists of official gene names of DEGs identified for
all the different categories analyzed though the paper. A: coupled; B: only
upregulated in translation; C: only downregulated in the subpolysomal fraction; D:
only downregulated in the polysomal fraction; E: only modulated in total RNA or
only upregulated in the subpolysomal fraction. Details of these DEGs are presented
in Table S5.
Table S2. Ingenuity Pathway Analysis (IPA) and DAVID results for coupled
DEGs common to Doxorubicin and Nutlin-3a treatments.
(A) We interrogated the 225 common DEGs for pathway enrichment. IPA analyzes
them according to their log2 fold change and p-value, combining up- and
downregulated DEGs. Having established the p53 pathway as the most significantly
enriched, we report the IPA output for 64 genes associated with the p53 pathway.
Column C (Log Ratio Doxo) and D (Log Ratio Nutlin) present the data from our array
analysis as log2 fold change. Column E (Findings) reports IPA terms based on
curated findings from the literature.
(B) DAVID output for the functional annotation analysis of the 64 DEGs in our
experiments that are associated with the p53 pathway according to IPA. The
annotation stringency was set to high. As expected, enriched GO terms are related
with cell cycle, apoptosis and DNA damage.
Table S3. Upstream regulators predicted by IPA.
Presented is the list of predicted upstream regulators that may be responsible for
the gene expression changes, based on the Ingenuity Pathway Analysis (IPA).
Table S4. Lists of RNA Binding Proteins (RBPs).
List of RBP genes found as uncoupled DEGs or coupled DEGs after Doxorubicin or
Nutlin-3a treatment. The last column corresponds to RBPs common to both
treatments for uncoupled and coupled DEGs together.
Table S5. Differential Expression microarray results.
Table containing detailed information on the whole set of genes represented on the
array. For each gene the table contains:
- Agilent probe ID
- HGNC symbol
- fold change for each RNA fraction (total, polysomal and subpolysomal) upon
Doxorubicin or Nutlin treatments (for a total of 6 pairwise comparisons)
- moderated t-test p-values, with and without multiple test correction (Benjamini
Hochberg), for each of the 6 pairwise comparisons
- normalized signal intensities for each hybridization
All the data are deposited in GEO under the following accession number: GSE50650
Table S6: Primers’ list
List of primers used with Sybr-Green qPCR assays. The genes that are not listed
were quantified by commercial Taqman assays.
Table S7. ΔCq of target genes analyzed by qPCR.
In the main figures, qPCR data is presented as relative fold of change and is
normalized to three reference genes as well as the results obtained in the mock
condition for each RNA preparation (tot, sub, pol). This normalization was chosen
because of our interest in defining the induced changes after p53 activation
separately for each RNA fraction. In order to present how these genes are
distributed among the three fractions in the mock condition, the column “ΔCq”
shows the Cq values for each gene after the normalization with the three reference
genes (Cq target - Cq reference genes).
A. MCF7vector 16hr-mock condition; B. MCF7shp53- 16hr mock condition; C.
MCF7vector 8hr mock condition; D. MCF7shp53 8hr mock condition.