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 sub-polysomal 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 ( 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 p-value, 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 up-regulation 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.