Download Supplementary Information (doc 30K)

Supplementary Figures Description
Fig. S1. Western blot analysis in HEp2 for stable USP7 depletion and HEp2 or H1299 cells to
compare expression of p53, Daxx and USP7. (A) Western blot representing USP7 depletion in HEp2
cells stably depleted by control or USP7 shRNAs. (B) Western blot analysis of p53, Daxx and USP7 in
HEp2 and H1299 cells. HEp2 cells express wild-type p53 while H1299 are p53 null. In spite of this
difference these cell lines express similar levels of Daxx and USP7.
Fig. S2. USP7 depletion results in stabilization of cyclin B1 (cycB) in HCT-116 isogenic cells in p53independent manner. (A) Western blot analysis of p53, Daxx and USP7 in isogenic HCT-116 cell lines
for p53 (HCT-116 parental and HCT-116 p53-/-). Actin immuno-blot is used for internal control.
Regardless the p53 status, Daxx and USP7 are expressed at the same levels in the two cell lines. (B)
Western blot analysis of cyclin B in HCT-116 parental (top) or HCT-116 p53-/- (bottom) cells that were
synchronized by double thymidine block (0 hr) and simultaneously transfected with siRNAs. Samples
were taken 72h post-transfection at the DTB (0) and 6, 8 and 10 hours after DTB release.
Fig. S3. Transient USP7 depletion destabilizes CHFR and induces accumulation of Aurora-A
protein levels in breast cancer cell line MDA-MB-468. (A) Western blot analysis of CHFR stability in
MDA-MB-468 cells transiently depleted by control or USP7 siRNAs and exposed to 1 µg/mL of
cyclohexamide (CHX) for 2, 4 or 6 hours. Numbers represent quantification of CHFR protein over
internal control (actin) and control-depleted at 0 hours. Depletion of USP7 causes reduction of CHFR
protein levels. (B) Western blot analysis showing levels of Aurora-A kinase in MDA-MB-468 cells
transiently depleted by control or USP7 siRNAs. Numbers below the blots represent quantification of
Aurora-A protein over actin.
Fig. S4. Transient depletion of either Daxx or USP7 causes accumulation of multipolar mitoses.
Graph represents the quantification of multipolar events over the total number of mitosis in control, Daxx
and USP7 siRNAs, in HEp2 and H1299 cells. For each experiment staining of Aurora-A, α-tubulin and
DNA was performed to label spindle poles and assess chromosomal segregation. At least one hundred
mitotic events were counted per experiment. Transient depletion of USP7 and Daxx in HEp2 and H1299
cells causes increase of multipolar spindles.
Fig. S5. USP7 depletion causes accumulation of Aurora-A kinase in HCT-116 isogenic cells in p53independent manner. Representative western blot analysis showing levels of Aurora-A kinase in HCT116 parental and p53 -/- cells, control or USP7 depleted. Cells were synchronized by DTB and releases in
growth media for 5 hours. Transient depletion of USP7 in these cell lines causes increase of Aurora-A
protein levels in a p53 independent manner.
1
Fig. S6. Aurora-A and CHFR z-scores in NCI-60. Scatter plot representing NCI-60 data for Aurora-A
and CHFR z-score across the NCI-60 cell lines separated according response to Taxol (means ± SEM).
Cell lines classified as resistant have negative z-scores for Taxol response, while sensitive cell lines have
positive z-scores (Table S2).
Table S1. Z-scores values for Daxx, USP7 and Taxol across the NCI-60 cell lines. Resistant cell lines
have negative z-scores for Taxol response, while sensitive cell lines have positive z-scores. Highlighted
Daxx z-score values correspond to semi-open circles or semi-open squares represented in Fig. 6 cell for
cell lines with negative or with positive Daxx z-scores respectively.
Table S2. Z-scores values for Aurora-A, CHFR and Taxol across the NCI-60 cell lines. Negative
Taxol z-scores represent resistant cell lines, while positive Taxol z-scores represent sensitive cell lines.
2