Cappello et al.
Supplementary Figure Legends
Supplementary Figure S1. Evaluation of in vivo tumor growth of various PyMT knockdown
clones.
(a) Confirmation of shRNA-mediated knockdown. qRT-PCR analysis of the expression of target
gene mRNA in mouse PyMT tumor cells transduced with lentiviral vector expressing control
scramble shRNA (PyMT-ctr) or shRNA specific for the indicated targets. Data were normalized
to gapdh mRNA levels and expressed relative to levels in control cells (set to 1.0). (b) KaplanMeier survival curves for syngeneic 129P2Ola mice injected in the tail vein with either PyMT-ctr
cells or the indicated PyMT knockdown clones. Significance: shStip1 p=0.28, shGrem1
p=0.0007, shAnln p=0.0007, shTrim24 p=0.18, shWnk2 p=0.0048, shPlk4 p=0.0057, shPrmt1
p=0.0048, shCdc2a p=0.0007, shCdc7 p=0.0007. Data of 5 mice per group. p values have been
determined by log-rank test.
Supplementary Figure S2. Nek2 knockdown induces aneuploidy in PyMT cells.
(a) Flow cytometric cell cycle analysis of PyMT-ctr and PyMT-shNek2 cells at the indicated
timepoints after release from nocodazole treatment. Red peaks, G1 and G2/M phases of diploid
cells; blue peaks, subG1 phase (dead cells); yellow peaks, G1 and G2/M phases of aneuploid
cells. Results are representative of three independent experiments. (b) Quantitation of the cell
cycle analyses in (a). (c) Quantitation of the percentages of the cells in (a) that were diploid
(DIP) or aneuploid (ANE). For (a), (b) and (c), representative data is shown out of three
independent experiments.
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Supplementary Figure S3. Nek2 knockdown induces aneuploidy in MDA-MB-231 cells.
(a) Flow cytometric cell cycle analysis of MDA-MB-231 cells expressing control shRNA
(MDA-MB-231-ctr) or shRNA against Nek2 (MDA-MB-231-shNek2) at the indicated times
after release from nocodazole inhibition. Data were analyzed as for Figure S2a and are
representative of three independent experiments. (b) Quantitation of the cell cycle analyses in
(a). (c) Quantitation of the percentages of the cells in (a) that were diploid (DIP) or aneuploid
(ANE). For (a), (b) and (c), representative data is shown out of three independent experiments.
Supplementary Figure S4. Nek2 knockdown impairs the proliferation of human breast cancer
cell lines.
(a) qRT-PCR analysis of Nek2 mRNA expression in human HCC1806, MCF-7 and T47D breast
cancer cell lines after transduction with lentiviral vectors expressing control scramble shRNA
(ctr) or shNek2. Data were normalized to GADPH mRNA levels and are expressed relative to
levels in control cells (set to 1.0). (b) Immunoblot of Nek2 protein in extracts of the cells in (a).
G6PDH, loading control. (c) Proliferative capacity of control and Nek2 knockdown HCC1806,
MCF-7 and T47D breast cancer cells at the indicated timepoints after release from either serum
starvation (-FBS) or nocodazole treatment (+NOC). Data represents the mean  SEM of two
independent experiments. * p < 0,05 and ** p < 0,01
Supplementary Figure S5. Loss of Nek2 induces cell cycle defects in the human breast cancer
cell line HCC1806.
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(a) Flow cytometric cell cycle analysis of HCC1806 cells expressing control shRNA (HCC1806ctr) or shRNA against Nek2 (HCC1806-shNek2) at the indicated timepoints after serum
starvation. Data were analyzed as for Figure S2a. (b) Quantitation of the cell cycle analyses in
(a). (c) Quantitation of cell cycle analysis of HCC1806-ctr and HCC1806-shNek2 cells at the
indicated times after release from nocodazole treatment. For (a), (b) and (c), representative data
is shown out of three independent experiments.
Supplementary Figure S6. Loss of Nek2 has no effect on the cell cycle but induces aneuploidy
in the human breast cancer cell line T47D.
(a) Flow cytometric cell cycle analysis of T47D cells expressing control shRNA (T47D-ctr) or
shRNA against Nek2 (T47D-shNek2) at the indicated timepoints after serum starvation. Data
were analyzed as for Figure S2a. (b) Quantitation of the cell cycle analyses in (a). (c)
Quantitation of cell cycle analysis of T47D-ctr and T47D-shNek2 cells at the indicated times
after release from nocodazole treatment. For (a), (b) and (c), representative data is shown out of
three independent experiments.
Supplementary Figure S7. Loss of Nek2 does has no effect on the cell cycle or on the induction
of aneuploidy in the human breast cancer cell line MCF7.
(a) Flow cytometric cell cycle analysis of MCF7 cells expressing control shRNA (MCF7 -ctr) or
shRNA against Nek2 (MCF7-shNek2) at the indicated timepoints after serum starvation. Data
were analyzed as for Figure S2a. (b) Quantitation of the cell cycle analyses in (a). (c)
Quantitation of a parallel cell cycle analysis of MCF7-ctr and MCF7-shNek2 cells at the
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indicated times after release from nocodazole treatment. For (a), (b) and (c), representative data
is shown out of three independent experiments.
Supplementary Figure S8. Nek2 knockdown reduces the migration but not invasion of MDAMB-231 cells.
(a) Real-time migration assay. The indicated numbers of MDA-MB-231-ctr and MDA-MB-231shNek2#3 cells were applied to an xCelligence real-time migration system. Cell migration is
expressed as the slope [1/h], where a higher slope indicates greater numbers of migrating cells.
(b) Transwell migration assay. The percentages of MDA-MB-231-ctr and MDA-MB-231shNek2#3 cells able to migrate. Data were normalized to the migration of control cells (set to
100%). (c) Transwell invasion assay. The percentages of MDA-MB-231-ctr and MDA-MB-231shNek2#3 cells able to invade. Data were normalized to the invasion of control cells (set to
100%).
Supplementary Movie Legends
Supplementary Movie S1. Normal division of a Nek2-expressing MDA-MB-231 cell into two
daughter cells. MDA-MB-231 cells expressing histone 2B conjugated to GFP (green nuclei), and
inducibly co-expressing shNek2 with RFP under the control of doxycycline (DOX), were serumstarved overnight. The culture medium was changed to DMEM containing 10% FBS (and
lacking DOX) and the cells were maintained/monitored in 5% CO2 at 37°C in the microscope
chamber of a spinning disc confocal microscope (Leica DMI 6000B) for 150 hrs. This movie
was generated by real-time microscopy and follows the mitosis of a MDA-MB-231 cell in which
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Nek2 knockdown was not induced. The movie shown is representative of five distinct fields
recorded with two or more cells examined per field.
Supplementary Movie S2. Abnormal division of a MDA-MB-231 cell lacking Nek2 expression.
MDA-MB-231 cells co-expressing DOX-inducible Nek2 shRNA plus RFP (red) along with
histone 2B-GFP (green nuclei) were serum-starved overnight. The culture medium was changed
to DMEM containing 10% FBS plus DOX to induce Nek2 knockdown, and the cells were
maintained/monitored in 5% CO2 at 37°C in the microscope chamber of a spinning disc confocal
microscope (Leica DMI 6000B) for 150 hrs. This movie shows the compromised mitosis and
multinuclear status of an MDA-MB-231 cell depleted of Nek2. Both daughter cells produced by
this abnormal cell division exhibit condensed chromatin and abnormal morphology. The movie
shown is representative of ten distinct fields with two or more cells examined per field.
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