Supplementary Figure Legends
Supplementary Figure 1. Id2 is degraded by APC/CCdh1 and binds to the core
subunits of APC/C and Cdh1. a, Proteins associated with Id2 in neuroblastoma
cells. Cellular extracts prepared from IMR-32 stably expressing Flag-HA-Id2 (FHId2) or the empty vector (Vec) were sequentially immunoprecipitated with Flag
and HA antibodies. Associated proteins were analyzed by Silver stain.
The
indicated proteins were identified by LC-MS/MS. b, Endogenous Id2 protein is
destabilized by Cdh1. U2OS cells were transfected with a Cdh1 expression
vector or the empty vector and analyzed after 12 h by Western blot (shown in
Fig. 1c). Parallel cultures were assayed by Flow cytometry. c, Immunoblot
analysis of U2OS cells that were engineered to produce Cdh1 after removal of
tetracycline (Tet). Where indicated the proteasomal inhibitor LLNL was added. d,
Lysates from cells treated with tet or released from tet for 12 h were
immunoprecipitated with E47 antibody and analyzed by Western blot for Id2
(E47-bound Id2). Total Id2, Cdh1 and actin are shown. e, Hela cells were
transfected with expression plasmids for Id2 (wild type Id2 or 15 amino acid Nterminal deletion mutant Id2) and E47 in the presence or absence of Cdh1 and
analyzed by Western blot. f, Interaction between Id2 and Cdh1. Hela cells were
transfected with the indicated expression plasmids and left untreated or treated
with MG-132 for 6 h before lysis. Lysates were reciprocally immunoprecipitated
with Flag and HA antibodies followed by Western blot with antibodies against HA
and Flag, respectively. g, Endogenous APC/C subunits and Cdh1 activator are
associated with Id2 in neuroblastoma cells. IMR-32 cells were treated with
1
MG132 before lysis. Lysates were immunoprecipitated with rabbit IgG or a
polyclonal antibody against Id2 and analyzed by Western blot for the indicated
APC/C
components.
Input,
0.5%
of
total
extract
used
for
each
immunoprecipitation.
Supplementary Figure 2. Id2 is unstable in cells withdrawing the cell cycle. a,
Immunoblot of Id2 in NIH3T3 cells deprived of serum for 12 h. b, CHX was added
and extracts were prepared at the indicated times and analyzed by immunoblot.
Double amount of total lysates was loaded for the serum deprived set and the
exposures of the Id2 blots were normalized to show comparable levels of Id2 at
the zero time point.
Supplementary Figure 3. Id2 protein stability in quiescent cells is regulated by
Cdh1. a, U2OS-Id2 cells in exponential phase of growth (0) were deprived of
serum for the indicated times in the absence (-) or in the presence (+) of MG-132
for 6 h and analyzed by immunoblot. b, Pulse-chase analysis of U2OS cells
stably expressing Id2 in the presence of serum (+ FBS) or after 48 h of serum
deprivation (- FBS). Id2 was recovered by anti-Id2 immunoprecipitation and
detected by autoradiography. c, Immunoblot of Id2 in SK-N-SH human
neuroblastoma cells treated with vehicle (Exp) or RA for 48 h before addition of
CHX for the indicated times (bottom panels). Cell extracts were analyzed by
immunoblot. The exposures of the Id2 blots were normalized to show
comparable levels of Id2 at the zero time point. d, U2OS-Id2 cells were
2
transfected with siRNA corresponding to Lamin, firefly luciferase (CTR), or Cdh1
mRNA and analyzed 16 h after transfection. Exogenous Id2 and endogenous Id1
were detected by immunoblot. e, U2OS-Id2 cells were cultured at low density
and after transfection with CTR or Cdh1 siRNA were grown in the presence of
serum or starved from serum for the indicated times. f, Quantification of band
intensities from Fig. 1g, showing the turnover of endogenous Id2 of quiescent
LAN-1 neuroblastoma cells in the presence and absence of Cdh1. The Id2 halflife is 3.7 minutes in control-treated cells and 36.6 minutes in Cdh1-knocked
down cells. g, Immunoblot of Hela cells transfected with Id2 in the presence or
absence of an Emi1 expression vector.
Supplementary Figure 4. Degradation of Id protein by APC/CCdh1 is dependent
on D box. a, Hela cells were transfected with wild type Id2 (Id2) or Id2-DB
carrying deletion of amino acids 100-107 in the presence or absence of Cdh1
and analyzed by immunoblot. Hela cells were transfected with Id2HLH (b), Id1
(c), Id3 (d) or Id4 (e) with or without Cdh1 and analyzed by immunoblot.
Supplementary Figure 5. Dual interaction modules of Id2 with Cdh1 and core
APC/C. a, Immunoblot for Cdh1, Cdc27, Apc1 and actin from binding assay
using GST, GST-Id2, GST-Id2-DBM and GST-Id2DB and extracts from Hela
cells. b, Hela cells were transfected with Flag-Id2 or Flag-Id2-DBM expression
plasmids. Lysates were immunoprecipitated with Flag antibody followed by
Western blot. Input, 0.5% of total extract used for each immunoprecipitation. c,
3
Immunoblot analysis of U2OS cells stably expressing Id2 or Id2-DBM after
treatment with TGF for 48 h. d, Autoradiogram of
35S-labelled
Id2 after in vitro
ubiquitination by immunopurified APC/C in the presence or in the absence of in
vitro translated Cdh1 at the indicated times. Arrowhead indicates unmodified Id2.
Supplementary Figure 6. Id2 does not affect the integrity and activity of
APC/CCdh1 a, IMR-32 cells were infected with Ad-vect or Ad-Id2 and cell extracts
were immunoprecipitated with Cdc27 antibody or mouse IgG followed by
immunoblot for the indicated proteins. Input is 0.5% of the lysate used in the
immunoprecipitation. b, Immunoblot of APC/CCdh1 targets from IMR-32 cells
infected with Ad-vect or Ad-Id2. c, Autoradiogram of
35S-labelled
cyclin B after in
vitro degradation assay by immunopurified APC/C in the presence (+) or in the
absence (-) of in vitro translated Cdh1 at the indicated times.
Supplementary Fig. 7. Id2 is a target of APC/CCdh1 for axonal growth. a, Id2
associates with APC/C core subunits in the brain. Extracts from mouse brain at
E16 were immunoprecipitated with Id2 antibody or rabbit IgG followed by
immunoblot. b, Cerebellar granule neurons express Id2 during in vitro axonal
elongation (1 to 4 days). 293T cells treated with CTR (-) or Id2 (+) siRNA are
shown as a control for Id2 immunoblot. c, Expression of Id2 in granule neurons is
markedly increased by proteasomal inhibition with MG-132. 293T are as in b. d,
Phase contrast microscopy of neuronally differentiated SK-N-SH after sequential
tretment with RA (5 days) and BDNF (4 days). e, Id2 associates with Cdh1 and
4
the core APC/C in terminally differentiated SK-N-SH. RA/BDNF treated SK-N-SH
were exposed to MG132 before lysis. Lysates were immunoprecipitated with
rabbit IgG or polyclonal anti-Id2 antibody and analyzed by Western blot for the
indicated APC/C components. f, Silencing of Cdh1 in neuronally differentiated
SK-N-SH stabilizes Id2 and Id1. RA/BDNF treated SK-N-SH were transfected
with siRNA corresponding to firefly luciferase (CTR) or Cdh1 mRNA and
analyzed 16 hours after transfection by Western blot for the indicated proteins. g,
Cerebellar granule neurons were transfected 24 h after plating with CTR or Cdh1
siRNA and analyzed by immunoblot (shown in Fig. 3c). Parallel culture were
immunostained for BrdU (top panels) and nuclei were counterstained with DAPI
(middle panels). Arrowheads indicate a BrdU positive glial cell as shown in the
bright field microphotograph in the bottom panels. h, Cerebellar granule neurons
were transfected with vector or Id2-DBM and GFP expression plasmids and
immunostained 2 days later with antibodies against GFP and MAP-2. MAP-2
staining is excluded from the long axonal projections (arrowheads, magnification
40X).
Supplementary Fig. 8. Degradation resistant Id2 promotes axonal growth of
cerebellar granule neurons in vitro and in vivo. a, Cerebellar granule neurons
were transfected with vector, Id2 or Id2-DBM and GFP expression plasmids.
Neurons were then grown in serum free medium and 3 days later were
immunostained with an antibody against GFP. Asterisks and arrowheads indicate
the cell body and axons, respectively. Scale bars equal to 50 m. b, Axonal
5
length was measured in granule neurons at DIV3. At least 600 cells were
analyzed for each transfection. Values represent mean±SEM of triplicate
experiments (*, p=<0.01). c, Axonal length was measured in cerebellar granule
neurons in vivo. Neurons from P6 rat pups were transfected in suspension with
the indicated plasmids and a GFP expression construct and plated on top of P9
cerebellar slices. Slices were analyzed 72 h later by immunohistochemistry for
GFP antibody to label transfected cells and Hoechst to reveal the anatomical
organization of cerebellar cortex. WM, white matter; IGL, internal granule layer.
d, Axonal length was measured in at least 150 neurons for each construct from
the experiments shown in panel c. Values represent mean±SEM of duplicate
experiments (*, p= <0.0001).
Supplementary Figure 9. A functional Cdh1-Id-bHLH pathway controls axonal
growth. a, Induction of axonal growth inhibitory genes by E47 in neuroblastoma
cells. SK-N-SH cells were infected with Ad-GFP or Ad-E47 and analyzed by
microarray 8 h and 20 h after infection. Analysis at 20 h is from two independent
experiments. b, RNA expression levels of E47 target genes was determined by
qRT-PCR in SK-N-SH harvested 20 h after infection with Ad-GFP and 8 h and 20
h after infection with Ad-E47. Bars represent the mean and SD of triplicate
experiments. Values are the fold changes above the Ad-GFP. c, Transcription
from an E-box-luciferase plasmid was measured after transfection of E47 and
increasing amounts of U6shCdh1 in SK-N-SH. Luciferase values were
normalized towards co-transfected galactosidase. d, RNA expression levels for
6
the indicated genes were determined by qRT-PCR in SF210 harvested 24 hours
after transfection with control siRNA or siRNA targeting Cdh1. Values represent
the mRNA of Cdh1 siRNA transfectants expressed as percentage of the
corresponding control. e, RNA expression levels for the indicated genes were
determined by qRT-PCR in SK-N-SH transfected with control siRNA or siRNA
targeting Cdh1 and then infected with Ad-GFP or Ad-E47. Values are plotted as
fold induction by Ad-E47 over Ad-GFP.
Supplementary Figure 10. Silencing of Cdh1 in cortical neurons stabilizes Id2
and Id1. DIV7 cortical neurons were transfected with siRNA corresponding to
firefly luciferase (CTR) or Cdh1 mRNA and analyzed by Western blot for the
indicated proteins.
7