Song et al. Supplemental Information
Validation of Notch-1 siRNA and γ-secretase inhibitor I: To knock down Notch-1 expression
we used a specific siRNA that we developed. The most effective 21-mer RNA oligonucletide
(siRNA) to Notch-1 selected from a preliminary screening was: 5’AAG TGT CTG AGG CCA
GCA AGA 3’. Sequence specificity was determined by BLAST searches for uniqueness and
validated using as control a scrambled siRNA with sequence (5’ AAC AGT CGC GTT TGC
GAC TGG 3’) that does not match any known mammalian GENEBANK sequence. The Notch-1
and scrambled sequences have been successfully used by 2 independent groups (Fan et al., 2004;
Purow et al., 2005). These sequences were used in all experiments where dsRNA was used for
RNA interference. The same sequences were cloned into pSuper RNAi expression vector
(Oligoengine).
Plasmid-encoded Notch-1 siRNA had identical specificity to dsRNA
oligonucleotides (see Supplemental Figure 1). The scrambled sequence had no detectable effects
on target proteins or housekeeping proteins, identical to empty pSuper. Therefore, empty pSuper
was used in all experiments where pSuper was used for RNA interference. For Western blot
analysis, all plasmid transfections were performed in 60 mm dishes with 2 μg DNA/dish; for
reporter assays, all plasmid transfections were performed in a 24-well-format with 0.2 μg/well
using FuGENE 6 (Roche Diagnostics). As shown in Supplemental Figure 1A (left panel), Notch1 specific siRNA selectively downregulated Notch-1, but not Notch-2 or -4 in CaSki cells.
Similar downregulation efficiency was observed in normal keratinocytes, and identical band
patterns were given by two different antibodies (Supplemental Figure 1A, right panel), a mouse
monoclonal antibody specific for Notch-1 ankyrin repeat 1 (Sigma N6786) and a polyclonal
antibody recognizing the C-terminal region of Notch-1 (Santa Cruz C-20). The effect of Notch-1
siRNA on Notch-1 expression was further visualized and confirmed by immunofluorescence
(Supplemental Figure 1B). The functional effect of Notch-1 siRNA on Notch activity was
assessed by monitoring CBF-1-dependent reporter activity. As shown in Supplemental Figure 1C,
the expression of Notch-1 siRNA led to a dose-dependent inhibition of CBF-1-dependent
luciferase activity in CaSki cells. Together, these data demonstrate that our Notch-1 siRNA can
be used as an effective approach to modulate the Notch-1 signaling in vitro.
In some experiments we also used a pharmacological pan-Notch inhibitor, namely, γsecretase inhibitor “GSI” (cbz-Leu-Leu-Nle-CHO). In our hands, this is the most potent γsecretase inhibitor commercially available, and inhibits Notch signaling in a variety of
transformed cells (Qin et al., 2004; Curry et al., 2005). As shown in Figure Supplemental Figure
1D, treatment with GSI led to a dose-dependent decrease of Notch-1 NIC as detected by a VLLSNIC-specific antibody; and accumulation of NTM as detected with the goat antibody to the C-
1
terminus in CaSki cells. Correspondingly, treatment with GSI caused dose-dependent inhibition
of γ-secretase-mediated Notch cleavage with an IC50 value of 0.43 μM in CaSki cells
(Supplemental Figure 1E), assessed by a GAL4-based Notch cleavage assay (Karlstrom et al.,
2002). In this assay, a Gal4-luciferase reporter and a DNA plasmid encoding fusion protein
Gal4VP16-Notch-1 E were co-transfected into CaSki cells; γ-secretase-mediated Notch
cleavage can be assessed by monitoring the Gal4VP16-Notch-1IC-driven luciferase activity.
Treatment with GSI also caused a dose-dependent suppression of CBF-1 reporter activity, with an
apparent IC50 between 1 and 2 μM (Supplemental Figure 1F). GSI is chemically similar to
proteasome inhibitor MG132. To determine whether the biological effects of GSI treatments were
in part due to proteasome inhibition, we measured cellular proteasome activity upon GSI
treatment by using the 20S Proteasome Activity Assay Kit from Chemicon. We did not observe
any proteasome inhibitory activities of GSI on intact cells in the concentration range we used as a
γ-secretase inhibitor (data not shown), suggesting that the effects of GSI in this concentration
range were primarily, if not exclusively, due to γ-secretase inhibition. Modest (< 20%)
proteasome inhibition was observed at concentrations one order of magnitude higher (10 μM).
Higher concentrations could not be tested due to excessive cytotoxicity.
Western blots and co-immunoprecipitation: For Western blots, equal amounts of protein (2030 g) were resolved on pre-cast 7% SDS gels and transferred to polyvinylidene difluoride
membranes (Bio-Rad, Hercules, CA) using InVitrogen’s buffering system. Membranes were
incubated with primary antibodies overnight at 4o C, washed, and visualized with horseradishconjugated secondary antibodies and chemiluminescent reagents (Roche Diagnostics).
For co-immunoprecipitation, 500-1000 g of total cell extract or nuclear extract was pre-cleared
with protein G Sepharose beads (Amersham Biosciences) at 4o C for 1 h with gentle rocking.
Pre-cleared-supernatants were incubated with primary antibodies or correspondent control IgG at
4o C for 2 h.
Antibody-protein complexes were then immunoprecipitated with protein G
Sepharose beads 1 h at 4o C with gentle rocking.
Bead-bound proteins were washed and
separated by centrifugation. Immune complexes were eluted with Laemmli Sample Buffer (BioRad), resolved on 7% SDS gels and analyzed by Western blotting. Quantitative
immunoprecipitation was performed as described (Vooijs et al., 2004), 48 h after transfecting
cells with full-length Notch-1/Renilla luciferase construct. Immunoprecipitation was carried out
as described above, but the readout was eluted Renilla luciferase activity normalized by total
(beads plus supernatant) luciferase activity. Specificity was verified using constructs encoding
2
GAPDH-Renilla luciferase (cytoplasmic control) and CDK1-Renilla luciferase (nuclear control).
Neither control was immunoprecipitated by Notch-1 antibodies (not shown).
IP-kinase assays: 300 μg whole cell extract were immunoprecipitated with antibodies to IKKα,
(Imegenex IMG-136A), IKKβ (Imegenex IMG-159A) or Notch-1 (C-20 Santa Cruz) overnight.
Immunoprecipitates were incubated with 20 μl Protein A/G agarose beads (Calbiochem IP05) for
2 h. Beads were washed 5 times with lysis buffer (20mM HEPES pH 7.4, 2 mM EGTA, 10%
glycerol, 1% Triton X-100, 1 mM DTT, 50 mM β-glycerophosphate, 1 mM sodium
orthovanadate, and protease inhibitor cocktail (Roche)) and 5 times with kinase buffer (50m M
HEPES pH 7.4, 20 mM MgCl2, 2 mM DTT, 10 μM ATP, 50 mM β-glycerophosphate, 1 mM
sodium orthovanadate) and then incubated with 23 μl kinase buffer containing 10 μCi γ-32P-ATP
and 2 μg GST-IκBα substrate (Upstate13-115) at 30 ºC for 30 min. The reaction was terminated
by adding 4X SDS-PAGE buffer and heating at 85 ºC for 5min. Proteins were resolved by 7%
SDS-PAGE. Gels were dried and subjected to autoradiography.
ChIP analysis: CaSki cells were transfected with Notch-1 siRNA or Scrambled siRNA. 24 h
post-transfection, cells were stimulated with TNF-α (10 ng/ml) for 30 min and the fixed with 1%
formaldehyde for 5 min. Chromatins from crosslinked cells were sheared by sonication and
incubated overnight with specific primary antibodies, followed by incubation with protein GSepharose saturated with salmon sperm DNA. Input and immunoprecipitated chromatins were
incubated at 65 oC overnight to reverse crosslinks. After proteinase K digestion, DNA was
extracted with phenol/chloroform and precipitated with ethanol.
Precipitated DNAs were
analyzed by PCR followed by agarose gel electrophoresis or by real-time Q-PCR using an
Applied Biosystems 730 Real-Time PCR system. Real-time PCR products were quantified using
a standard curve generated from a titration of input chromatin from 1 to 0.005. 60 μl input
chromatin corresponds to 1 (Wu et al., 2005). The following human promoter-specific primers
were used: 5’-GACGACCCCAATTCAAATCG and 5’-TCAGGCTCGGGGAATTTCC-3’ for
IκBα
(Yamamoto
et
al.,
2003);
AGCAAGGACAAGCCCAGTCT-3’
for
5’-GTGTGTGTGGTTATTACCGC-3’
c-IAP-2
(Hoberg
et
al.,
and
5’-
2004);
5’-
CGCGACCTCAGATCAGACGT-3’ and 5’-GCTTTGGGCTGCATTCCCAA-3’ for ribosomal
RNA
promoter
output
control;
5’-TGCACTGTGCGGCGAAGC-3’
and
5’-
TCGAGCCATAAAAGGCAA-3’ for β-actin input control (Yamamoto et al., 2003); and 5'AGCTCAGGCCTCAAGACCTT-3' and 5'-AAGAAGATGCGGCTGACTGT-3' for GAPDH
input control (Yeung et al., 2004).
3
Supplemental Figure Legends:
Supplemental Figure 1: (A) Empty vector (EV) or plasmid encoding Notch-1-siRNA were
transiently transfected in either CaSki or primary human keratinocytes. The efficiency and
specificity of Notch-1 down-regulation were verified by Western blotting with two different
Notch-1 antibodies (Santa Cruz: C-20; Sigma: N6786).
(B) CaSki cells were transiently
transfected with the same plasmids as (A) and Notch-1 expression was visualized by
immunofluorescence 48 h post-transfection. (C) CaSki cells in 24-well plate format were
transfected with CBF-1-luciferase reporter plasmid plus pRL-Null plasmid as internal control;
cells were also transfected with either empty vector or expression plasmids encoding Notch-1
siRNA in a dose-dependent manner. Total DNA in each well was equalized with corresponding
control DNA. Dual-luciferase assay was performed 48 h post-transfection. Relative luciferase
activity was calculated considering 1.0 as the activity of cells transfected with empty vector. *
indicates P ≤ 0.005 compared with control group. (D) CaSki cells were treated with the indicated
concentrations of GSI for 18 hr. The effect of GSI on Notch-1 cleavage was analyzed by Western
blot for Notch-1TM (Santa Cruz C-20), and Notch-1 NIC (Abcam Ab8925). (E) CaSki cells were
co-transfected with Gal4-luciferase reporter and DNA plasmid encoding fusion protein
Gal4VP16-Notch-1 E. Twenty-four h later, cells were treated with various concentrations of
GSI, and γ-secretase cleavage rates were assessed by monitoring Gal4VP16-Notch-1IC-driven
luciferase reporter activity. Results are reported as means 
F) Cells were
transfected with CBF-1-luciferase reporter plasmid plus pRL-Null plasmid as an internal control.
Twenty-four h post-transfection, cells were treated with the indicated concentrations of GSI
overnight (18hr) and dual-luciferase assay was performed. Data were analyzed as in (C). *
indicates P ≤ 0.01; (G), cells were transduced with Notch-2 shRNA-encoding lentivirus
pLenti4/TO/V5-DEST or empty vector. The sequence used was shN2-up:5’-GAT CAG TGA
GCG AAC CCC ATT GAT CTC GAC GGC CAT GGC TGA AGC TTG TCC CAT GGC CGT
CGA TCA ATG GGG TCT GCC TAC T-3’ shN2-down:5’TCG AAG TAG GCA GAC CCC
ATT GAT CGA CGG CCA TGG GAC AAG CTT CAG CCA TGG CCG TCG AGA TCA ATG
GGG TTC GCT CAC T-3’. NF-κB and CBF-1 reporter activity were determined by dualluciferase assays. Knockdown was verified by Western blotting using Notch-2 antibody
Abcam8927; (H), cells were transduced with Notch-4 siRNA or scrambled sequence cloned in
pSUPER vector (OligoEngine, Seattle, WA) and analyzed as described in (G). The Notch-4
4
sequence used was 5’-AAC CCT GTG CCA ATG GAG GCA-3’. Knockdown was verified by
Western blotting using Santa Cruz H-225 antibody.
Supplemental Figure 2: Recruitment of IKKα to the IκBα promoter is Notch-1 dependent.
CaSki cells were transfected with either empty vector control (EV) or plasmid encoding Notch-1siRNA. Cells were stimulated with TNF-α (10 ng/ml) for 30 min. Cross-linked lysate was
immunoprecipitated with IKK antibody and subjected to ChIP for the IκB
same cross-linked lysates were also analyzed without IP for the -actin promoter (input control).
Output controls (human ribosomal RNA promoter) were generated after the IP process to control
for loss of DNA during washes. “C” indicates input control in which IκB promoter amplicon
was amplified from cross-linked lysate sample without IP. Note that Notch-1 siRNA abolishes the
presence of IKKα on this promoter.
5
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