Supp. Fig. 1:
A: w/t and p52/100 null MEF cells were fractionated by centrifugal elutriation and
stained with propidium iodide for analysis by FACS. A representative distribution of
cells at different cell cycle stages is shown.
B: Western blot showing NF-kB subunit levels in wild type, nfkb2 (p52/p100) -/- and
rela -/- MEFs.
C: Quantitative RT-PCR analysis was performed using primers specific to Cyclin D1,
Skp2, c-Myc and GAPDH control, using total RNA prepared from wild type and p52
null MEF cells following centrifugal elutriation.
D: U2OS cells were fractionated by centrifugal elutriation and stained with propidium
iodide and with antibodies to total p100, S866 phosphorylated p100 or an IgG control
as indicated, for analysis by FACS.
E: Semi-quantitative PCR analysis of Bcl-xL, Bcl-2, A20 and Bcl-3 mRNA levels in
fractions from elutriated U2OS cells.
F: U2OS cells were transfected with an siRNA targeting Bcl-3. mRNA expression
were analyzed as shown.
Supp. Fig. 2.
A & B: Analysis of Cyclin D1, Skp2, c-Myc and p52/p100 protein and mRNA
expression in U2OS (passage 13 and 26), MCF7 and HUT78 cells.
C: Table summarizing FACS analysis data showing the cell cycle distribution of
Cyclin D1, c-Myc and Cyclin B1 protein in U2OS (passage 13 and 26), MCF7 and
HUT78 cells. Cells were stained with antibodies to the indicated proteins and with
propidium iodide for DNA content. Numbers shown are the percentage of cells
scored by propidium iodide staining as being at a particular cell cycle stage that also
showed staining for the target protein.
D: U2OS (passage 14 and 26), MCF7 and HUT78 cells were separated by elutriation
and fractions were analysed by western blotting for the indicated proteins.
Supp. Fig. 3:
Analysis of NF-B subunit, coactivator and repressor recruitment to the promoters of
NF-B target genes in U2OS cells. ChIP analysis of the Cyclin D1, Skp2, c-Myc and
GAPDH promoters was performed by Quantitative PCR using antibodies to the
indicated protein, in U2OS cells following centrifugal elutriation.
Supp. Fig. 4:
A: w/t and RelA null MEF cells were fractionated by centrifugal elutriation and
stained with propidium iodide for analysis by FACS. A representative distribution of
cells at different cell cycle stages is shown.
B: Analysis of NF-B subunit, coactivator and repressor recruitment to the promoters
of NF-B target genes in wild type, nfkb2 null and RelA null MEF cells following
centrifugal elutriation. ChIP analysis of the Cyclin D1, Skp2, c-Myc and GAPDH
promoters was performed by Quantitative PCR using antibodies to the indicated
Supp. Fig. 5:
A: Western blot analysis of whole cell MEF extracts following centrifugal elutriation
for S468 and S536 phosphorylated RelA.
B: IKK or IKK were immunoprecipitated from whole cell lysates prepared from
elutriated U2OS cells. The immunoprecipitate was incubated with whole cell lysate
from serum starved U2OS cells. These lysates were then analysed by western
blotting with antibodies to S866 phosphorylated p100 and S468 or S536
phosphorylated RelA as indicated
C: U2OS Nuclear, cytoplasmic and whole cells protein extracts following centrifugal
elutriation were pooled according to the cell cycle stage and subjected to western blot
analysis using antibodies to Chk1.
Supp. Fig. 6.
Control quantitative PCR analysis showing ATR, ATM, Chk1, Chk2 and IKK
mRNA levels following treatment of U2OS cells with the indicated siRNAs.
Supp. Fig. 7.
Chk1 inhibition of IKK activity requires Chk1 kianse activity. Experiment was
performed as in Figure 8B, except that either the Chk1 inhibitor Go6976 was included
(A) or purified Chk1 was inactivated by heat treatment (B). An additional control, the
inclusion of a non-hydrolysable ATP analogue, is also shown.
Supp. Fig. 8.
A: Analysis of NF-B subunit, HDAC1 and CBP recruitment to the promoters of NF-
B target genes in each cell cycle stage following Chk1 and Akt pathway inhibition.
ChIP analysis of the Cyclin D1, Skp2, c-Myc and GAPDH promoters, was performed
in U2OS following centrifugal elutriation of U2OS cells with or without Gö6976 or
B: RNA extracts were prepared from elutriated U2OS cells treated with Gö6976 or
LY294002 and subjected to semi-quantitative PCR analysis as indicated.
Supp. Fig. 9.
Cell cycle analysis of all elutriated U2OS cell fractions used in each Figure.
Supp. Fig. 10. Summary of the primers used for ChIP assays in U2OS cells.
Schematic diagram of the human Cyclin D1 (A), c-Myc (B), Skp2 (C), Bcl-2 (D) and
Bcl-xL (E) promoters. The relative positions of previously described NF-B binding
sites () are shown.
A: 3 NF-B binding sites (-858, -749 and -39) have been described on the Cyclin D1
promoter (Guttridge et al, 1999; Joyce et al, 1999; Westerheide et al, 2001).
B: Two NF-B binding sites (-1095 and +449) have been described on the human c-
Myc gene (Duyao et al, 1990; Kessler et al, 1992).
C: The NF-B binding sites on Skp2 promoter have not been characterized but it was
previously shown that ChIP primers over the transcription initiation site can detect
NF-B subunit binding (Schneider et al, 2006) and these were used in this analysis.
D: Three NF-B binding sites have been described on Bcl2 gene, one in the upstream
promoter (-2445) (P1) and the others are in the P2 region (-212 and -174) (Catz &
Johnson, 2001; Xiang et al, 2006).
E: The primers used to the 1 NF-B binding site on the Bcl-xL promoter (Chen et al,
2000) have been described previously (Campbell et al, 2004)
F: Three NF-kB binding sites have been described on the IkBa promoter (-304, -50
and -19) (Ito et al, 1994).
Supplementary Information: Materials and methods
IKK inhibitors BAY 11-7082 (10M) and IKKi IV ([5-(p-Fluorophenyl)-2-
ureido]thiophene-3-carboxamide), together with the Chk1 inhibitor Go6976 were
purchased from Merck Biosciences. Caffeine and aphidicolin were purchased from
Sigma Aldrich. PI-3 kinase inhibitor LY 294002 was purchased from Promega.
Concentrations used were: Go6796: 1μM on the cells and 0.1μM for the kinase
assay; LY294002: 50μM; Bay 11-7082: 10μM; IKKi IV: 50nM; caffeine:1mM;
Western blots were performed essentially as described previously (Rocha et al, 2003).
For assays involving detection of phosphorylated proteins, 50-150g of protein
extracts were loaded per well.
Flow cytometric analysis of cell cycle distribution and proteins.
Adherent and detached cells were harvested, pooled, washed once in phosphate-
buffered saline (PBS), and fixed by incubation in 1% paraformaldehyde/PBS (pH 6.8)
for 10 min. Cells were washed once in PBS 0.1% tween-20 and then permeabilised in
70% ethanol (vol/vol). Cells were then washed twice in PBS (plus 0.1% (vol/vol)
tween-20) and resuspended in PBS containing 0.1% (vol/vol) tween-20 and 1% BSA
(blocking buffer). After incubation at room temperature for 15 min, cells were
incubated with blocking buffer containing antibody for 20 min. The dilution of the
antibodies used was 1:200 for anti-p52, anti-phospho-serine p100, anti-Cyclin D1,
anti-Cyclin B1 and anti-c-Myc antibodies. All secondary antibodies (labelled with
FITC or Cy-5) were purchased from Jackson Immunoresearch and used at 1:200
dilution. The cells were resuspended in PBS containing 0.1% (vol/vol) tween-20, 50
µg/ml of propidium iodide and 50 µg/ml of RNase A for 20 min at room temperature,
and analyzed for cell cycle distribution with a FACS Calibur flow cytometer and
CellQuest software (Becton Dickinson). For all antibodies used in this study,
appropriate controls were performed. Cells were either stained with primary
antibodies without secondary antibodies to control for auto-fluorescence or stained
with secondary antibodies alone to control for background staining.
Chromatin Immunoprecipitation (ChIP)
Cells, either grown to 70% confluency or harvested and subjected to elutriation, were
cross-linked with 1% formaldehyde at room temperature for 10 min. Cells were
washed twice with 10 ml of ice-cold phosphate-buffered saline and then scraped into
0.5 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1, 1 mM
PMSF, 1 g/ml leupeptin, 1 g/ml aprotinin) and left on ice for 10 minutes. Samples
were then sonicated at 4°C seven times. Each sonication was for 20 seconds with a
40 seconds gap between each sonication. Supernatants were recovered by
centrifugation at 12,000 rpm in an eppendorf microfuge for 10 min at 4°C before
being diluted 4-8 fold in dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM
NaCl, 20 mM Tris-HCl, pH 8.1). Samples were then pre-cleared for 2 hours at 4°C
with 2 g of sheared salmon sperm DNA and 20 l of protein A or G-Sepharose
(50% slurry). At this stage, 20l of the material was kept as Input material.
Immunoprecipitations were performed overnight with specific antibodies (1g), with
the addition of NP-40 detergent to a final concentration of 0.5%. The immune
complexes were captured by incubation with 20 l of protein A or G-Sepharose (50%
slurry) and 2 g salmon sperm DNA for 1 hour at 4°C. The immunoprecipitates were
washed sequentially for 5 minutes each at 4°C in TSE 1 (0.1% SDS, 1% Triton X-
100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), TSE 2 (0.1% SDS, 1%
Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), and Buffer 3
(0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl,
pH 8.1). Beads were washed twice with TE buffer (10mM Tris-HCl, 1mM EDTA)
and eluted with 500 l of Elution Buffer (1% SDS, 0.1 M NaHCO3). For ReChIP
experiments 25µl of ReChIP buffer (Dilution Buffer, 10mM DTT) was added to
beads following washes and incubated at 37°C for 50 minutes. The sample was then
diluted 40 times in dilution buffer and immunoprecipitations, washes and elution were
performed as before.
To reverse the crosslinks, samples, including 'Input', were incubated at 65 °C
overnight. DNA was precipitated using classical Phenol-Chloroform procedures. For
PCR, 5l of DNA was used from an 80µl DNA preparation and subjected to 30
cycles of PCR amplifications. Control regions for GAPDH gene were subjected to 35
cycles of PCR Amplification.
Quantitative PCR analysis
Total RNA was extracted with the Nucleospin RNA II isolation system (Macherey
Nagel; 740955), according to the manufacturer’s directions. For reverse transcriptase
PCR (RT-PCR), 1g RNA sample were reverse transcribed with Quantitect Reverse
Transcription Kit (QIAgen; 205313). The cDNA stock was diluted by 200 and 5l
was used for PCR with GoTaq flexi DNA polymerase (Promega; M8305).
Quantitative PCR data was generated on a Rotor-Gene 3000 (Corbett Research) using
the following experimental settings: Hold 50°C for 3 min; Hold 95°C 10 min; Cycling
(95°C for 20 sec; 55°C for 20 sec; 72°C for 20 sec with fluorescence measurement) ×
45; Melting Curve 50–99°C with a heating rate of 1°C every 5 sec. All values were
calculated relative to untreated levels and normalised to GAPDH levels using the
Pfaffl method (Pfaffl, 2001). Each RNA sample was assayed in triplicate and the
results shown are representative of three separate experiments.
Cells were elutriated as indicated previously. Pellets were lysed in lysis buffer (25
mM Hepes (pH 7.9), 300 mM KCl, 3 mM EDTA, 0.2 mM EDTA, 10% glycerol
(vol/vol), 0.5% NP-40, 0.5 mM PMSF, 1 mM sodium orthovanadate, 1 µg of
leupeptin/ml, 1 µg of aprotinin/ml, 10 mM p-nitrophenyl phosphate, 10 mM sodium
fluoride). Supernatants were diluted further in lysis buffer and precleared with
Protein G-sepharose for 1hr. Precleared extracts were immunoprecipitated with 1 µg
of antibody overnight at 4°C. Protein G-Sepharose beads (Amersham Pharmacia
Biotech) were then added to each tube, and the samples were rotated for 60 min at
4°C. Afterward, samples were washed twice in lysis buffer, once in TE buffer (10mM
Tris-HCl, 1mM EDTA) and once in kinase buffer (50 mM HEPES (pH 7.7), 20 mM
MgCl2, 200 mM NaCl2, 10% glycerol, 10 µM ATP). The kinase activity was assayed
in kinase buffer by incubating with 25 µg whole cell extracts from serum starved cells
for 15 min at 30°C, supplemented with purified recombinant GST-Chk1 if required
(provided by Dr John Rouse, Dundee).
Antibodies used were: anti-p21 (sc-397, Santa Cruz), anti-p52 monoclonal and anti-
p52 polyclonal (05-361 & 06-413, Upstate Biotechnology), anti-p52 polyclonal (sc-
848, Santa Cruz), anti-p50 NF-B (sc-114, Santa Cruz), anti-RelA (sc-372, Santa
Cruz), anti-RelB (sc-226, Santa Cruz), anti-c-Rel (sc-71, Santa Cruz), anti-
IKK(sc-7607, Santa Cruz), anti--actin (A5441, Sigma), anti-Cyclin D1 (556470,
BD Pharmingen), anti-Skp2 (sc-7164, Santa Cruz), anti-c-Myc (sc-42, sc764, sc789;
Santa Cruz), anti-p21WAF1 (PC55-100ug, Oncogene Research Product), anti-Cyclin
A (CC02-20UG, Oncogene research product), anti-Cyclin B1 (sc752, Santa Cruz;
V152, Corning), anti-Chk1 (sc-7898, Santa Cruz), anti-Akt (sc-8312, Santa Cruz),
Cyclin E (CC05-20UG, Oncogene Research Product), CDC25C (sc-327, Santa Cruz)
Antibodies used in ChIP assays were: anti-p52 polyclonal (sc-848, Santa Cruz), anti-
p50 NF-B (sc-114, Santa Cruz), anti-RelA (sc-372, Santa Cruz), anti-RelB (sc-226,
Santa Cruz), anti-c-Rel (sc-71, Santa Cruz), anti-Gal4 (sc-510, Santa Cruz), anti-CBP
(sc-369, Santa Cruz), anti-Bcl-3 (sc-185, Santa Cruz), anti-mSin3A (sc-767, Santa
Cruz), and anti-HDAC1 (sc-6298, Santa Cruz).
Phospho-antibodies used in ChIP assays, western blot and co-immunoprecipitation
were S866-p100 (4881S, Cell Signaling; ab31474-100, Abcam), S180/1-
IKK(2681, Cell Signaling), S280-Chk1 (2347S, Cell Signaling), S317-Chk1
(2344S, Cell Signaling), S345-Chk1 (2341S, Cell Signaling), T308-Akt (4056, Cell
Signaling), S473-Akt (4058, Cell Signaling), S468-RelA (3039L, Cell Signaling),
S536-RelA (3031S, Cell Signaling). The Goat T505-RelA phospho-specific antibody
was raised in collaboration with Active Motif and has been described previously
(Campbell et al, 2006). The rabbit T505-RelA antibody has been described
previously (Rocha et al, 2005).
quantitative pcr primer set
semi quantitative pcr primer set
Cyclin D1-Murine: FOR-CTCTGCTACTGCGCCGACA
GAPDH-Human: FOR- CGGTGCGTGCCCAGTTG
Cyclin D1-Human: FOR-GTGCTGCGAAGTGGAAACC
Cyclin D1-Murine: FOR-CAGAAGTGCGAAGAGGAGGTC
Cyclin E-Human: FOR-TGAAGTGGTTAAGAAAGCCTCAG
Cyclin A-Human: FOR-ACATTACAGATGATACCTACACCAAG
GAPDH-Murine : FOR-GCTACACTGAGGACCAGGTTG
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