Dynamic NF-κB and E2F interactions control the priority and timing of inflammatory signalling and cell proliferation

  1. John M Ankers
  2. Raheela Awais
  3. Nicholas A Jones
  4. James Boyd
  5. Sheila Ryan
  6. Antony D Adamson
  7. Claire V Harper
  8. Lloyd Bridge
  9. David G Spiller
  10. Dean A Jackson
  11. Pawel Paszek
  12. Violaine Sée
  13. Michael RH White  Is a corresponding author
  1. Institute of Integrative Biology, United Kingdom
  2. Faculty of Life Sciences, United Kingdom
  3. University of Swansea, United Kingdom
18 figures and 4 tables

Figures

NF-κB dynamics following TNFα treatment in HeLa and SK-N-AS cells: Mapping the NF-κB response over the cell cycle in synchronized HeLa cells.

(A,B,C and D) The dynamics of RelA-dsRedxp following 10 ng/ml TNFα treatment in transiently transfected SK-N-AS (A), or following 30 pg/ml TNFα treatment in SK-N-AS, and 10 ng/ml TNFα treatment in …

https://doi.org/10.7554/eLife.10473.003
Figure 2 with 3 supplements
Mapping the NF-κB response over the cell cycle through virtual synchronization.

(A) Selected images from time-lapse imaging of RelA-dsRedxp transiently expressing Hela cells treated with 10 ng/ml TNFα. (B) Virtual synchronization of HeLa cells treated with 10 ng/ml TNFα. Cells …

https://doi.org/10.7554/eLife.10473.004
Figure 2—figure supplement 1
Analysis of cell cycle duration and G1/S timing in HeLa and SK-N-AS cells.

(A) Time series for FUCCI expression in single representative HeLa and SK-N-AS cells. White arrows mark cells before and after the fluorescence levels were detectable. (B) Analysis of cells in (A), …

https://doi.org/10.7554/eLife.10473.005
Figure 2—figure supplement 2
Statistical analysis of NF-κB translocation in HeLa cells at inferred cell cycle stages following 10 ng/ml TNFα stimulation.

(A) Analysis of dynamics of initial RelA-dsRedxp translocation with respect to cell cycle phase, using virtual synchronization in HeLa cells. Data were analyzed using nonparametric Anova analysis …

https://doi.org/10.7554/eLife.10473.006
Figure 2—figure supplement 3
Statistical analysis of NF-κB translocation in SK-N-AS cells at inferred cell cycle stages following 30 pg/ml TNFα stimulation.

(A) Correlation of estimated cell cycle timing with RelA-dsRedxp N:C peak amplitude following 30 pg/ml TNFα treatment (n=138). (B) Analysis of dynamics of initial RelA-dsRedxp translocation with …

https://doi.org/10.7554/eLife.10473.007
Cell cycle length and variability is modified by TNFα addition at G1/S.

Analysis of the timing and variability of mitosis (parameter 1 plus 5 from Figure 2B) following 10 ng/ml TNFα treatment of asynchronous untransfected HeLa cells, compared to subsets of those cells …

https://doi.org/10.7554/eLife.10473.008
Figure 4 with 2 supplements
Physical and functional interaction between NF-κB and E2F-1 systems.

(A) NF-κB-dependent transcription was assessed by luciferase reporter assay (NF-luc), in SK-N-AS cells (n=3, +/- s.d) expressing EGFP-E2F-1, RelA-dsRedxp or both. (B) IκBα and IκBε mRNA levels in …

https://doi.org/10.7554/eLife.10473.009
Figure 4—figure supplement 1
E2F-1 modulates NF-κB dynamics in the absence of stimulus in SK-N-AS cells.

(A) Time-lapse confocal microscopy of representative SK-N-AS cells transiently transfected with RelA-dsRedxp and EGFP-E2F-1. (B) Trajectories of three representative cells expressing different …

https://doi.org/10.7554/eLife.10473.010
Figure 4—figure supplement 2
E2F-1 modulates NF-κB dynamics in the absence of stimulus in HeLa cells.

(A) Representative HeLa cells transiently transfected with combinations of RelA and E2F-1 fluorescent fusion proteins. (B) Time-lapse confocal microscopy of representative HeLa cells transiently …

https://doi.org/10.7554/eLife.10473.011
Interaction of E2F-1 with RelA.

(A) Representative cell demonstrating co-localisation of E2F1-EGFP and RelA-dsRedxp upon transient transfection. (B) Co-Immunoprecipitation of E2F-1 with RelA pull down in HeLa cells synchronized in …

https://doi.org/10.7554/eLife.10473.012
Mathematical modelling predicts an additional key component for NF-κB - cell cycle interactions: E2F-4 identified as a putative candidate.

(A) Model simulations of RelA-dsRedxp dynamics when co-expressed with EGFP-E2F-1 in cells treated with TNFα. (B) Dynamics analysed in representative SK-N-AS cells treated with 10 ng/ml TNFα …

https://doi.org/10.7554/eLife.10473.013
Figure 7 with 1 supplement
E2F-4 directly interacts with NF-κB and perturbs RelA dynamics in response to TNFα stimulation.

(A) Single cell trajectories from groups of HeLa cells expressing RelA-dsRedxp and different levels of EGFP-E2F-4 showing the dynamics of RelA-dsRedxp after 10 ng/ml TNFα treatment (n=60 cells). (B) …

https://doi.org/10.7554/eLife.10473.014
Figure 7—figure supplement 1
Analysis of RelA-dsRedxp dynamics in HeLa and SK-N-AS cells co-expressing EGFP-E2F-4 following TNFα stimulation.

The effects of different EGFP-E2F-4 expression levels on the amplitude and timing of the first peak of RelA translocation in HeLa and SK-N-AS cells treated with 10 ng/ml and 30 pg/ml TNFα, …

https://doi.org/10.7554/eLife.10473.015
Figure 8 with 4 supplements
Effect of cell cycle timing on RelA-dsRedXP translocation in dual BAC HeLa cells (C1-1 line) that co-express E2F-1-Venus fusion protein.

(A) Selected images from time-lapse experiment of dual BAC transfected HeLa stable clone 1-1 showing translocation of RelA-dsRedXP and E2F-1-Venus expression at different cell cycle phases. Cells …

https://doi.org/10.7554/eLife.10473.016
Figure 8—figure supplement 1
Virtually synchronized HeLa C 1-1 cells.

Normalised E2F-1-Venus expression at the time of TNFα stimulation of C1-1 cells (data also shown in Figure 8B). E2F-1-Venus expression was normalised to its peak expression. The time axis represents …

https://doi.org/10.7554/eLife.10473.017
Figure 8—figure supplement 2
Physiological and functional expression of E2F-1-Venus in stable BAC-transduced HeLa cells.

(A) HeLa cells stably expressing an E2F-1-Venus fluorescent fusion protein from a 5KB endogenous E2F-1 promoter (Green), transiently transfected with a FUCCI reporter for SCF (SKP-2) activity …

https://doi.org/10.7554/eLife.10473.018
Figure 8—figure supplement 3
Analysis of the expression of E2F-1-Venus and RelA-DsRedxp translocation in single C1-1 HeLa cells stimulated with 10ng/ml TNFα at different cell cycle phases.

Grey line shows the E2F-1-Venus expression level plotted agains the right y-axis. The red, green blue and orange lines show the timecourse of RelA-dsRedxp localization in exemplar cells in the G1, …

https://doi.org/10.7554/eLife.10473.019
Figure 8—figure supplement 4
Expression and interaction of RelA-dsRedxp and E2F-1-Venus.

(A) Western blot of RelA and α-tubulin levels in dual BAC stable C1-1 and WT HeLa showing exogenous expression of RelA-dsRedxp. (B) Western blot of E2F-1 and cyclophilin A levels in dual BAC stable …

https://doi.org/10.7554/eLife.10473.020
Schematic representation of NF-κB – E2F interactions.

(A) Predicted mechanisms for NF-κB interaction with E2F proteins over the G1/S transition (B) Model simulations of single cell behaviour.

https://doi.org/10.7554/eLife.10473.021
Appendix 1—figure 1
Oscillations in the NF-κB system.

(A) Dynamics of RelA-dsRedxp in transiently transfected SK-N-AS cells following 10 ng/μl TNFα stimulation, plotted over 450 and 150 min respectively. (B) Dynamics of RelA-dsRedxp in transiently …

https://doi.org/10.7554/eLife.10473.022
Appendix 1—figure 2
Use of double-Thymidine block to synchronize HeLa cells at G1/S.

Flow cytometric analysis of the distribution of DNA content of non-synchronized HeLa cells and cells harvested at relevant times post-release from Thymidine block.

https://doi.org/10.7554/eLife.10473.023
Appendix 1—figure 3
Effect of Double-Thymidine block on tagged and endogenous RelA levels HeLa cells.

Endogenous RelA and tagged RelA-DsRedxp expression levels in unsynchronised and synchronised WT-HeLa and double BAC stable cells. Synchronized fractions at 0, 2 and 4 hr post release of thymidine …

https://doi.org/10.7554/eLife.10473.024
Appendix 1—figure 4
Negative Co-IP. 

(A) Co-Immunoprecipitation of E2F-1 with RelA (pulled down with a RelA antibody) in asynchronous HeLa cells showing no detectable band of E2F-1. (B) Co-Immunoprecipitation of E2F-4 with RelA (pulled …

https://doi.org/10.7554/eLife.10473.025
Appendix 1—figure 5
A typical simulation protocol of the NF-κB:E2F-1 mathematical model.

Simulation protocol of a live cell imaging experiment involving transfection and TNFα stimulation.

https://doi.org/10.7554/eLife.10473.026
Appendix 1—figure 6
Simulations from the NF-κB:E2F model.

Blue lines represent E2F1= 0, TR= 1. Black lines represent E2F1= NFkB= 0.1 and TR=0. Red lines represent E2F1= NFkB= 0.1 and TR=1.

https://doi.org/10.7554/eLife.10473.031
Appendix 1—figure 7
Cell Cycle length of Clonal HeLa BAC population.

(A) Analysis of cell cycle duration in populations of dual BAC stable cell line (C1-1) with wild type HeLa cells. (B) Analysis of the effects of TNFα treatment in C1-1 and WT HeLa cells.

https://doi.org/10.7554/eLife.10473.032
Appendix 1—figure 8
FCCS control.

FCCS assays between transiently transfected RelA-dsRedxp and IκBα-EGFP (red line) and Empty-DsRedxp and Empty-EGFP (blue line) in single live SK-N-AS cells (+/- s.e.m based on 10 measurements in …

https://doi.org/10.7554/eLife.10473.033
Appendix 1—figure 9
FCCS autocorrelation analysis.

Autocorrelation lines for RelA/IkBα, RelA/E2F-1 and RelA/E2F-4 FCCS studies in single live SK-N-AS cells (+/- s.e.m based on 10 measurements in each of 10+ cells per condition).

https://doi.org/10.7554/eLife.10473.034

Tables

Appendix 1—table 1

A Initial conditions for the NF-κB:E2F mathematical model. Concentrations of NF-κB and E2F-1 added to mimic cell transfection are also included.

https://doi.org/10.7554/eLife.10473.027
SpeciesBiological nameInitial Conditions
Equilibration stage(μM)
Initial Conditions
TNFα stimulation
(μM)
NFkBCytoplasmic RelA00.004 (+0.1)
nNFkBNuclear RelA00.015
E2F1Cytoplasmic E2F100 (+0.1)
nE2F1Nuclear E2F100
tIkBaIκBα mRNA01e-005
IkBaCytoplasmic IκBα00.017
nIkBaNuclear IκBα00.004
IKKnNeutral IKK0.10.1
IKKActive IKK00
IKKiInactive IKKi00
tA20A20 mRNA01e-005
A20A2000.001
pIkBaphospho-IκBα00
pIkBaNFkBphospho-IκBα RelA complex00
NFkBE2F1cyto. RelA E2F-1 complex00
nNFkBE2F1nuclear RelA E2F-1 complex00
IkBaNFkBcyto IκBα RelA complex0.10.091
nIkBaNFkBnuclear IκBα RelA complex00.001
tE2F4E2F-1 target E2F-4 mRNA00
E2F4E2F-1 target E2F-400
E2F4NFkBE2F-4 RelA complex00
E2F4IkBaNFkBE2F-4 IκBα RelA complex00
Appendix 1—table 2

NF-κB:E2F-1 model equations Symbol ‘n’ denotes nuclear variables,‘t’ denotes mRNA transcripts, ‘p’ denotes phosphorylated form of IκBα. Symbols denoting cytoplasmic localisation were omitted.

https://doi.org/10.7554/eLife.10473.028
ddtNFκB(t)=ka1a×IκBα(t)×NFκB(t)+kd1a×(IκBα:NFκB)(t)ki1×NFκB(t)+ke1×nNFκB(t)+kt2a×(pIκBα:NFκB)(t)+c5a×(IκBα)(t)+kd2e×(NFκB:E2F1)(t)ka2e×E2F1(t)×NFκB(t)+c8ne×(NFκB:E2F1)(t)ka3e×NFκB(t)×E2F4(t)+kd3e×(E2F4:NFκB)(t)+c4x×(E2F4:NFκB)(t)\eqno(1)
ddtnNFκB(t)=+ka1a×nIκBα(t)×nNFκB(t)+kd1a×(nIκBα:nNFκB)(t)+ki1×kv×NFκB(t)ke1×kv×nNFκB(t)+kd2e×(nNFκB:nE2F1)(t)ka2e×nE2F1(t)×nNFκB(t)+c9ne×(nNFκB:nE2F1)(t)\eqno(2)
ddtE2F1(t)=kie×E2F1(t)kee×kv×nE2F1(t)c6e×E2F1(t)+kd2e×(NF κB:E2F1)(t)ka2e×nE2F1(t)×NF κB(t)+kdis×(NF κB :E2F1)(t)×IκBα(t)\eqno(3)
ddtnE2F1(t)=+kie×kv×E2F1(t)kee×kv×nE2F1(t)c7e×nE2F1(t)+kd2e×(nNF κB:nE2F1)(t)ka2e×nE2F1(t)×nNF κB(t)+kdis×(nNF κB :nE2F1)(t)×nIκBα(t)\eqno(4)
ddttIκBα(t)=+c1a×nNFκBh(t)nNFκBh(t)+khc3a×tIκBα(t)\eqno(5)
ddtIκBα(t)=kd1a×(IκBα:NFκB)(t)ka1a×IκBα(t)×NFκB(t)+c2a×tIκBα(t)c4a×IκBα(t)ki3a×IκBα(t)+ke3a×nIκBα(t)kc1a×IKK(t)×IκBα(t)kdis×(NFκB:E2F1)(t)×IκBα(t)ka3e×(NFκB:E2F4)(t)×IκBα(t)+kd3e×(E2F4:IκBα:NFκB)(t)\eqno(6)
ddtnIκBα(t)=kd1a×(nIκBα:nNFκB)(t)ka1a×NIκBα(t)×nNFκB(t)c4a×nIκBα(t)+ki3a×kv×IκBα(t)ke3a×kv×nIκBα(t)kdis×(nNFκB:nE2F1)(t)×nIκBα(t)\eqno(7)
ddtIKKn(t)=kp×(kbA20kbA20+TRA20×A20(t))×IKKi(t)TR×ka×IKKn(t)\eqno(8)
ddtIKK(t)=TR×ka×IKKn(t)ki×IKK(t)\eqno(9)
ddtIKKi(t)=ki×IKK(t)kp×kbA20kbA20+TRA20×A20(t)×IKKi(t)\eqno(10)
ddttA20(t)=+c1×nNFκBh(t)nNFκBh(t)+khc3×tA20(t)\eqno(11)
ddtA20(t)=c2×tA20(t)c4×A20(t)\eqno(12)
ddtpIκBα(t)=kc1a×IKK(t)×IκBα(t)kt1α×pIκBα(t)\eqno(13)
ddt(pIκBα:NFκB)(t)=κc2α×IKK(t)×(IκBα:NFκB)(t)κt2α×(pIκBα:NFκB)(t)\eqno(14)
ddt(NFκB:E2F1)(t)=ka2e×E2F1(t)×NFκB(t)kd2e×(NFκB:E2F1)(t)kine×(NFκB:E2F1)(t)+kene×(nNFκB:nE2F1)(t)c8ne×(NFκB:E2F1)(t)kdis×(NFκB:E2F1)(t)×IκBα(t)\eqno(15)
ddt(nNFκB:nE2F1)(t)=ka2e×nE2F1(t)nNFκB(t)kd2e×(nNFκB:nE2F1)(t)+kine×kv×(NFκB:E2F1)(t)kene×kv×(nNFκB:nE2F1)(t)c9ne×(nNFκB:nE2F1)(t)kdis×(nNFκB:nE2F1)(t)×nIκBα(t)\eqno(16)
ddt(IκBα:NFκB)(t)=ka1a×IκBα(t)×NFκB(t)kd1a×(IκBα:NFκB)(t)c5a×(IκBα:NFκB)(t)+ke2a×(nIκBα:nNFκB)(t)kc2a×IKK(t)×(IκBα:NFκB)(t)+(kdis)×(NFκB:E2F1)(t)×IκBα(t)ka3e×(IκBα:NFκB)(t)×E2F4(t)+kd3e×(E2F4:IκBα:NFκB)(t)+c4x×(E2F4:IκBα:NFκB)(t)\eqno(17)
ddt(nIκBα:nNFκB)(t)=ka1a×nIκBα(t)×nNFκB(t)kd1a×(nIκBα:nNFκB)(t)ke2a×kv×(nIκBα:nNFκB)(t)+kdis×(nNFκB:nE2F1)(t)×nIκBα(t)\eqno(18)
ddttE2F4(t)=+clx×nE2F1h(t)nE2F1h(t)+khc3x×tE2F4(t)\eqno(19)
ddtE2F4(t)=c2x×tE2F4(t)c4x×E2F4(t)ka3e×NFκB(t)×E2F4(t)+kd3e×(E2F4:NFκB)(t)ka3e×E2F4(t)×(IκBα:NFκB)(t)+kd3e×(E2F4:IκBα:NFkB)(t)\eqno(20)
ddt(E2F4:NFκB)(t)=ka3e×NFκB(t)×E2F4(t)kd3e×(E2F4:NFκB)(t)c4x×(E2F4:NFκB)(t)+c5a×(E2F4:IκBα:NFκB)(t)ka3e×(NFκB:E2F4)(t)×IκBα(t)+kd3e×(E2F4IκBαNFκB)(t)\eqno(21)
ddt(E2F4:IκBα:NFκB)(t)=+ka3e×(IκBα:NFκB)(t)×E2F4(t)kd3e×(E2F4:IκBα:NFκB)(t)c5a×(E2F4:IκBα:NFκB)(t)+ka3e×(NFκB:E2F4)(t)×IκBα(t)kd3e×(E2F4:IκBα:NFκB)(t)c4x×(E2F4:IκBα:NFκB)(t)\eqno(22)
Appendix 1—table 3

Model reactions and associated parameters.

https://doi.org/10.7554/eLife.10473.029
ReactionSymbolValueReferences
Spatial parameters
Total cell volumetv2700 µm3Measured
C:N ratiokv3.3Measured
Conversion to nuclear volumenv×(kv+1)-
Conversion to cytoplasmic volumecv×(1/kv+1)-
Initial concentration
Total NF-κBNF0.08 µMInitialized as cytoplasmic IκBα·NF-κB
Total IKK-0.08 µMInitialized as IKKn
Complex formation & dissociation
IκBα + NF-κB → IκBα·NF-κB
nIκBα + nNF-κB → nIκBα·NF-κB
ka1a0.5 µM-1s-1(Hoffmann et al., 2002)
IκBα·NF-κB → IκBα + NF-κB
nIκBα·nNF-κB → nIκBα + nNF-κB
kd1a0.0005 s-1(Hoffmann et al., 2002)
NF-κB + E2F (1 or 4) → NF-κB·E2F
nNF-κB + nE2F → nNF-κB·nE2F
ka2e0.5 µM-1s-1fitted, same as IκBα + NF-κB
NF-κB·E2F → NF-κB + E2F
nNF-κB·nE2F → nNF-κB + nE2F
kd2e0.0005 s-1fitted, same as IκBα + NF-κB
NF-κB·E2F1 + IκBα→ IκBα·NF-κB + E2F1
nNF-κB·nE2F1 + nIκBα → nIκBα·NF-κB + nE2F1
kdis0.001 s-1fitted
Transport
NF-κB → nNF-κBki10.0026 s-1Measured fitting range: Average 0.0026 ± 0.0018s-1
nNF-κB → NF-κBke10.000052 s-1ki1/50 (Carlotti et al., 2000)
E2F1→ nE2F1kie0.0026 s-1fitted, same as NF-κB
nE2F1 → E2F1kee0.000052 s-1fitted, same as NF-κB
IκBα → nIκBαki3a0.00067 s-1Measured fitting range:
Average 0.00043 ± 0.00024 s-1
nIκBα → IκBαke3a0.000335 s-1ki3a/2 (Carlotti et al., 2000)
nIκBα·nNF-κB → IκBα·NF-κBke2a0.01 s-1Fitted
NF-κB·E2F1 → nNF-κB·nE2F1kine0.0026 s-1fitted, same as NF-κB
nNF-κB·nE2F1 → NF-κB·E2F1kene0.000052 s-1fitted, same as NF-κB
Protein synthesis & degradation
nNF-κB → nNF-κB + tIκBα
Order of hill function, h=2
Half-max constant, k=0.065h(fitted)
c1a1.4×10-7
 µM-1s-1
Fitted (constrained):
1.07×10-7 – 8.2×10-7µM-1s-1 (Femino et al., 1998);
(Cheong et al., 2006)
tIκBα→ tIκBα + IκBαc2a0.5 s-1(Lipniacki et al., 2004)
NF-κB·IκBα→ NF-κBc5a0.000022 s-1(Pando and Verma, 2000;
Mathes et al., 2008)
nNF-κB·nIκBα→ nNF-κB-0 s-1Assumed (O'Dea et al., 2007;
Mathes et al., 2008)
nNF-κB → nNF-κB + tA20
Order of hill function, h=2 Half-max constant, k=0.065h
c11.4×10-7 
µM-1s-1
Assumed to be the same as IκBα
nE2F1 → nE2F1 + tE2F4
Order of hill function, h=2 Half-max constant, k=0.065h
c1x9.8×10-7
µM-1s-1
Fitted
tA20→ tA20 + A20c20.5 s-1-
tE2F-4→ tE2F-4 + E2F4c2x0.5 s-1-
tIκBα→ Sinkc3a0.0003 s-1Fitted (constrained): 0.00077-0.00029 s-1
(Blattner et al., 2000)
tA20→ Sinkc30.00048 s-1Fitted, constrained >tIκBα
turnover (Ashall et al., 2009)
tE2F4→ Sinkc3x0.00048 s-1Fitted
IκBα→ Sinkc4a0.0005 s-1Fitted (constrained): 0.000105 – 0.002 s-1
(Pando and Verma, 2000; O'Dea et al., 2007;
Mathes et al., 2008)
A20 → Sinkc40.0045 s-1Fitted
E2F4 → Sinkc4x0.00016 s-1Fitted
E2F1 → Sinkc6e0.00016 s-1Fitted
nE2F1 → Sinkc7e0.00016 s-1Fitted
NF-κB·E2F1 → Sinkc8ne0.00016 s-1Fitted
nNF-κB·nE2F1 → Sinkc9ne0.00016 s-1Fitted
TNFα stimulation
TNFαTR1/0on/off (Lipniacki et al., 2004)
IKK parameters
IKKn → IKKaka0.004 s-1Fitted, as above
IKKa → IKKiki0.003 s-1Fitted, as above
IKKi → IKKnkp0.0006 s-1Fitted
A20 inhibition rate constantkbA200.0018Fitted, scales kp dependent on
receptor state kbA20×TR
IKKa + IκBα → pIκBαkc1a0.074 s-1Assumed (0.037×2) (Heilker et al., 1999)
IKKa + IκBα·NF-κB → pIκBα·NF-κBkc2a0.37 s-1Assumed (0.037×5×2)
(Heilker et al., 1999;
Zandi and Karin, 1999)
pIκBα → Sinkkt1a0.1 s-1Fitted
pIκBα·NF-κB → NF-κBkt2a0.1 s-1Fitted
Appendix 1—table 4

Simulation protocols used throughout the manuscript. TNFα stimulation is invoked via TR=0/1. E2F refers to levels of 'transfection' (in μM). E2F4 off/on refers to whether its transcription is …

https://doi.org/10.7554/eLife.10473.030
FigureModel conditions
3ETR=0, E2F1= (0.05, 0.1, 0.15), E2F4 off
4ATR=1, E2F1 = 0.1, E2F4 off
4CTR=1, E2F1 = 0.1, E2F4 on
4I (G1, G2)TR=1, E2F1= 0, E2F4 on (but unaffected)
4I (G1/S)TR=1, E2F1 = 0.2, E2F4 on
4I (S)TR=1, NFkBIkBa = 0.1, E2F1 = 0, E2F4= 0.1

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