(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 …
(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 …
(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), …
(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 …
(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 …
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 …
(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 …
(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 …
(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 …
(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 …
(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 …
(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) …
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, …
(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 …
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 …
(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 …
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, …
(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 …
(A) Predicted mechanisms for NF-κB interaction with E2F proteins over the G1/S transition (B) Model simulations of single cell behaviour.
(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 …
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.
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 …
(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 …
Simulation protocol of a live cell imaging experiment involving transfection and TNFα stimulation.
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.
(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.
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 …
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).
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.
Species | Biological name | Initial Conditions Equilibration stage(μM) | Initial Conditions TNFα stimulation (μM) |
---|---|---|---|
NFkB | Cytoplasmic RelA | 0 | 0.004 (+0.1) |
nNFkB | Nuclear RelA | 0 | 0.015 |
E2F1 | Cytoplasmic E2F1 | 0 | 0 (+0.1) |
nE2F1 | Nuclear E2F1 | 0 | 0 |
tIkBa | IκBα mRNA | 0 | 1e-005 |
IkBa | Cytoplasmic IκBα | 0 | 0.017 |
nIkBa | Nuclear IκBα | 0 | 0.004 |
IKKn | Neutral IKK | 0.1 | 0.1 |
IKK | Active IKK | 0 | 0 |
IKKi | Inactive IKKi | 0 | 0 |
tA20 | A20 mRNA | 0 | 1e-005 |
A20 | A20 | 0 | 0.001 |
pIkBa | phospho-IκBα | 0 | 0 |
pIkBaNFkB | phospho-IκBα RelA complex | 0 | 0 |
NFkBE2F1 | cyto. RelA E2F-1 complex | 0 | 0 |
nNFkBE2F1 | nuclear RelA E2F-1 complex | 0 | 0 |
IkBaNFkB | cyto IκBα RelA complex | 0.1 | 0.091 |
nIkBaNFkB | nuclear IκBα RelA complex | 0 | 0.001 |
tE2F4 | E2F-1 target E2F-4 mRNA | 0 | 0 |
E2F4 | E2F-1 target E2F-4 | 0 | 0 |
E2F4NFkB | E2F-4 RelA complex | 0 | 0 |
E2F4IkBaNFkB | E2F-4 IκBα RelA complex | 0 | 0 |
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.
Model reactions and associated parameters.
Reaction | Symbol | Value | References |
---|---|---|---|
Spatial parameters | |||
Total cell volume | tv | 2700 µm3 | Measured |
C:N ratio | kv | 3.3 | Measured |
Conversion to nuclear volume | nv | ×(kv+1) | - |
Conversion to cytoplasmic volume | cv | ×(1/kv+1) | - |
Initial concentration | |||
Total NF-κB | NF | 0.08 µM | Initialized as cytoplasmic IκBα·NF-κB |
Total IKK | - | 0.08 µM | Initialized as IKKn |
Complex formation & dissociation | |||
IκBα + NF-κB → IκBα·NF-κB nIκBα + nNF-κB → nIκBα·NF-κB | ka1a | 0.5 µM-1s-1 | (Hoffmann et al., 2002) |
IκBα·NF-κB → IκBα + NF-κB nIκBα·nNF-κB → nIκBα + nNF-κB | kd1a | 0.0005 s-1 | (Hoffmann et al., 2002) |
NF-κB + E2F (1 or 4) → NF-κB·E2F nNF-κB + nE2F → nNF-κB·nE2F | ka2e | 0.5 µM-1s-1 | fitted, same as IκBα + NF-κB |
NF-κB·E2F → NF-κB + E2F nNF-κB·nE2F → nNF-κB + nE2F | kd2e | 0.0005 s-1 | fitted, same as IκBα + NF-κB |
NF-κB·E2F1 + IκBα→ IκBα·NF-κB + E2F1 nNF-κB·nE2F1 + nIκBα → nIκBα·NF-κB + nE2F1 | kdis | 0.001 s-1 | fitted |
Transport | |||
NF-κB → nNF-κB | ki1 | 0.0026 s-1 | Measured fitting range: Average 0.0026 ± 0.0018s-1 |
nNF-κB → NF-κB | ke1 | 0.000052 s-1 | ki1/50 (Carlotti et al., 2000) |
E2F1→ nE2F1 | kie | 0.0026 s-1 | fitted, same as NF-κB |
nE2F1 → E2F1 | kee | 0.000052 s-1 | fitted, same as NF-κB |
IκBα → nIκBα | ki3a | 0.00067 s-1 | Measured fitting range: Average 0.00043 ± 0.00024 s-1 |
nIκBα → IκBα | ke3a | 0.000335 s-1 | ki3a/2 (Carlotti et al., 2000) |
nIκBα·nNF-κB → IκBα·NF-κB | ke2a | 0.01 s-1 | Fitted |
NF-κB·E2F1 → nNF-κB·nE2F1 | kine | 0.0026 s-1 | fitted, same as NF-κB |
nNF-κB·nE2F1 → NF-κB·E2F1 | kene | 0.000052 s-1 | fitted, 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) | c1a | 1.4×10-7 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 + IκBα | c2a | 0.5 s-1 | (Lipniacki et al., 2004) |
NF-κB·IκBα→ NF-κB | c5a | 0.000022 s-1 | (Pando and Verma, 2000; Mathes et al., 2008) |
nNF-κB·nIκBα→ nNF-κB | - | 0 s-1 | Assumed (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 | c1 | 1.4×10-7 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 | c1x | 9.8×10-7 µM-1s-1 | Fitted |
tA20→ tA20 + A20 | c2 | 0.5 s-1 | - |
tE2F-4→ tE2F-4 + E2F4 | c2x | 0.5 s-1 | - |
tIκBα→ Sink | c3a | 0.0003 s-1 | Fitted (constrained): 0.00077-0.00029 s-1 (Blattner et al., 2000) |
tA20→ Sink | c3 | 0.00048 s-1 | Fitted, constrained >tIκBα turnover (Ashall et al., 2009) |
tE2F4→ Sink | c3x | 0.00048 s-1 | Fitted |
IκBα→ Sink | c4a | 0.0005 s-1 | Fitted (constrained): 0.000105 – 0.002 s-1 (Pando and Verma, 2000; O'Dea et al., 2007; Mathes et al., 2008) |
A20 → Sink | c4 | 0.0045 s-1 | Fitted |
E2F4 → Sink | c4x | 0.00016 s-1 | Fitted |
E2F1 → Sink | c6e | 0.00016 s-1 | Fitted |
nE2F1 → Sink | c7e | 0.00016 s-1 | Fitted |
NF-κB·E2F1 → Sink | c8ne | 0.00016 s-1 | Fitted |
nNF-κB·nE2F1 → Sink | c9ne | 0.00016 s-1 | Fitted |
TNFα stimulation | |||
TNFα | TR | 1/0 | on/off (Lipniacki et al., 2004) |
IKK parameters | |||
IKKn → IKKa | ka | 0.004 s-1 | Fitted, as above |
IKKa → IKKi | ki | 0.003 s-1 | Fitted, as above |
IKKi → IKKn | kp | 0.0006 s-1 | Fitted |
A20 inhibition rate constant | kbA20 | 0.0018 | Fitted, scales kp dependent on receptor state kbA20×TR |
IKKa + IκBα → pIκBα | kc1a | 0.074 s-1 | Assumed (0.037×2) (Heilker et al., 1999) |
IKKa + IκBα·NF-κB → pIκBα·NF-κB | kc2a | 0.37 s-1 | Assumed (0.037×5×2) (Heilker et al., 1999; Zandi and Karin, 1999) |
pIκBα → Sink | kt1a | 0.1 s-1 | Fitted |
pIκBα·NF-κB → NF-κB | kt2a | 0.1 s-1 | Fitted |
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 …
Figure | Model conditions |
---|---|
3E | TR=0, E2F1= (0.05, 0.1, 0.15), E2F4 off |
4A | TR=1, E2F1 = 0.1, E2F4 off |
4C | TR=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 |