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Multi-parameter analysis of the kinetics of NF-B signalling and transcription in single living cells

¹ School of Biological Sciences, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK
² AstraZeneca R&D Charnwood, Molecular Biology, Bakewell Road, Loughborough, Leicestershire, LE11 5RH, UK
³ Kinetic Imaging Ltd, 2 Brunel Road, Wirral, CH62 3NY, UK

View larger version (20K): [in a new window]   Fig. 5. Long term NF-B response to TNF. (A) Long term widefield fluorescence microscopy of p65-localisation in response to TNF. Cells were treated at t=0 hours with 10 ng/ml TNF or carrier (control). Mean nuclear fluorescence intensities were determined for each cell at each time point. Results show mean values obtained from two experiments. (B) Transcriptional regulation of NF-B-sensitive promoter. Luciferase expression monitored in living cells transfected with pNF-B-luc. Cells were transfected in 24-well microtitre plates. 1 mM luciferin was added to cells 12 hours prior to addition of TNF (10 ng/ml final concentration) or carrier added at t=0 hours [n=4, results show means±s.d.(n-1)].

View larger version (38K): [in a new window]   Fig. 1. Nuclear accumulation of p65-EGFP in response to TNF stimulation. (A) Time series images of p65-EGFP fluorescence at stated times (in minutes) after addition of TNF. Inhibition of translocation was observed in cells treated with 18 µM SN50 for 15 minutes prior to addition of TNF. (B) Quantification of p65-EGFP translocation in response to TNF. Mean nuclear and cytoplasmic fluorescent intensities were determined for fluorescent cells at each time point and plotted as a ratio relevant to the initial ratio at t=0 minutes. Translocation is shown in the absence of inhibitor or in the presence of 18 µM SN50, 18 µM SN50M or 12.5 mM Bay11-7082. Confocal microscopy was carried out in cells transfected with p65-EGFP 24 hours prior to their use. Cells were treated with appropriate inhibitor for 15 or 30 minutes (see text) prior to stimulation with 10 ng/ml TNF at t=0 minutes. Results show mean values±s.d.(n-1) from two experiments with at least six cells per experiment plotted for each treatment.

View larger version (53K): [in a new window]   Fig. 2. Degradation of IB-EGFP in response to TNF stimulation. (A) Time series images of IB-EGFP fluorescence at stated times (in minutes) after addition of TNF. The degradation was prevented in cells treated with 12.5 µM Bay11-7082 for 30 minutes prior to addition of TNF. (B) Degradation of IB-EGFP in response to TNF. Mean cellular fluorescence intensities were determined for fluorescent cells at each time point and plotted as a percentage of the fluorescence values at t=0 minutes. Confocal microscopy was carried out in cells transfected with IB-EGFP 24 hours prior to their use. Cells were treated with appropriate inhibitor for 15 minutes (for SN50 and SN50M) or 30 minutes (for Bay11-7082) prior to stimulation with 10 ng/ml TNF at t=0 minutes. Results show mean values±s.d.(n-1) from two experiments with at least six cells per experiment plotted for each treatment.

View larger version (31K): [in a new window]   Fig. 3. Combined imaging of p65-dsRed localisation and IB-EGFP degradation following TNF stimulation of dual-transfected cells. (A) Quantification of IB-EGFP degradation in dual-transfected cells also expressing either p65-dsRed or the control expression vector pdsRedN1. IB-EGFP fluorescence was determined as described in Fig. 2. (B) Quantification of translocation of p65-dsRed in dual-transfected cells also expressing either IB-EGFP or the control EGFP-N1. The nuclear: cytoplasmic ratio of p65-dsRed fluorescence was determined as described in Fig. 1. Results show mean values±s.d.(n-1) from two experiments, using at least four cells from each experiment.

View larger version (112K): [in a new window]   Fig. 4. Confocal microscopy of p65-dsRed and IB-EGFP in living cells. Time series images of IB-EGFP and p65-dsRed fluorescence at stated times (in minutes) after addition of 10 ng/ml TNF. Green and red fluorescence were recorded as separate images and then merged for visualisation. Green IB-EGFP and red p65-dsRed co-localisation are represented as yellow in the presence of higher IB-EGFP:p65-dsRed ratios, and orange in the presence of higher p65-dsRed:IB-EGFP ratios.

View larger version (35K): [in a new window]   Fig. 6. Quantitative analysis of p65 translocation, IB degradation and NF-B-dependent transcription in single living cells. (A) Dual confocal microscopy images of p65-dsRed and IB-EGFP before and 40 minutes after TNF stimulation. (B) Quantitative analysis of the time course of p65-dsRed translocation, IB-EGFP degradation and cell luminescence (luciferase activity) in three cells, a-c (A,C). p65-dsRed translocation is shown as nucleo-cytoplasmic ratio and IB degradation is shown as EGFP fluorescence. Luminescence activity is shown as luminescence counts from successive luminescence images with background counts removed. (C) Luminescence images of cells following TNF stimulation. Luminescence directed by NF-B-promoter-directed luciferase activity. Cells marked a-c correspond to those marked in A and used for quantification in B.

View larger version (60K): [in a new window]   Fig. 7. Quantification of differential accumulation of p65-dsRed and IB-EGFP in the nucleus following treatment of dual-transfected cells with leptomycin B. (A) Cells transfected with p65-dsRed and IB-EGFP were treated for 2 hours with 10 ng/ml LMB and monitored by confocal microscopy. (B) Confocal microscopy of long term LMB-treated cells. The top panel was treated for 8 hours with 10 ng/ml LMB. The bottom panel was incubated with 10 ng/ml LMB followed 1 hour later by stimulation with 10 ng/ml TNF. The cells were then analysed for a further 8 hours following TNF stimulation.

View larger version (49K): [in a new window]   Fig. 8. Quantitative analysis of p65 translocation, IB degradation and NF-B-dependent transcription in single living cells. (A) Dual confocal microscopy images of p65-dsRed and IB-EGFP at different time intervals following 1 hour LMB treatment followed by TNF stimulation. Images are shown before and after the 1 hour LMB treatment and 40 minutes after TNF stimulation. (B) Quantitative analysis of the time course of p65-dsRed translocation, IB-EGFP degradation and cell luminescence (luciferase activity) in three cells a-c (A,C) as described in Fig. 6. (C) Luminescence images of cells following TNF stimulation. Luminescence directed by NF-B-promoter-directed luciferase activity. Cells marked a-c correspond to those marked in A and used for quantification in B.