Fig. 5
Comparison of extent of co-expression of c-fos-YFP and NFAT-RFP in the same cells. a Representative flow cytometry plots of c-fos-driven YFP and NFAT-driven RFP expression in non-transfected cells (labelled Blank), transfected but unstimulated cells (labelled Rest), cells challenged with 30 nM thapsigargin and cells stimulated with 100 nM thapsigargin in high K+ solution. Plots were divided into four sections (Q1–Q4), representing high RFP expression (Q1), high RFP and YFP expression (Q2), high YFP expression (Q3) and low expression of both proteins (Q4). For blank, % cells in Q1 was 0.044, Q2 was 0.009, Q3 was 0.005 and Q4 was 99.9. For rest, % cells in Q1 was 0.46, Q2 was 0.1, Q3 was 0.63 and Q4 was 98.7. For 30 nM thapsigargin, % cells in Q1 was 50.9, Q2 was 2.16, Q3 was 0.32 and Q4 was 46.6. For 100 nM thapsigargin in high K+ solution, % cells in Q1 was 60.4, Q2 was 5.1, Q3 was 0.23 and Q4 was 34.2. b Histograms plot the fluorescence of YFP for each condition. Median values are stated in each histogram and the percentages denote the % of cells exhibiting a fluorescence intensity of ≥103. c Histograms plot the fluorescence of RFP for the conditions shown. Median and % values have the same meaning as in panel b. d Time-course of c-fos-eYFP fluorescence is compared at different times after stimulation. The fraction of cells expressing c-fos at different times is compared (denoted % responders) as is the fluorescence intensity of these individual responding cells (43–133 cells for each condition). e Time-course of NFAT-driven RFP fluorescence is shown at different times after stimulation (1–182 cells for each time point; only 1 out of 579 non-stimulated (basal) cells were NFAT-positive). For d and e, expression was monitored using epifluorescence microscopy. f Histograms, derived from the FACS data, with fitted distributions shown in red for non-responders and responders as described in the methods. Blue dashed lines indicate the two normal distributions that are summed to form bimodal cases
