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. 2016 Sep 30;5:e18458. doi: 10.7554/eLife.18458

Figure 3. Msn2 and Msn4 exhibit distinct gene regulatory functions in single cells in response to 30-min inhibitor inputs.

(A) (i) A scatter plot showing the relationship of the fast kinetics promoter PDCS2 reporter expression with Msn2 and Msn4 activation at the single cell level. Each dot represents a single cell. Single-cell time traces were tracked over a 3-hr period in which the reporter fluorescence in most cells has already reached the plateau. The x and y axes represent the peak values of Msn4 and Msn2 nuclear translocation (the maximal values in the first 30 min of translocation time traces), respectively; and the dot color represents the maximal level of gene expression as indicated in the color bar. To cover the full dynamic range of TF translocation, the data from the experiments using 30 min inhibitor pulses with 0.1, 0.25, 0.5, 0.75 and 1 μM doses have been combined (n: 444 cells). (ii) Plots show the relationships between PDCS2 reporter expression and (left) Msn2 or (right) Msn4, respectively. Single cells are binned based on their Msn2 or Msn4 nuclear level as indicated in the x-axis and the average of reporter expression is calculated for each binned groups of single cells and shown in the bar graphs. (B) Scatter plots and bar graphs showing the relationship of the slow kinetics promoter PSIP18 reporter expression with Msn2 and Msn4 activation at the single cell level. The data analysis and presentation schemes are consistent with those in (A) (n: 595 cells). Single-cell data used in these plots are provided in the source data files.

DOI: http://dx.doi.org/10.7554/eLife.18458.009

Figure 3—source data 1. Source data for Figure 3A.
DOI: http://dx.doi.org/10.7554/eLife.18458.010
elife-18458-fig3-data1.csv (9KB, csv)
DOI: 10.7554/eLife.18458.010
Figure 3—source data 2. Source data for Figure 3B.
DOI: http://dx.doi.org/10.7554/eLife.18458.011
elife-18458-fig3-data2.csv (12.1KB, csv)
DOI: 10.7554/eLife.18458.011

Figure 3.

Figure 3—figure supplement 1. Single-cell distributions of reporter gene expression versus nuclear TF levels in response to 30-min inhibitor inputs.

Figure 3—figure supplement 1.

(A) Single-cell scatter plots showing the relationships between PDCS2 reporter expression with (left) Msn2 or (right) Msn4 nuclear level, respectively. Single-cell data are from Figure 3A. (B) Single-cell scatter plots showing the relationships between PSIP18 reporter expression with (left) Msn2 or (right) Msn4 nuclear level, respectively. Single-cell data are from Figure 3B.
DOI: http://dx.doi.org/10.7554/eLife.18458.012
Figure 3—figure supplement 2. The relationship between the probability of reporter gene expression and nuclear TF levels in response to 30-min inhibitor inputs.

Figure 3—figure supplement 2.

(A) Bar graphs showing the relationships between the probability of PDCS2 reporter expression with (left) Msn2 or (right) Msn4 nuclear level, respectively. Single-cell data are from Figure 3A. Single cells are binned based on their Msn2 or Msn4 nuclear level as indicated in the x-axis and the proportion of 'responder' cells (green and red cells in Figure 3), instead of the average of reporter expression, is calculated for each binned groups of single cells and shown in the bar graphs. (B) Bar graphs showing the relationships between the probability of PSIP18 reporter expression with (left) Msn2 or (right) Msn4 nuclear level, respectively. Single-cell data are from Figure 3B.
DOI: http://dx.doi.org/10.7554/eLife.18458.013
Figure 3—figure supplement 3. Relationship between PSIP18 reporter gene expression and the ratio of nuclear Msn2 versus Msn4 in response to 30-min inhibitor inputs.

Figure 3—figure supplement 3.

(A) Scatter plot showing the single-cell distribution of PSIP18 reporter expression with the ratio of nuclear Msn2 versus Msn4. Single-cell data are from Figure 3B. (B) Bar graph shows the relationship between PSIP18 reporter expression and the ratio of nuclear Msn2 versus Msn4. Single cells are binned based on their ratio of nuclear Msn2 versus Msn4 as indicated in the x-axis and the average of reporter expression is calculated for each binned groups of single cells and shown in the bar graphs.
DOI: http://dx.doi.org/10.7554/eLife.18458.014
Figure 3—figure supplement 4. Msn2 and Msn4 exhibit similar gene regulatory functions in single cells in response to 60-min inhibitor inputs.

Figure 3—figure supplement 4.

(A) (i) A scatter plot showing the relationship of the slow kinetics promoter PSIP18 reporter expression with Msn2 and Msn4 activation at the single cell level. To cover the full dynamic range of TF translocation, the data from the experiments using 60 min inhibitor pulses with 0.1, 0.25, 0.5, 0.75 and 1 μM doses have been combined (n: 702 cells). (ii) Single-cell scatter plots showing the relationships between PSIP18 reporter expression with (left) Msn2 or (right) Msn4 nuclear level, respectively. Single-cell data are from (i). (B) Plots show the relationships between PSIP18 reporter expression and (left) Msn2 or (right) Msn4, respectively. Single cells are binned based on their Msn2 or Msn4 nuclear level as indicated in the x-axis and the average of reporter expression is calculated for each binned groups of single cells and shown in the bar graphs.
DOI: http://dx.doi.org/10.7554/eLife.18458.015
Figure 3—figure supplement 5. Relationship between reporter gene expression and the area-under-the-curve (AUC) of nuclear TF levels in response to 30-min inhibitor inputs.

Figure 3—figure supplement 5.

Single-cell data from Figure 3 were analyzed to show the relationship of (A) the fast kinetics promoter PDCS2 or (B) the slow kinetics promoter PSIP18 reporter expression with the AUC of Msn2 and Msn4 nuclear translocation. The AUC is calculated as the sum of TF nuclear levels for each single-cell time trace (data points taken every two minutes). (i) Scatter plots showing single-cell distributions; (ii) Bar graphs showing the average level of gene expression in single cells binned based on their TF AUCs.
DOI: http://dx.doi.org/10.7554/eLife.18458.016
Figure 3—figure supplement 6. Single-cell distributions of reporter gene expression versus the speed of TF nuclear import or export.

Figure 3—figure supplement 6.

(A) Scatter plots showing single-cell distributions of PDCS2 reporter expression versus (i) the speed of nuclear import or (ii) the speed of nuclear export of Msn2 and Msn4. The speed of nuclear translocation is quantified by the time needed to reach half maximum of nuclear translocation of Msn2 or Msn4 in response to stimulation (nuclear import speed) or upon the removal of stimulation (nuclear export speed). Single-cell data are from Figure 3A. The means of reporter expression are calculated for grouped single cells and shown using black solid lines. The mean and standard deviation of nuclear translocation times in single cells are calculated and shown above each plot. (B) Scatter plots showing single-cell distributions of PSIP18 reporter expression versus (i) the speed of nuclear import or (ii) the speed of nuclear export of Msn2 and Msn4. Single-cell data are from Figure 3B.
DOI: http://dx.doi.org/10.7554/eLife.18458.017