This data set is from an HCS experiment to characterize
how camptothecin affects cell cycle progression.
Cells are treated with camptothecin for 24 hours at a gradient of
Three cellular parameters are monitored:
- Channel 1: DNA stain (nuclear stain intensity; DNA content).
- Channel 2: Cellular target 1, p53, (nuclear stain intensity).
The response of p53 is that its level in nuclei
goes up or down.
- Channel 3: Cellular target 2, pRb, (nuclear stain intensity). Its
response is just like that of p53; its level in nuclei goes up or
Since we are looking at the effects of a cell cycle drug
the first thing to look at would be the DNA content,
our first channel.
In the following 27-second video,
we create a trellis of histograms of DNA content.
It is clear from the trellis graph of DNA content in the video
that camptothecin has different effects on cell cycle progression
at different concentrations.
The "2N" and "4N" peaks are quite distinct
in the the upper 5 panels.
The distribution of DNA content in each panel also spreads out
quite a lot.
This is because the particular cell line used in this experiment
shows a wide range of chromosome numbers.
The 2nd Channel, p53
In the following 12-second video, we
use a boxplot to visualize the effect of camptothecin on p53.
The boxplot in the video clearly shows that the level of p53
in nuclei increases markedly at higher concentrations of camptothecin.
The median of the H1 well is 4 times bigger than the median
of the A1 well.
The 3rd Channel, pRb
Much like we did for the second channel,
we can get a boxplot with Well
as the X variable and Nucleus Intensity (pRb)
in log scale as the Y variable:
This boxplot clearly shows
that as the concentration of camptothecin goes up
the level of pRb goes down
Point to PonderIf the expression level or phosphorylation state
of a cellular protein changes in different phases of
does it make sense not to examine a target protein
in terms of the phases of cell cycle?
In the rest of this tutorial,
we'll take cell cycle into account to see how p53 and pRb
are affected by camptothecin.
Dividing Cells into 3 Categories Based on DNA Content
Here we will divide all cells in this experiment
into 3 categories:
- Those in the 2N phase of cell cycle,
- Those in the 4N phase of cell cycle, and
- Those in the S phase of cell cycle.
In the following 23-second video,
we will do all of the division graphically
by making a histogram of DNA content of all
cells and using a paint brush:
What we just demonstrated in the above video is essentially equivalent to
creating on the fly a new categorical variable with 3 categories:
- Blue (cells in the 2N phase of cell cycle)
- Green (cells in the S phase of cell cycle)
- Red (cells in the 4N phase of cell cycle)
Visualizing p53 Responses in Different Phases of Cell Cycle
Now we have divided all cells into 3 groups, each corresponding
to one phase of cell cycle.
We can visualize this information on p53 responses,
phases of cell cycle, and drug concentration with a trellis
of boxplots. Because Panmo treats each plot as a viewport
into the data space and allows us to retrieve any data visible
in a viewport, we can use the data in the histogram
at the end of last video
to make this trellis of boxplots.
The following video runs for 1 minute.
It's clear the patterns in the G1 and H1 panels (subject to the
highest camptothecin concentrations) are distinctly different from
those in other panels.
Visualizing pRb Responses in Different Phases of Cell Cycle
Following similar steps, we can easily get the following
trellis of boxplots:
Note that the pattern in the H1 (subject to the highest camptothecin
concentration) panel is distinctly different from those
in other panels.
Visualizing the Relationship Between p53 and pRb Conditioning
on Drug Concentration and Phases of Cell Cycle
Finally, we are going to take drug concentration and phases of cell
cycle into account to explore the relationship between p53 and pRb.
We do so by making a trellis of scatterplots of p53 versus pRb
in the following 25-second video: