Comparison of Differently Measured Changes in Gene Expression within and between Two ESC Lines Grown for 0, 24, 72, and 96 h in +LIF ± RA or –LIF ± DMSO

<div><p>(A) Comparison of Q–RT–PCR and microarray results for the R1 line (r = 0.76).</p><p>(B) Comparison of Q–RT–PCR and microarray results for the J1 line (r = 0.82).</p><p>(C) Comparison of results between the R1 and J1 lines (r = 0.87).</p></div

Comparison of Biological and Molecular Changes in ESC Stimulated to Differentiate by Exposure to AA

<div><p>(A) Changes in EB frequency.</p><p>(B) Changes in the levels of transcripts for seven ESC signature change genes measured by Q–RT–PCR. +LIF (▪), +LIF + AA (•), and –LIF + AA (▴).</p></div

Transcriptome Plots of Estimated Expression Changes, Based on Fitting Models to Each Dataset

<div><p>All plots have density shading to demonstrate the number of points (genes) in different regions. Lines illustrate examples of some of the requirements that make up the definition of the ESC signature change and observed gene expression patterns that fulfilled all requirements are marked as ♦. Experiment-specific implementations of requirements are explained below.</p><p>(A) DMSO/RA dataset. The requirement for large absolute changes is illustrated by the solid blue lines. Consistency across conditions implied that genes must exhibit a change in the same direction in both treatments (bottom left or top right quadrant).</p><p>(B) R1–LR dataset. Note that the <i>y</i>-axis is the change seen at 72 h relative to that seen at 18 h. The requirement for large absolute changes is illustrated by the solid blue lines. The criterion for consistency was applied by requiring that the change 18 h after LIF removal be in the same direction as that after 72 h (i.e., in the lower left or upper right quadrants), regardless of its magnitude</p><p>(C) M–LR dataset. The requirement for large absolute changes is illustrated by the dashed blue lines. To meet the consistency criterion, we required that a temporal gene expression trend either increase or decrease continuously over the duration of the experiment. This requirement was relaxed slightly to retain trends with a direction change occurring either very early (red line) or very late (green line).</p></div

The Effect of LIF Removal with or without Addition of DMSO or RA on the Maintenance and Differentiation of ESC

<p>(A) CFC frequency and (B) EB-forming ability of cells from the ESC cultures assessed at varying times after initiation of the treatment (+LIF controls =▪ , –LIF = •, –LIF + DMSO = ▴, +LIF + RA = ▾). * denotes the data for the +LIF sample are significantly different from all other treatments. (C) Gene expression of differentiation markers was monitored by Q–RT–PCR after 96 h of treatment. Results shown are relative to the +LIF control cells.</p

Two-way Pareto Front Analysis applied to CVs from the R1–LR Dataset and the M–LR Dataset

<div><p>(Left) Shows the CVs for all comparable genes with the first five Pareto fronts highlighted (red, first PF; blue, second PF; green, third PF; gold, fourth PF; black, fifth PF).</p><p>(Right) Shows a magnification of the dashed box in the left panel. Here the red gene is said to dominate all other genes because, although it has an M–LR CV equal to that of several other genes, it has the highest R1–LR CV. Thus it lies on the first PF. In the same way, the blue gene dominates all genes except the red gene and thus lies on the second PF. It is not possible to choose between the two green genes because they each have larger CVs in one of the two experiments. They, therefore, lie on the same (third) PF. The highlighted yellow gene does have a larger CV in the R1–LR dataset than the green gene on the left but it falls on a lower PF because it is completely dominated by the green gene on the right.</p></div