Calibrated experimental immunoblot data.

<p>This figure shows calibrated analyte amounts for 40 of our 230 samples that we analyzed with immunoblots. The others were qualitatively similar. Gray bars represent results from the 2-step method, red bars represent results from the 1-step method, and error bars represent standard error values. On average, there were 3.4 calibrated measurements for each sample with the 2-step method and 4.9 for the 1-step method. Note that the 1-step method results have smaller standard errors.</p

Comparison of the 1-step and 2-step methods using artificial data.

<p>A. Sample analyte amounts for an artificial data set. Here and in subsequent panels, black features represent the true analyte amounts, gray features represent results from the 2-step method, and red features represent results from the 1-step method. Error bars represent standard errors. (B-D) Comparison of computed sample analyte amounts, <i>a</i> sensitivity coefficients, and <i>b</i> sensitivity coefficients with their true values for the same artificial data set. (E-G) Histograms of errors between fit values and true values for computed sample analyte amounts, <i>a</i> sensitivity coefficients, and <i>b</i> sensitivity coefficients for 1000 artificial data sets. Note that the 1-step method yields more accurate data calibration. See main text for details.</p

Comparison of workflow for 2-step and 1-step calibration methods, illustrated for calibrating band intensities on immunoblots.

<p>A. Illustration of samples 1 (the standard) through 7, run on 5 different immunoblots with variable replication. The band intensities shown depend on the sample, blot, and experimental noise. B. Tabulated data showing assigned band intensities for each sample and blot. C. Direct comparison of the conventional 2-step calibration method (left) with the 1-step calibration method (right). D. Plots of the calibrated estimates of analyte amounts in each sample using the different methods. Error bars represent the standard error of the mean (precision of estimate) and numbers above the bars represent the number of calibrated measurements of each sample.</p

Hsp90-buffered variation contributed to predicted selection responses.

<p>Fold-increase in the predicted response to truncation, directional or stabilizing selection of the ‘populations’ of 9 <i>RI</i> line genotypes in the <i>P582i</i> mutant relative to equivalent ‘populations’ of genotypes in the <i>Sami</i> control flies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000075#pone-0000075-t005" target="_blank">Table 5</a>).</p

Model for biological control of canalization and evolvability. In any individual the strength of signaling through Hsp90 target pathways is directly proportional to the level of Hsp90 function (top panel), but the phenotypic effects of changes in signaling strength are often highly nonlinear, creating thresholds (the inflection point, second from top).

<div><p>A simple consequence of steep non-linearity is that sensitivity to all kinds of perturbation and stochastic effects is expected to be highest at the inflection point (second panel from bottom).</p> <p>This simple relationship between sensitivity to variation and the steepness of the signaling-phenotype relationship at the inflection point, the inverse of canalization (bottom panel), suggests why all variation increases in concert for individuals and/or populations near trait thresholds.</p></div

Experimental design partitions genetic and purely-environmental components of variation.

<p>Females heterozygous for <i>P582i</i> (<i>Sam1;Sam2;P582i/Sami</i>; left) were crossed to males from each of 9 highly inbred (95% homozygous) <i>RI</i> line backgrounds (different colors) to create matched “clones” of Hsp90 mutant and control male progeny from the same vial and maternal environments and hybrid for <i>Sam</i> and each <i>RI</i> line background.</p

Hsp90 controlled environmental variation specific to previously invariant or canalized traits.

<div><p>Comparison of mean-normalized components of purely-environmental variation (z-axis) across the <i>RI</i> line backgrounds (x-axis).</p> <p>Developmental stability was calculated as the averaged (unsigned) deviations of left and right from the mean, i.e. (L+R)/2, within each individual (<i>V_e within</i>) or on the averaged (unsigned) deviations of each individual (L+R) from their clone means for each <i>RI</i> line genotype (<i>V_e among</i>).</p> <p>There was generally no effect of Hsp90 allele (<i>Sami</i>, blue or <i>P582i</i>, yellow) on the variable traits, and a highly significant effect on either measure of <i>V_e</i> for canalized bristle traits but not wing area, which was highly canalized independent of Hsp90 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000075#pone-0000075-t004" target="_blank">Table 4</a>).</p></div

Regression of early fecundity and viability onto penetrance in 1,432 recombinant isogenic lines.

<p>If genes for <i>dfe</i> had effects on viability and survival, a negative relationship would be expected, however penetrance was positively correlated with fitness in recombinant lines from <i>HE1</i> and <i>HE3</i>.</p

Hsp90 controlled phenotypic variation of most invariant quantitative traits.

<div><p>(<b>A</b>) Effects of <i>P582i</i> (yellow) and <i>Sami</i> (blue) alleles of Hsp90 in the isogenic <i>Sam</i> background on <i>V_P</i> of mutant and control sets of 450 males across the 9 <i>RI</i> line backgrounds.</p> <p>(<b>B and C</b>) Effects of 3^rd chromosomes carrying null (<i>P582</i>), and dominant-negative (<i>9J1</i>) Hsp90 mutations or wild-type Sam alleles introgressed into the <i>Sam</i> background in mutant and control sets of 225 male or 225 female sibs.</p> <p> <i>TM6B</i> contains the dominant <i>Humeral</i> (<i>Hu</i>) mutation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000075#pone.0000075-FlyBase1" target="_blank">[32]</a>, which increases humeral TH bristle numbers.</p> <p>Therefore, for comparison of TH bristles between TM6B and the other genotypes we used TH-HU, indicating that TH was scored only for the remaining 18 non-humeral TH bristle types.</p> <p>All experiments were conducted under temperature, density and humidity controlled conditions.</p> <p>Environmental effects were further controlled by direct comparisons between flies from the same vial and maternal environments.</p> <p>Coefficients of variation (<i>CV</i> = standard deviation/mean) are shown to enable between-trait comparisons. Statistical tests of the significance of Hsp90 effects on phenotypic variability and <i>P</i>-values are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000075#pone-0000075-t001" target="_blank">Table 1</a> (for A) and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000075#pone-0000075-t002" target="_blank">Table 2</a> (for B and C).</p></div

Representative tests of penetrance, absolute and relative fitness.

<div><p>High (red) and low (blue) lines, genetically differentiated for <i>dfe</i> penetrance were not differentiated by tests of viability and fitness.</p> <p>Controls (grey) were either <i>Canton-S</i> (<i>CS</i>) flies tested at the same time or published values for wild-type flies (WT; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000076#pone.0000076-Ashburner1" target="_blank">[21]</a>).</p> <p>Experiments showing the most extreme between line differences are displayed.</p> <p>Error bars indicate the standard errors of the mean.</p> <p><b>A.</b> Penetrance of eye deformity in <i>dfe</i> lines and <i>CS</i> flies reared at 25°C.</p> <p><b>B.</b> Hatch rate of timed embryo collections measured at 36 hours post egg-laying.</p> <p><b>C.</b> Egg-to-adult survival measured as the number of adult flies emerging versus eggs laid.</p> <p><b>D.</b> Fractional survivial of <i>dfe</i> progeny in competition with <i>CS</i> flies co-cultured at high density.</p> <p><b>E.</b> Lifetime female production of eggs (wild-type values; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000076#pone.0000076-Ashburner1" target="_blank">[21]</a>) or viable adult offspring (high and low line <i>dfe</i> females).</p></div