Image-based profiles of RNAi sequences are highly reproducible.

<p>Using the 95^th percentile of the null distribution (A) as a threshold to define significant correlations, 92% of replicate correlations in B are seen to be significant. Correlations are computed between profiles of individual wells. The percentage of correlations above the defined threshold is indicated; dotted line indicates 95^th percentile of the null distribution (A). The difference between means of A and B is highly significant (<i>P</i>-value < 10⁻¹⁵; two-sided Student's <i>t</i>-test).</p

Different RNAi reagents targeting the same gene rarely produce similar profiles, whereas RNAi reagents sharing a seed sequence do.

<p>shRNAs targeting the same gene have very low correlation (B), whereas those containing the same seed sequence have a much higher correlation (C). Using the 95^th percentile of a null distribution (A) as a threshold to define significant correlations, only 10% of correlations in B are significant, compared to 73% in C. This indicates that the phenotypes induced by RNAi knockdown are dominated by seed effects. Correlations are computed between profiles of sequences, obtained by median-averaging profiles of replicate wells. The percentage of correlations above the defined threshold is indicated for each group; dotted line indicates 95^th percentile of the null distribution (A). The difference between means of B and C is highly significant (<i>P</i>-value < 10⁻⁵; two-sided Student's <i>t</i>-test).</p

Image-based profiles of RNAi sequences are highly reproducible.

<p>Using the 95^th percentile of the null distribution (A) as a threshold to define significant correlations, 92% of replicate correlations in B are seen to be significant. Correlations are computed between profiles of individual wells. The percentage of correlations above the defined threshold is indicated; dotted line indicates 95^th percentile of the null distribution (A). The difference between means of A and B is highly significant (<i>P</i>-value < 10⁻¹⁵; two-sided Student's <i>t</i>-test).</p

Cell Painting assay.

<p>U2OS cells prepared for this study were stained using the Cell Painting assay protocol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131370#pone.0131370.ref034" target="_blank">34</a>], with six stains imaged across five channels, revealing eight cellular components/structures. Scale bar 25 μm.</p

Workflow for generating morphological profiles using Cell Painting.

<p>U2OS cells are treated with shRNAs and transferred to 384-well plates in which they are stained and then imaged. The images are analyzed and ~1400 features are extracted from each cell. These data are then transformed to generate multivariate profiles.</p

Visual example of seed effects dominating morphological profiles.

<p>Only nuclear shape features were used in this example in order to yield visually interpretable phenotypes. Two of the shRNA sequences targeting CDCA7L and BECN1 share a seed sequence (sequences 299864 and 17937 have a common seed GAATGA at nucleotides 12–17). The Spearman correlation between the morphological profiles of these shRNAs is high (seed correlation = 0.44, red bar). For each of these two shRNAs, a same-gene shRNA with a dissimilar phenotype is also shown (same-gene shRNA correlation = -0.31 and -0.11, green bars). All four shRNAs have high replicate correlation and are dissimilar from untreated cells. We specifically chose an example where the seed correlation is high and the same-gene shRNA correlations are low; however this phenomenon is seen globally (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131370#pone.0131370.g003" target="_blank">Fig 3</a>). Images have been zoomed in, showing only 2% of the imaged region.</p

Image_6_Designed Surface Topographies Control ICAM-1 Expression in Tonsil-Derived Human Stromal Cells.PNG

<p>Fibroblastic reticular cells (FRCs), the T-cell zone stromal cell subtype in the lymph nodes, create a scaffold for adhesion and migration of immune cells, thus allowing them to communicate. Although known to be important for the initiation of immune responses, studies about FRCs and their interactions have been impeded because FRCs are limited in availability and lose their function upon culture expansion. To circumvent these limitations, stromal cell precursors can be mechanotranduced to form mature FRCs. Here, we used a library of designed surface topographies to trigger FRC differentiation from tonsil-derived stromal cells (TSCs). Undifferentiated TSCs were seeded on a TopoChip containing 2176 different topographies in culture medium without differentiation factors, then monitored cell morphology and the levels of ICAM-1, a marker of FRC differentiation. We identified 112 and 72 surfaces that upregulated and downregulated, respectively, ICAM-1 expression. By monitoring cell morphology, and expression of the FRC differentiation marker ICAM-1 via image analysis and machine learning, we discovered correlations between ICAM-1 expression, cell shape and design of surface topographies and confirmed our findings by using flow cytometry. Our findings confirmed that TSCs are mechano-responsive cells and identified particular topographies that can be used to improve FRC differentiation protocols.</p

Image_4_Designed Surface Topographies Control ICAM-1 Expression in Tonsil-Derived Human Stromal Cells.JPEG

<p>Fibroblastic reticular cells (FRCs), the T-cell zone stromal cell subtype in the lymph nodes, create a scaffold for adhesion and migration of immune cells, thus allowing them to communicate. Although known to be important for the initiation of immune responses, studies about FRCs and their interactions have been impeded because FRCs are limited in availability and lose their function upon culture expansion. To circumvent these limitations, stromal cell precursors can be mechanotranduced to form mature FRCs. Here, we used a library of designed surface topographies to trigger FRC differentiation from tonsil-derived stromal cells (TSCs). Undifferentiated TSCs were seeded on a TopoChip containing 2176 different topographies in culture medium without differentiation factors, then monitored cell morphology and the levels of ICAM-1, a marker of FRC differentiation. We identified 112 and 72 surfaces that upregulated and downregulated, respectively, ICAM-1 expression. By monitoring cell morphology, and expression of the FRC differentiation marker ICAM-1 via image analysis and machine learning, we discovered correlations between ICAM-1 expression, cell shape and design of surface topographies and confirmed our findings by using flow cytometry. Our findings confirmed that TSCs are mechano-responsive cells and identified particular topographies that can be used to improve FRC differentiation protocols.</p

Image_5_Designed Surface Topographies Control ICAM-1 Expression in Tonsil-Derived Human Stromal Cells.JPEG

<p>Fibroblastic reticular cells (FRCs), the T-cell zone stromal cell subtype in the lymph nodes, create a scaffold for adhesion and migration of immune cells, thus allowing them to communicate. Although known to be important for the initiation of immune responses, studies about FRCs and their interactions have been impeded because FRCs are limited in availability and lose their function upon culture expansion. To circumvent these limitations, stromal cell precursors can be mechanotranduced to form mature FRCs. Here, we used a library of designed surface topographies to trigger FRC differentiation from tonsil-derived stromal cells (TSCs). Undifferentiated TSCs were seeded on a TopoChip containing 2176 different topographies in culture medium without differentiation factors, then monitored cell morphology and the levels of ICAM-1, a marker of FRC differentiation. We identified 112 and 72 surfaces that upregulated and downregulated, respectively, ICAM-1 expression. By monitoring cell morphology, and expression of the FRC differentiation marker ICAM-1 via image analysis and machine learning, we discovered correlations between ICAM-1 expression, cell shape and design of surface topographies and confirmed our findings by using flow cytometry. Our findings confirmed that TSCs are mechano-responsive cells and identified particular topographies that can be used to improve FRC differentiation protocols.</p

Image_3_Designed Surface Topographies Control ICAM-1 Expression in Tonsil-Derived Human Stromal Cells.JPEG

<p>Fibroblastic reticular cells (FRCs), the T-cell zone stromal cell subtype in the lymph nodes, create a scaffold for adhesion and migration of immune cells, thus allowing them to communicate. Although known to be important for the initiation of immune responses, studies about FRCs and their interactions have been impeded because FRCs are limited in availability and lose their function upon culture expansion. To circumvent these limitations, stromal cell precursors can be mechanotranduced to form mature FRCs. Here, we used a library of designed surface topographies to trigger FRC differentiation from tonsil-derived stromal cells (TSCs). Undifferentiated TSCs were seeded on a TopoChip containing 2176 different topographies in culture medium without differentiation factors, then monitored cell morphology and the levels of ICAM-1, a marker of FRC differentiation. We identified 112 and 72 surfaces that upregulated and downregulated, respectively, ICAM-1 expression. By monitoring cell morphology, and expression of the FRC differentiation marker ICAM-1 via image analysis and machine learning, we discovered correlations between ICAM-1 expression, cell shape and design of surface topographies and confirmed our findings by using flow cytometry. Our findings confirmed that TSCs are mechano-responsive cells and identified particular topographies that can be used to improve FRC differentiation protocols.</p