Additional file 1 of A systematic evaluation of nucleotide properties for CRISPR sgRNA design

Supplementary Information. The pdf document that contains all supplementary notes, figures and tables. Figures S1-S2 plot the top 10 most informative features ranked by BIC and variable importance scores, respectively. Tables S1-S3 contain the results from randomforest in binary outcome model. Tables S4-S6 contain the results from gbm in binary outcome model. Tables S7-S9 contain the results from elastic net in continuous outcome model. Tables S10-S12 contain the results from randomforest in continuous outcome model. Tables S13-S15 contain the results from gbm in continuous outcome model. Tables S16-S18 contain the results comparing 30bp and 40bp sequences. Tables S19-S20 contain the results from leave-one-gene out prediction. (PDF 151 kb

Classification of human HCC based on expression of RXR-α and Wnt pathway genes.

<p>HCC samples in the Boyault dataset[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118480#pone.0118480.ref012" target="_blank">12</a>] were clustered based on expression of 138 RXR-α and Wnt pathway genes. Beneath the heatmap are four rows, showing for each HCC sample (1) cluster assignment to the two major groups found by unsupervised clustering of all genes (2) relative prognosis based on the 65-gene signature of Kim et al.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118480#pone.0118480.ref024" target="_blank">24</a>], ranging from red = poor, white = neutral, green = good; (3) grey bars indicate activating mutation in <i>CTNNB1</i>; (4) average expression of the 5 genes known to be overexpressed in <i>CTNNB1</i>-mutant HCC cells; red = expression, green = less expression of the 5-gene signature associated with <i>CTNNB1</i> mutation[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118480#pone.0118480.ref012" target="_blank">12</a>].</p

Gene expression and biochemical changes in RXR-α and Wnt pathways during liver development.

<p>(A) Clustering of RXR-α and Wnt pathway gene expression profiles of normal mouse liver samples taken from four different periods of development. (B) Reporter assays for Wnt and RXR pathways comparing hepatoblasts (E14) to immature hepatocytes (E18). Both TCF (p = 0.016) and (C) RXR-α activities (p = 0.021) were significantly different (D) RNA and (E) protein expression of RXR-α in hepatoblasts compared to immature hepatocytes.</p

9-cis retinoic acid activation of RXR-α suppresses β-catenin signaling.

<p>(A) Effect of 9-cis retinoic acid on Wnt signaling activity in Huh-7 and HLE cell lines as measured using a TCF transcriptional reporter. Labeling as in Panel B. (* = p < 0.05; ** = p < 0.001). (B) Effect of 9-cis retinoic acid on β-catenin protein levels in Huh-7 cells. (C) Effect of 9-cis retinoic acid on clonogenic growth of Huh-7 cells transfected with either mutationally activated <i>CTNNB1</i> (S33Y; red column) or empty vector (blue column) ((p = 0.0037 and 0.0041). In all panels, error bars indicate standard deviations.</p

Effects of lowering RXR-α protein levels in hepatocytes on Wnt signaling and tumorigenicity.

<p>(A) Validation of two independent shRNAs for their ability to lower RXR-α protein levels in hepatoctyes as determined by immunoblotting using Ras Gap protein expression as a loading control and shRNA directed against luciferase as a vector control. B) Tumor growth following subcutaneous injection in nude mice of MYC/p53^-/-; E18 hepatocytes infected with either shluc (red column), RXRA sh1 (purple column), or RXRA sh2 (blue column). Error bars indicate standard deviations. Tumor incidence is noted above columns for each condition. <b>C</b>) Survival curves of nude mice after intrasplenic injections of <i>MYC/p53^-/-</i>; E18 hepatocytes transfected with either shluc (red line), or RXRA sh1 (purple line), or RXRA sh2 (line), n = 10 injections. D) Images of livers taken from mice following transplantation of <i>MYC/p53^-/-</i> E18 hepatocytes transfected with shluc, RXRA sh1, or RXRA sh2. The five panels are from left to right, intact livers, GFP-imaging of livers, hematoxylin and eosin staining of liver tissue sections, PCNA immunohistochemical staining, GFP immunohistochemical staining. Size bar = 200 μm.</p

Comparative genomic analysis of human and mouse model HCC.

<p>(A) Frequency plots of copy number alterations in human HCC. Frequency of gains (red) and losses (green) as determined by ROMA array CGH analysis of 101 human HCC samples. (B) Frequency plots of copy number alterations in mouse model HCC as determined by ROMA array CGH analysis in 38 mouse model HCC samples. (C) Unsupervised hierarchical clustering of expression profiles of various mouse HCC tumors and normal stages of liver development. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118480#pone.0118480.s008" target="_blank">S3 Table</a> for a description of the different samples.</p

Pathways that distinguish the two major clusters of mouse HCC.

<p>Top ten most significantly different pathways based on gene expression of the two major clusters of mouse HCC and normal liver samples determined by GSEA (A) and Ingenuity (B).</p