Additional file 6: of Landscape of transcriptional deregulation in lung cancer

Figure S5. Consistency among the normal lung tissues from the four datasets: TCGA-LUAD, TCGA-LUSC, LUAD2 and LUSC2. The PC1 and PC2 axes from Principal Component Analysis (PCA) together explained 91.8% of total variance. A good consistency of these normal lung tissues justified the assumption that they could be pooled together for comparison with SCLC cancer samples. (PDF 42 kb

Additional file 1: of Landscape of transcriptional deregulation in lung cancer

Figure S1. Co-expression between JUND or its neighborhood and its known target gene GADD45A. Three of JUND’s neighborhood genes with strongest co-expression with GADD45 were chosen for display. (PDF 93 kb

Additional file 3: of Landscape of transcriptional deregulation in lung cancer

Figure S2. Down-regulation of TFs in LUSC. (A) Consistency of down-regulated TFs identified in the LUSC and LUSC2 datasets. (B) Clustering of down-regulated TFs shared in the two LUSC datasets. Cluster membership was determined using Fisher’s exact test (p<0.05). (C) DMRT3 expression grouped by DMRT3 copy number status (deletion vs. non-deletion) (Wilcoxon signed-rank test). (D) DMRT3 loss status in relation to TP63 expression (Wilcoxon signed-rank test). (E) DMRT3 loss status in relation to SOX2 expression (Wilcoxon signed-rank test). (F) SOX2 (red) and DMRT3 (blue) binding motifs on the TP63 promoter (-10kb to +10kb of TSS). Genomic coordinates are according to the hg19 assembly. (PDF 92 kb

Additional file 5: of Landscape of transcriptional deregulation in lung cancer

Figure S4. The complete version of Fig. 7c, showing the global TF deregulation patterns across the five datasets: LUAD, LUAD2, LUSC, LUSC2 and SCLC. Colors reflected the log2 scaled number of a TF’s targets, with up-regulated TFs in red and down-regulated in blue. (PDF 22 kb

Continuous activation of ER-K-ras^G12D by 500 µg tamoxifen treatment induced lung hyperplasia and adenoma formation in P53 L/L, LSL-ER-K-ras mice.

<p>A) Continuous activation of ER-K-ras^G12D by 500 µg tamoxifen I.P. daily injection for 12 weeks is sufficient to drive lung adenoma formation in P53 L/L, LSL-ER-K-ras mice after adeno-Cre treatment. The adenoma were positive for Ki67 immunostaining. B) The incidence of lung hyperplasia and/or adenoma formation in <i>P53L/L</i>, <i>LSL-ER-K-ras^G12D</i> mice was shown after adeno-Cre treatment in the absence or presence of tamoxifen treatment.</p

Distribution of accuracy versus recall by different combinations of quality score (QUAL) and read depth (DP) values in two sets (tiers 1 and 2) of SNVs and indels.

<p>(a) Tier One SNVs. (b) Tier Two SNVs. (c) Tier One Indels. (d) Tier Two Indels. For each variant set (panel), each node represents a combination of cutoff values for QUAL and DP. Specifically, the QUAL cutoff was selected by an integer value in the range of 15 to 35 with an increment of 1 each time, and the DP cutoff by an integer value in the range of 3 to 15 with an increment of 1 each time. Then, we evaluated the accuracy, recall, and <i>F</i> score (see text) for each cutoff combination. Note that many nodes are overlapped on the panel and shown by jitter (i.e., points at the same locations are slightly shifted for visibility). The combination of values that could generate the highest <i>F</i> score was selected (shown in red points).</p

Sustained activation of ER-K-ras^G12D induced by tamoxifen treatment is important for cell proliferation, anchorage-independent cell growth, cell invasiveness and tumor maintenance.

<p>A) Cell number counting of indicated MEFs after withdrawal of tamoxifen after 6 passages in the presence of tamoxifen. The PEK-C MEFs always without tamoxifen treatment and the PEK-C MEFs always kept in tamoxifen were used as control. B) Tamoxifen were withdrawn from the PEK-C MEFs at day 5, 10 and 28 after continuous tamoxifen treatment in soft agar. C) Tamoxifen withdrawal from the PEK-C MEFs after indicated days of tamoxifen treatment in cell invasiveness assay in matrigel. D) Tamoxifen withdrawal for indicated time in the <i>P53L/L</i>, <i>LSL-ER-K-ras^G12D</i> mice previously treated for 12 weeks of tamoxifen. The typical lung pathology and cleaved caspase-3 immunostaining were shown.</p

Treatment of tamoxifen at an optimal dose induced the activation of ER-K- ras^G12D and downstream signaling.

<p>A) The <i>P53−/−</i>, <i>ER-K-ras^G12D</i> MEFs (PEK-C) derived from male embryo was counted for cell number after indicated dosage of tamoxifen treatment after 5 passages. B) Relative expression level of <i>K-ras</i> in indicated MEFs with or without tamoxifen treatment. PEK: <i>P53L/L</i>, <i>Loxp-Stop-Loxp ER-K-ras^G12D</i>; PEK-C: <i>P53−/−</i>, <i>ER-K-ras^G12D</i>; PK: <i>P53L/L</i>, <i>Loxp-Stop-Loxp-K- ras^G12D</i>; PKC: <i>P53−/−</i>, <i>K- ras^G12D</i>. (C) Detection of the ER-K-ras^G12D expression in PEK-C cells with or without 0.05 µM tamoxifen treatment. D) Detection of K-ras and ER-K-ras protein level in MEFs infected with or without adeno-Cre in the presence or absence of 0.05 µM tamoxifen treatment in indicated MEFs. β-actin serves as internal control. E) The activation of Ras effector PI3K pathway signaling was confirmed by western blot.</p

Detection of spurious genes.

<p><i>RPE:</i> the number of Reads Per Exon after adjusting the length of the exon and the overall sequencing depth per sample. <i>P_HQR</i>: the Proportion of High-Quality Reads for each exon. Each point represents an exon. The grey points represent all the exons in one sample. The red points indicate the distribution of the 13^th exon of the gene <i>CDC27</i> in all 36 samples, and purple points indicate the distribution of the 42^nd exon of the gene <i>MLL3</i> in all 36 samples, both of which are representative spurious genes and failed to be validated by experiments. The vertical dash line is set <i>RPE</i>  = 1.5 and the horizontal dash line is set <i>P_HQR</i>  = 0.4.</p

Comparison of validation of 159 SNVs and 22 indels by different parameter setting in variant calling.

<p>QUAL: quality score for SNVs and indels. DP: read depth. Definition of Tiers One and Two is provided in text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038470#pone-0038470-g001" target="_blank">Figure 1</a>. TP: true positive; FP: false positive; FN: false negative; TN: true negative;; .</p