Figure 1.
RNA-Seq analysis of MCF10A and DCIS models (A) Differential interference contrast (DIC) images of 12-day 3D rBM overlay cultures of MCF10A, MCF10.DCIS, SUM102, and SUM225 cells.
Scale bar, 200 µm. (B) Unsupervised hierarchical clustering of the transcript profile from the eight RNA-Seq samples. (C) Cluster dendrogram of RNA-Seq samples based on differentially expressed genes. (D) Venn diagram of differential expression results (with a p-value <0.001 and cut-off fold change of 4) showing the overlap between the genes expressed by different models of DCIS in comparison to MCF10A. There are a total of 295 genes that are consistently differentially expressed between the MCF10A and the three DCIS models: MCF10.DCIS, SUM102 and SUM225. The expansion shows that 156 transcripts were decreased, 63 transcripts were increased, and 76 transcripts were differentially expressed in all three models but the direction of change was variable.
Table 1.
Genes with the largest fold change in expression identified in the list of those differentially expressed.
Table 2.
Significant canonical pathways associated with differentially expressed genes.
Table 3.
The molecular and cellular functions and diseases and disorders associated with the consistently differentially expressed genes derived from RNA-Seq data analysis.
Table 4.
Significant consistently differentially expressed genes involved in various types of cancers.
Figure 2.
Significant biological processes associated with the differentially expressed genes as identified by Genomatix.
The differentially expressed genes in all models of DCIS in comparison to MCF10A were analyzed by WebGestalt2 tool. A Fisher’s exact test was used to determine statistical significance with a significance level of 0.05. The p-values and the number of genes associated with a particular function or process are indicated in bold in the respective boxes.
Figure 3.
Cellular components related to significantly differentially expressed genes.
The differentially expressed genes in all models of DCIS in comparison to MCF10A were analyzed by WebGestalt2 tool. The p-values and the number of differentially expressed genes associated with a particular cellular component are indicated in bold in the respective boxes.
Table 5.
Network pathway analysis of differentially expressed genes by Genomatix.
Figure 4.
Comparison of the changes in expression of selected genes detected by RNA-Seq and qRT-PCR.
The values represent log2fold change in MCF10.DCIS, SUM102 and SUM225 compared to expression in MCF10A. Positive values indicate up-regulation (highlighted in green) and negative values indicate down-regulation (highlighted in red).
Figure 5.
ALDH5A1 is over-expressed in DCIS models.
Whole cell lysates from 3D rBM overlay cultures of MCF10A, MCF10.DCIS, SUM102 and SUM225 were probed for ALDH5A1 (upper panel) and for GAPDH (lower panel) as a loading control. Densitometry indicated that ALDH5A1 levels in the lysates of MCF10.DCIS, SUM102, and SUM225 were 2.7, 3.1, and 7.3-fold over that in MCF10A in this representative experiment. In three independent analyses of MCF10.DCIS 3D rBM cultures, the mean increase in ALDH5A1 over control was 3.8-fold.
Figure 6.
ALDH5A1 as a target in DCIS models.
(A, B) Effect of DSF and VPA on proliferation of MCF10A (open circles), MCF10.DCIS (closed circles), SUM102 (triangles) and SUM225 (squares) 3D rBM cultures. Cells were incubated for 72 hours with the indicated concentrations of drugs or with DMSO as a vehicle control and subjected to an MTT assay. Sigmoidal dose-response curves were plotted using nonlinear regression analysis. Data represent the mean ± SEM of three independent experiments (each performed in triplicate). (C) Effect of DSF and VPA on 3D rBM cultures of SUM102-mRFP cells. Cells were incubated for 8 days with the indicated concentrations of drugs or with DMSO as a vehicle control and DIC images are shown. Size bar, 90 µm.