3D profile of expression differences among genes related to the DNA-damage response pathway following NPAS2 silencing in MCF-7 cells as measured by pathway specific PCR array (SuperArray). 22 of the 81 genes measured (27.2%), had greater than 2-fold reduced expression and 73 (91.4%) were down-regulated. Of the 7 genes with increased expression, none was greater than 1.31 fold. The data are a result of 2 replicates from 2 independent experiments.

Reduced NPAS2 expression leads to changes in cell cycle distribution among mutagen treated and mock treated MCF-7 cells. Cells were either treated with scrambled negative or NPAS2-targeting siRNA oligos, and either 0.015% (v/v) MMS or PBS. Cell cycle distribution was determined 24 hours after MMS/PBS treatment and 72 hours after siRNA treatment. Data was collected on an average of 47,060 cells per sample (range 40,335 to 50,609), and cell phases were determined by the software using the Watson pragmatic algorithm. The cell phase distributions were: 44% G1, 35% S and 21% G2/M for cells treated with siRNA-/MMS- (A); 51% G1, 20% S and 29% G2/M for cells treated with siRNA+/MMS- (B); 61% G1, 13% S and 26% G2/M for cells treated with siRNA-/MMS+ (C); and 47% G1, 32% S and 22% G2/M for cells treated with siRNA+/MMS+ ^† (D). The cell phase distribution was significantly different between treatments siRNA-/MMS- and siRNA-/MMS+ (χ²₂=13.777; p= 0.001), but not between siRNA+/MMS- and siRNA+/MMS+ (χ²₂ = 3.830; p=0.147), indicating an improper response to mutagen treatment among cells with reduced NPAS2 expression. Normal cells respond to mutagen treatment with a significant G1/G2 delay, as expected, while NPAS2 knockdown cells do not display a significant change in cell cycle distribution (E).
^†Percentages may not sum to 100 due to rounding.

DNA damage repair capacity assessed by the Comet Assay. (A) Representative cell images from each of the four treatments. Mean Olive tail moment (B) determined from 50 cells from each of the four treatment groups was 0.292±0.037 for siRNA-/MMS-, 1.003±0.164 for siRNA-/MMS+, 0.204±0.026 for siRNA+/MMS-, and 1.654±0.235 for siRNA+/MMS+. All pairwise comparisons were examined using Fisher’s PLSD. Among the samples not treated with mutagen, the difference in tail moment between cells with reduced and normal NPAS2 expression was not significant (p=0.668), indicating that without inducing damage, there is no difference in repair. However, among the mutagen-treated populations, cells with reduced NPAS2 had a significantly longer mean tail moment (p=0.0018) as depicted in Figure 4B, indicating decreased DNA repair capacity compared to cells with normal NPAS2 expression. As expected, all other comparisons were significant (p<0.001).

Representative flow cytometry results for each of the 4 treatments (A). Live cells are found in the lower left quadrant, early apoptotic cells are in the lower right quadrant, and necrotic cells occupy the upper area. Cells were stained with PI (y axis) along with Annexin V (x axis) and 10,000 cells were counted. Cells were then categorized as live, dead, apoptotic, or ambiguous. (B): Percentage of live cells for each treatment from 2 independent experiments. In both mutagen treated and non-treated populations, cells with reduced NPAS2 expression were significantly less viable (p=0.001, and p=0.003, respectively). (C): Percentage of the total population undergoing early apoptosis for each treatment from 2 independent experiments. Comparisons are between MMS and PBS treated cell populations in each siRNA treatment group, and asterisks denote significant differences (p<0.05). Both normal and NPAS2 knockdown cells exhibit increased apoptosis following mutagen exposure (p=0.027, and p=0.037, respectively), suggesting that NPAS2 itself does not directly hinder the apoptotic response.