File S1 - Suppression of Akt-mTOR Pathway-A Novel Component of Oncogene Induced DNA Damage Response Barrier in Breast Tumorigenesis

Supporting information figures and tables. Figure S1, Cyclin E levels in MCF10A parental (P) or cyclin E over expressing stable cell clones {MCF10A CyE (B10) and MCF10A CyE (C3) (a) measured at RNA level by QPCR using primers that span cyclin E and Flag sequence thus show the levels of transfected flag tagged cyclin E in breast cells. (b) measured by immunofluorescence (IF) by using cyclin E antibody that picked up both the endogenous and transfected cyclin E levels. Figure S2, Cyclin E over expression does not cause cell death through apoptosis. (a) Bar diagram showing the % of cells undergoing early apoptosis, late apoptosis and necrosis in cyclin E over expressing cell clones {MCF10A CyE (B10) and MCF10A CyE (C3) as compared to MCF10A parental (P). 100,000 cells were plated in 6-well plate and next day cells were stained with Annexin V-FITC and PI and were sorted by flow cytometry. The values shown are mean + standard error obtained by repeating the cell cycle assay 3 times. Figure S3, Transient over expression of Cyclin E suppresses AKT-mTOR pathway in MCF10A parental (P) cells. (a) levels of indicated proteins were measured by western blotting. Table S1, P values for pair wise comparisons of the three groups. Table S2, The heterogeneity (e) and 95% CI of the biomarkers.
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补充信息附图与附表。
S1图：MCF10A亲本细胞（P）或过表达细胞周期蛋白E（Cyclin E）的稳定细胞株{MCF10A CyE (B10)与MCF10A CyE (C3)}中Cyclin E的表达水平。(a) 以覆盖Cyclin E与Flag标签序列的引物为扩增引物，通过实时定量PCR（QPCR）检测RNA水平的表达量，以此反映乳腺癌细胞中转染的Flag标记型Cyclin E的表达水平；(b) 使用可同时识别内源性与转染型Cyclin E的Cyclin E抗体，通过免疫荧光（IF）检测蛋白水平的Cyclin E表达量。
S2图：过表达Cyclin E不会通过凋亡途径诱导细胞死亡。(a) 柱状图对比了MCF10A亲本细胞（P）与过表达Cyclin E的细胞株{MCF10A CyE (B10)、MCF10A CyE (C3)}中早期凋亡、晚期凋亡及坏死细胞的占比。实验方法为：将1×10^5个细胞接种于6孔板中，次日采用膜联蛋白V-异硫氰酸荧光素（Annexin V-FITC）与碘化丙啶（PI）对细胞进行染色，通过流式细胞术进行分析；图中数值为重复3次实验所得的均值±标准误。
S3图：在MCF10A亲本细胞（P）中，Cyclin E的瞬时过表达可抑制AKT-mTOR信号通路。(a) 通过蛋白质印迹法检测目标蛋白的表达水平。
S1表：三组间两两比较的P值。
S2表：生物标志物的异质性（e）与95%置信区间（CI）。
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