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. 2016 Mar 10;7(16):22590–22604. doi: 10.18632/oncotarget.8026

Figure 6. APE1 N-terminal domain or its acetylation regulates expression of multiple genes and essential for sustained cell proliferation.

Figure 6

A. Heat Maps (Bioconductor/R; Limma) generated from micro-array based GeneChip analysis shows differential gene expression profile of control siRNA, APE1-siRNA 2 (80nM), TSA-treated control and TSA-treated APE1 siRNA-transfected A549 cells (p =0.001). Three independent biological replicates of each group are represented. Color intensities correspond to relative signal levels as measures of mRNA expression on a logarithmic scale. B. Real Time RT-PCR analysis showing steady-state mRNA level in relative quantitation (RQ) with respect to HPRT1 of randomly selected cell proliferation-related APE1/AcAPE1-regulated genes in control vs. APE1-siRNA1 and APE1-siRNA2 knockdown A549 cells. Error bars denote SD (N=2-3). C. Heat Maps (Bioconductor/R; Limma) generated from micro-array based GeneChip analysis shows differential gene expression profile of ectopic WT, NΔ42 and K6R/K7R (RR) mutant APE1-expressing BEAS-2B cells (p = 0.00001). Three independent biological replicates of each group are represented. D. Real Time RT-PCR analysis showing steady-state mRNA level in relative quantitation (RQ) with respect to HPRT1 of randomly selected cell proliferation-related APE1/AcAPE1-regulated genes in ectopic WT vs. RR APE1 and WT vs. NΔ42 APE1 expressing BEAS-2B cells. Error bars denote SD (N=2-3) E. Endogenous APE1 was downregulated with doxycycline (Dox) treatment in HEK293T cell line stably expressing APE1 siRNA from a doxycycline-inducible promoter HEK293TAPE1siRNA. Approximately 500 cells were plated on 60-mm dishes and allowed to grow for two weeks until visible colonies appear. The number of viable colonies was counted in cells in which endogenous APE1 was downregulated and in which FLAG-tagged WT APE1 or acetylation defective (mutations of Lys6,7,27,31&32 to non-acetylable arginine (K5R) or glutamine (K5Q) or N-terminal deletion mutants was ectopically expressed(APE1 levels in cell extracts are shown in inset) Error bars denote SD (N=3). F. Soft-agar assay for anchorage-independent growth of BEAS-2B cells with stably expressing ectopic WT or N-terminal 42 aa deleted (NΔ42) mutant APE1.