Volume 17, Issue 3, Pages 463-470 (February 2005) A Vital Role for Ape1/Ref1 Protein in Repairing Spontaneous DNA Damage in Human Cells  Hua Fung, Bruce Demple  Molecular Cell  Volume 17, Issue 3, Pages 463-470 (February 2005) DOI: 10.1016/j.molcel.2004.12.029

Figure 1 Downregulation of Ape1 Expression by siRNA (A) Cells were infected with the indicated retroviral vectors and after 2 days subjected to selective medium containing puromycin. Abbreviations: Con, uninfected cells; H1, H2, and H7, APE1-specific siRNA vectors; LUC, luciferase-specific siRNA vector; RevLUC, retrovirus expressing luciferase; and Apn1, transfection with the yeast APN1 gene (see text). After an additional 5 days (HCT116 cells), 10 days (MCF7 cells), or 6 days (TK6 cells), cell samples were subjected to immunoblotting with an Ape1-specific monoclonal antibody, which was quantified by phosphorimaging and normalized to immunoblotting for β-actin (bar graphs). (B) Cell-free extracts of HCT116 cells infected with the retroviral vector H1 as for (A) were assayed for AP endonuclease activity (top, the 12-mer indicates the AP endonuclease cleavage product). Three independent assays were performed across a range of protein concentrations and quantified by phosphorimaging (bottom). Abbreviations: BSA, bovine serum albumin (negative control); Ape1, purified recombinant Ape1 protein. Standard deviations are shown, and an asterisk indicates significant difference from the uninfected control, with p < 0.05. Molecular Cell 2005 17, 463-470DOI: (10.1016/j.molcel.2004.12.029)

Figure 2 Effect of Ape1 Downregulation on Cell Proliferation and Outgrowth of Ape1-Expressing Cells Cells were infected with siRNA retroviral vectors (APE1-specific H1 for HCT116 cells and H2 for MCF7 cells; LUC, luciferase-specific; and control, uninfected cells), incubated 2 days in normal growth medium, then incubated in selective medium containing puromycin a further 5 days (HCT116) or 8 days (MCF7). At various times thereafter (starting at 1d), the cultures were sampled to assay for Ape1 expression by immunoblotting ([A and E]; UT, untreated) or for quantification of cell numbers (B, C, D, F, and G). (A–D), HCT116 cells. (E–G), MCF7 cells. The results are from three independent experiments. Error bars correspond to standard deviations, and an asterisk indicates significant difference from control or LUC-infected cells, with p < 0.05. Suppression of Ape1 levels after activation of an inducible siRNA vector ([H], top panels show immunoblotting and quantitation versus days of inducing treatment) was accompanied by suppressed cell proliferation in U2OS-derived cells ([H], bottom). The Ape1 level shown is relative to day 0. Molecular Cell 2005 17, 463-470DOI: (10.1016/j.molcel.2004.12.029)

Figure 3 Apn1 Expression Rescues Ape1-Deficient Human Cells (A) HCT116 cells were transfected with the APN1 expression vector (or the empty vector as a control), incubated 2 days in nonselective medium, infected with the indicated siRNA retrovirus, and incubated 14 days in double-selective medium containing both G418 (for the APN1 vector) and puromycin (for the retroviral vectors). Samples were analyzed by immunoblotting for Ape1, Apn1, or β-actin as a control. Abbreviations: YE, yeast whole-cell extract (50 μg); siAPE1, APE1-specific H1 siRNA retrovirus; siLUC, luciferase-specific siRNA retrovirus. (B) Cultures grown in double-selective medium as described in (A) were replated at low density and incubated an additional 14 days for single colonies, which were isolated by using cloning cylinders and then expanded another 10 days in double-selective medium. Expression of Ape1 and Apn1 was detected by immunoblotting and normalized to β-actin. The APN1-transfected clones that expressed Apn1 protein are indicated in the last column. (C) Cell proliferation was quantified in three clones isolated as described in (B). Control, HCT116 cells infected with the control (LUC) retroviral vector. The error bars correspond to standard deviations (n = 3). Molecular Cell 2005 17, 463-470DOI: (10.1016/j.molcel.2004.12.029)

Figure 4 Apoptosis and Abasic DNA Damage in Ape1-Deficient Cells (A and B) Cells were transiently transfected with the APN1 expression vector and in vitro-generated siRNA precursor transcripts as indicated; the cells were cotransfected with a vector encoding membrane bound green fluorescent protein (mGFP). At the indicated times after transfection, samples were analyzed for DNA content by flow cytometry with gating for mGFP-positive cells. (A) sub-G1 cells in HCT116. (B) sub-G1 cells in MCF7. (C) HCT116 cells infected with control (LUC, a–c) or APE1-specific (d–f) siRNA retrovirus vectors were subjected for 5 days to selective medium containing puromycin then analyzed by immunostaining for Ape1 or DAPI staining for DNA by using a protocol that retains both apoptotic and nonapoptotic cells. The arrows in (d) and (f) indicate cells with low Ape1 content coincident with DNA condensation. (D) TK6 cells infected with siRNA vector H1 and APN1 expression vector as indicated, analyzed for sub-G1 DNA content. CMV-LUC, luciferase-expressing vector. (E) Cleavage of poly(ADP-ribose) polymerase (PARP) in TK6 cells infected with H1 siRNA retrovirus and cultured 3 days in nonselective medium. Analysis by immunoblotting. Days after infection are indicated by 2d, 3d, and 4d. 116 KD and 85 KD denote, respectively, intact and caspase-cleaved PARP. (F) HCT116 cells transfected with the APN1 or control expression vector and infected with the indicated siRNA retrovirus vectors were incubated 5 days in selective medium containing puromycin and G418, followed by the isolation of chromosomal DNA, which was then analyzed for abasic damage by using an aldehyde-reactive probe. (G) Abasic DNA damage in TK6 cells infected with APE1-specific siRNA or luciferase-specific siRNA (siRNA-LUC), or coinfected with the retroviral Apn1 or LUC expression vectors. The flow cytometry (A, B, and D) and abasic damage (F and G) data are based on at least three independent experiments. Standard deviations are shown, and an asterisk indicates significant difference from the control, with p < 0.05. Molecular Cell 2005 17, 463-470DOI: (10.1016/j.molcel.2004.12.029)