Titles and legends to supplemental figures

Supplementary Movie 1 Alteration in the localization of AChES during apoptosis. HeLa cells were transfected with the plasmids expressing (A) AChES-GFP (green) or (B) Tubulin-GFP control protein (green). After 24 h, cells were treated with 100 μM H2O2. Images were analyzed by a Leica AS MDW live cell image acquisition system. Frames were taken every 2 min for 290 min. The experiments were repeated twice with similar results.

Supplementary Figure 1. Subcellular location of the AChE proteins during apoptosis. (A) Confocal images showing endogenous AChE and activated caspase-3 in HeLa and PC-12 cells treated with or without H2O2 (50 µM) for 24 h, using the antibodies against AChE (N-19) (Santa Cruz, sc-6431) and cleaved caspase-3 (ASP175) (Cell Signaling, #9661), respectively. (B) Cells from each group mentioned in (A) were collected and subjected to nuclear-cytoplasmic separation followed by immunoblot analysis. Nuclear and cytoplasmic fractions were identified with antibodies against histone H3 (Santa Cruz, sc-8654) and α-tubulin (Santa Cruz, sc-5286), respectively.

Supplementary Figure 2.hAChE-T547 cleaves other plasmid DNA and naked genomic DNA isolated from HeLa cells. (A)Agarose gel electrophoresis images showing three DNA plasmids (150 ng) incubated with hAChE-T547 (0.5 μM) at 37°C for 6 h. EcoRI-digested DNA (0.5 U/μL)was used to demarcate the linearized plasmid.(B)Images of genomic DNA (150 ng) incubated with hAChE-T547 (0.5 μM) and three restriction enzymes (0.5 U/μL) at 37°C for 6 h.(C)hAChE-T547 digested genomic DNA in a dose-dependent manner. The genomic DNA (150 ng) was incubated with hAChE-T547 at the indicated concentration at 37°C for 6 h. (D)Images of genomic DNA (150 ng) digested by hAChE-T547 (0.5 μM) at 37°C for the indicated time-periods.(E) Images showing degradation of genomic DNA (150–600 ng) by 0.5 μM hAChE-T547 at 37°C for 6 h.(F)hAChE-T547 cleaves linearized plasmids into smears in a dose-dependent manner. pcDNA 3.1 (150 ng) was incubated with hAChE-T547 at the indicated concentration at 37°C for 6 h.

Supplementary Figure 3. mAChE-T548 digests DNA in vitro.(A) Silver staining and Western blot analysis showing the purified mAChE-T548; Lanes 2 and 2’.(B)Agarose gel images of pEGFP-c1 plasmid DNA (150 ng) incubated with mAChE-T548 at the indicated concentration at 37°C for 6 h. 0.5 U/μL EcoRI-digested DNA was used to demarcate the linearized plasmid.(C) Agarose gel images of pEGFP-c1 plasmid DNA (150 ng) incubated with 2 μM mAChE-T548 at 37°C for the indicated time-periods. EcoRI-digested DNA (0.5 U/μL) was used to demarcate the linearized plasmid, and plasmid incubated with 2 μM BSA was used as a negative control. (D) Agarose gel images showing other plasmids (150 ng) of different sizes incubated with mAChE-T548 (3 μM) at 37°C for 6 h. (E) Images showing naked genomic DNA (150 ng) incubated with mAChE-T548 at the indicated concentration at 37°C for 6 h. The DNA incubated with EcoRI or BSA was used as a control.

Supplementary Figure 4. Thecholinesterase active center of AChES is neither relevant to its DNA cleavage activity nor to its pro-apoptotic function. (A)AChE inhibitors inhibit cholinesterase activity of AChES polypeptide.mAChE-T548 (3 μM) was pre-incubated with or without AChE inhibitors (10 μM huperzine A, 1 μM tacrine and 13 μM donepezil) at 37°C for 5 min. Acetylcholinesterase activity was then assayed by the Ellman method, with measurement of the change in OD 405 nm. Iso-OMPA (13 μM), a specific inhibitor of BChE, was used as a negative control. Data are presented as the mean of triplicate samples from the representative one of three independent experiments. (B) AChE inhibitors fail to affect the DNA cleavage activity of mAChE-T548. "+" and "-"mean the hydrolysis system was pre-treated with or without the indicated AChE inhibitors.Data shown are representative of three independent experiments.(C) Western blot analysis of fusion protein expression. HeLa cells were transfected with the NLS-AChES-GFP expression plasmid as a control or the pEGFP-NLS-mtAChES (S234A, E365A, H478A) plasmid. At 24 h after transfection, cells were harvested. A whole-cell extract was prepared, resolved by SDS-PAGE, and blotted with anti-GFP antibody. Lane 1, NLS-AChES-GFP; Lane 2, NLS-mtAChES-GFP. (D) Ellman assay for cholinesterase activity of NLS-mtAChES-GFP. GFP and NLS-AChES-GFP were used as the controls. (E) Growth or apoptosis curve of GFP-positive HeLa cells expressing the indicated proteins during 72 h after sorting. The relative number of cells at the indicated time-points was determined by MTT assay. Data are presented as the mean ± S.D. of triplicate samples from one representative of three independent experiments. (F) Inverted phase-contrast fluorescence microscopy images showing the morphology of GFP-positive HeLa cells at 72 h after sorting. Scale bar, 30 μm.(G)Confocal images showing TUNEL staining in HeLa cells over-expressing the indicated proteins for 48 h. Cells over-expressing GFP were used as a negative control. Scale bar, 10 μm.

Supplementary Figure 5. Analysis of the DNase activity of the synthesized AChE peptides. (A) Analysis of the purity of the synthesized hAChE-T107 using Dionex Summit HPLC (Dionex, USA). Conditions: Column, PLRP-S 100A (5 μm) 250 × 4.6 mm (Varian, USA); Mobile phase, A = 0.1% TFA in 100% water and B = 0.1% TFA in 100% acetonitrile; Gradient, 25% ACN-65% ACN in 25 min; Volume, 5 uL; Wavelength, 220 nm; Flow rate, 1.0 mL/min. The peak corresponding to hAChE-T107 is indicated by *. (B) Analysis of the purity of the synthesized hAChE-T41 using Dionex Summit HPLC. Conditions: Column, 250 × 4.6 mm Venusil XBP-C18 (Agela, USA); Mobile phase, A = 0.1%TFA in 100% water and B = 0.1%TFA in 100% acetonitrile; Gradient, 32% ACN-57% ACN in 25 min; Volume, 5 uL; Wavelength, 220 nm; Flow rate, 1.0 mL/min. The peak corresponding to hAChE-T41 is indicated by #. Peak No., Peak number; Ret Time, Retention time; Conc., Concentration.(C) Agarose gel images showing genomic DNA (150 ng) digested by hAChE-T107 at the indicated concentration at 37°C for 6 h.The DNA (150 ng) incubated with hAChE-T41 (10 μM) was used as a negative control.(D) Images of genomic DNA (150 ng) digested by 10 μM hAChE-T107 for the indicated time-periods. (E) hAChE-T107 cleaves linearized plasmids into smears at a higher dose. pcDNA 3.1 (150 ng) was incubated with hAChE-T547 at the indicated concentration at 37°C for 6 h.

Supplementary Figure 6. Nuclear localized AChES cleaved genomic DNA in a caspase- and EndoG-independent manner. HeLa cells were treated with 150 μM Z-VAD-FMK or 150 μM Z-FA-FMK (negative control of Z-VAD-FMK) at 37°C for 1 h prior to transfection with pEGFP-NLS-AChES. After 48 h, immunofluorescence assays were performed. (A) Inverted phase-contrast fluorescent microscopy images showing the blockage of caspase-3 activation by Z-VAD-FMK. Scale bar, 60 μm. (B) Confocal microscopy images showing the blockage of Endo G nuclear translocation by Z-VAD-FMK. Scale bar, 10 μm.(C) Analysis of the average fluorescence intensity of TUNEL staining in (b) by Leica Confocal Software. TUNEL staining in the nuclei of randomly selected GFP-positive cells (n = 50) in each group was quantified. (Z-FA-FMK)-treated HeLa cells expressing NLS-AChES-GFP were used as the control group. Values represent the mean ± S.D. ** P < 0.01 vs. the control group. Two-tailed Student’s t-test. Data are representative of two independent experiments.

Supplementary Figure 7.Endogenous AChES participates in DNA cleavage via aa 32–138 during apoptosis. AChE+/+ and AChE-/- MEFs were infected by lentivirus expressing the indicated proteins. After 48 h, cells were treated with 40 µM MMC for 36 h, and TUNEL staining assay was then performed. Scale bar, 150 μm. The experiments were repeated twice with similar results.

Supplementary Figure 8. Established HeLa stable cell lines. (A) Immunoblotting assay for protein expression in HeLa cells stably expressing the indicated proteins. Lane 1, AChES-GFP, used as a control; Lane 2, NLS-AChES Δ (aa 32–138)-GFP. (B) Inverted phase-contrast fluorescence microscopy showing stable cells expressing the indicated proteins. Scale bar, 60 μm.(C) Stereo fluorescence microscopy (Olympus Corporation; Tokyo, Japan) showing GFP-positive subcutaneous tumors in Balb/c nude mice formed by the indicated HeLa stable cell lines. Scale bar, 2 mm.

Supplementary Figure 9. Nuclear translocation of endogenous AChES in mouse or cellular models of AD.(A) Identification of B6C3-Tg (APPswe, PSEN1dE9) 85Dbo/J mice by reverse transcription PCR. 1, PSEN1dE9; 2, APPswe. (B) Serial tissue sections of the brains from the indicated mouse showing the distribution of AChE protein. Upper panel: Congo red (Sigma-Aldrich) staining for Aβ amyloid in the brain section of the indicated 1-year-old mice. Scale bars, 15 μm. Middle panel: AChE staining in serial brain sections. Lower panel: In the detailed image, the areas indicated by the arrow head in the middle panel are magnified 3×. (C) Confocal images showing the changes in AChES protein distribution and TUNEL staining in isolated primary hippocampal neurons exposed to amyloid β-protein fragment 25–35 (Aβ (25–35)) or its sequence-reversed peptide (35–25) (Sigma-Aldrich, USA) for 18 h. In the detailed images, cells indicated by the white arrow on the left are magnified 2.5× on the right in form of separated channels. Scale bars, 25 μm in the left panel, 10 μm in the right panel. These experiments were repeated three times with similar results.

1