Functional regulation of the Apurinic/apyrimidinic endonuclease APE1 by Nucleophosmin (NPM1): Impact on tumor biology
Carlo Vascotto*, Lisa Lirussi*, Mattia Poletto*, Mario Tiribelli#, Daniela Damiani#, Dora Fabbro*, Giuseppe Damante*, Bruce Demple§,
Emanuela Colombo@ and Gianluca Tell*
SUPPLEMENTARY INFORMATION
List of contents:
- Page 2 to 3, Supplementary Materials;
- Page 4 to 6, Supplementary Figure legends;
- Page 7 to 14, Supplementary Figures;
Supplementary Materials
Preparation of cell extracts
Cell nuclear and cytoplasmic extracts were prepared starting from 5x106 cells. After trypsinization, cells were collected, centrifuged at 250g for 5 min at 4°C. The supernatant was removed, and the pellet was washed once with ice-cold phosphate-buffered saline (PBS) and then centrifuged again. Then cells were resuspended in 100 L of Lysis Buffer A (10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM MgCl2, 0.1 mM EDTA pH 8.0, 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail). Nuclei were collected by centrifugation at 500 g for 15 min at 4°C. The supernatant was considered as the cytoplasmic fraction. Nuclei were then washed with the same volumes of Buffer A to minimize cytoplasmic contamination and collected as previously reported. Nuclear proteins were extracted with 100 L of Lysis Buffer B (10 mM Hepes pH 7.9, 400 mM NaCl, 1.5mM MgCl2, 0.1 mM EDTA pH 8.0, 0.5 mM PMSF and protease inhibitor cocktail). After incubating for 30 min at 4°C, samples were centrifuged at 19000 g for 20 min at 4°C. For preparation of total cell lysates cell pellets were resuspended at a cell density of 107 cells/mL in Lysis Buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM EDTA and 1% (vol/vol) Triton X-100 supplemented with 0.5 mM PMSF and protease inhibitor cocktail) and incubated for 30 min at 4°C. After centrifugation at 19000 g for 20 min at 4°C, the supernatant was collected as total cell lysate. Cytoplasmic, nuclear and total cell extracts were then stored at -20°C in aliquots.
Cell transfection
One day before transfection, HeLa cells were seeded in 6-multiwells plate at a density of 4.5x105 cells/plate. Cells were then transiently transfected with 2 g of plasmidic DNA per well, using 6 l of Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer’s instructions. Cells were harvested 24h after transfection. For reconstitution experiments MEF-NPM1-/- were seated in Petri dishes at a density of 3.5x106 cells/plate and transiently transfected with 12 g of plasmidic DNA per well, using 36 l of Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer’s instructions.
Protein quantification
A protein assay, based on the method of Bradford, was used for the accurate determination of the concentration of solubilized proteins in total, cytoplasmic and nuclear extracts. Comparison to a standard curve obtained with BSA provided relative measurement of protein concentration. Bio-Rad Protein Assay reagent was used in accordance with manufacture’s instruction (Biorad).
Western blotting analysis
The indicated amounts of cell extracts were electrophoresed onto a 12% SDS-PAGE. Proteins were then transferred to nitrocellulose membranes (Schleicher & Schuell). Membranes were saturated by incubation at RT for 1h with 5% (wt/vol) non fat dry milk in PBS–0.1% (vol/vol) Tween 20 and then incubated with the primary antibody (-APE1 (Novus) monoclonal, 3h at 25°C, dilution 1:2000; -NPM1 (Zymed) monoclonal, 3h at 25°C, dilution 1:1000;-LSD1 (Abcam) rabbit monoclonal, o/n at 4°C, dilution 1:5000;-Actin (Sigma) polyclonal, 2h at 25°C, dilution 1:2000; -Tubulin (Sigma) monoclonal, 2h at 25°C, dilution 1:2000). After three washes with PBS–0.1% (vol/vol) Tween 20, membranes were incubated with an -Mouse or -Rabbit antibody coupled to peroxidase (Sigma) for 2h at 25°C. Then, membranes were washed three times with PBS–0.1% (vol/vol) Tween 20 and blots were developed using the ECL enhanced chemiluminescence procedure (GE Healthcare). Blots were quantified by using a ChemiDoc XRS video densitometer (Bio-Rad).
Incision assay on abasic dsDNA
The determination of APE1 AP endonuclease activity was performed using an oligonucleotide cleavage assay. Reported amount of nuclear cell extracts (NCE) were incubated with 2.5 pmol of a 5’-32P-end-labeled 26-mer oligonucleotide containing a single tetrahydrofuranyl (THF) artificial AP site at position 14, which is cleaved to a 14-mer in the presence of AP endonuclease activity. NCE were incubated at 37°C for the reported time with radiolabeled THF oligonucleotide in presence of 50 mM HEPES, 50 mM KCl, 10 mM MgCl2, 1 g/L bovine serum albumin, and 0.05% (vol/vol) Triton X-100 in a final volume of 10 L. Reactions were halted by adding an equal volume of 96% (vol/vol) formamide and 10 mM EDTA, with xylene cyanol and bromophenol blue as dyes. AP assay products (5 L) were separated on a 20% polyacrylamide gel containing 7 M urea. Gels were wrapped in Saran wrap and exposed to film for autoradiography.
Supplementary Figure Legends
Supplementary Figure S1
Schematic representation of human NPM1 WT and c+ mutant primary structure and their functional domains.
Supplementary Figure S2
As reported in M&M section, MEF NPM1+/+ and NPM1-/- were processed to obtain total cell extracts (WCE), cytoplasmic (CCE) and nuclei (NCE) enriched fractions. 20 g of total cell extracts and 10 g of cytoplasmic and nuclei enriched fractions of each cell line were separated onto 12% SDS-PAGE and transferred to a nitrocellulose membrane. Western blotting analysis was carried out to evaluate the quality of the enrichment procedure. Tubulin and LSD1 were used as cytoplasmic and nuclear markers, respectively. Actin was used as loading control. Western blotting with an antibody -NPM1 confirmed the absence of the protein in MEF NPM1-/- cells.
Supplementary Figure S3
Reported amounts of nuclear cell extracts (NCE) from MEF-NPM1+/+ and NPM1-/- cells were separated in SDS-PAGE and transferred into a nitrocellulose membrane. APE1 amount present in the nuclear extract of the two cell lines was evaluated through Western blotting analysis using a specific monoclonal antibody -APE1. As loading control, an antibody -Actin was used. In the diagram below are reported the absolute chemiluminescence intensity of APE1 signal from MEF-NPM1+/+ and NPM1-/- NCE normalized for Actin. Absence of NPM1 leads to a significant increase (~40%) of nuclear APE1’s expression in NPM1-/- cells. Each value represents the mean±SD values of three independent experiments.
Supplementary Figure S4
MEF NPM1-/- cells were transiently transfected with pCMV5.1 empty vector (Empty) or a NPM1encoding plasmid (NPM1 rec). 48h after transfection cells were fixed with 4% PFA and immunofluorescence analysis was carried out using antibodies -APE1 and -NPM1 proteins as described on M&M section. Empty cells showed an uniform APE1 nuclear staining (red) without NPM1 positivity (green). On the contrary, in NPM1 expressing cells (NPM1 rec.) APE1 colocalizes with NPM1 within nucleoli. Images werecaptured using the same setting parameters (488nm laser at 36% of intensity and PMT1 at 800V; 543nm laser at 78% of intensity and PTM2 at 830V). Fluorescence intensity analysis was carried out on a 20m line (white line) and relative histograms showed an uniform fluorescence intensity of APE1 within the nuclear compartment in Empty cells (~140 average fluorescence arbitrary unit) (Upper panel). On the contrary, APE1 colocalization and accumulation with NPM1 within nucleolar structure was clearly visible in MEF NPM1-/- after re-expression of NPM1 (~190 average fluorescence arbitrary unit) (Lower panel).
Supplementary Figure S5
Absence of NPM1 negatively affect cell viability making MEF-NPM1-/- cells more sensitive to oxidative stress and DNA damage inducing agents. Panel A:Cell viability of MEF-NPM1+/+ and NPM1-/- cells was evaluated through MTS assay after treatment with increasing amount of H2O2 for 1h.Panel B:Survival cell number of cytotoxicity experiment of Fig. 1G is reported in this panel.
Supplementary Figure S6
PARP inhibitor PJ34 and UVC were used as DNA damaging agents that do not require BER to be repaired. In both cases MEF NPM1+/+ and NPM1-/- cell lines did not display differential sensitivity.Panel A:Cell viability of MEF-NPM1+/+ and NPM1-/- cells after treatment with PARP inhibitor PJ-34 was evaluated through WST-1 Cell Proliferation Reagent (Roche). 3x103 cells were seeded onto 96 wells plates and allowed to adhere for 24 hours. MEFs were then incubated with the indicated amounts of PJ-34 for 48h and then viability was measured following manufacturer’s instructions.Panel B:1 x 104 cells were seeded into 6 wells plates, 24 hours later cells were exposed to the indicated amounts of UVC light. Surviving cell fraction was evaluated after 48 hours through cell counting using a Cell Counter (Beckman).
Supplementary Figure S7
HeLa cell lines stably expressing a FLAG-tagged APE1 WT and N33 mutant have been used to verify the specificity of Proximity Ligation Assay. Cells grown on glass cover slips have been incubated with an -FLAG (FITC conjugated) primary antibody and an -NPM1 primary antibody. Then, PLA protocol has been followed as reported in M&M section. Technical control (Ctrl PLA), represented by the omission of NPM1 primary antibody, results in the complete loss of PLA signal. In the biological control, represented by the expression of N33APE1 form which lost its binding ability to NPM1 (17), no signal is present within the nucleus while a few dots are visible in correspondence of the cytosol. The analysis of APE1 WT expressing cells showed the accumulation of APE1 within the nucleoli and, as expected, the presence of several red dots in correspondence of this sub-nuclear compartment.
Supplementary Figure S8
Functional inhibition of APE1 in NPM1WT cells phenocopies the response of NPM1c+ to genotoxic damage, but does not increase the sensitivity of NPM1c+ cells. CellTiter Glo Luminescent Cell Viability assay was used to evaluate the sensitization of OCI/AML2 and OCI/AML3 cells to MMS after the functional inhibition of APE1, using specific APE1 DNA-repair inhibitors (Methoxyamine) or a redox inhibitor (E3330). Cells were pre-treated with the indicated amounts of the APE1 inhibitors for 16 hr at 37°C and then incubated with MMS (0.3mM) in the presence of APE1 inhibitors for further 8 h. The specific inhibition of the APE1 DNA-repair function sensitizes OCI/AML2 cells (expressing NPM1wt) to MMS-induces DNA damage, while no substantially increased sensitivity was observed for NPM1c+ cells. Mean±SD values are the result of three independent experiments.
PLA Movie
Starting from the top of the cell, 25 z-stack images of APE1 WT and N33 cells have been acquired and sequentially mounted. Nuclei are blue, PLA signals are represented by red dots, while green fluorescence indicates APE1 localization. To view the movie launch the Power Point presentation and click on the picture.
Supplementary Figure S1
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