Human NSCLC cell lines A549, H1299 and PC-9 from ATCC were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycinneomycin (PSN) Antibiotic Mixture (Invitrogen, USA) at 37°C in a humidified 5% CO₂/95% air environment.

For the yeast two-hybrid assay, the bait plasmid pGBKT7-SIRT3 was generated by inserting a PCR-amplified cDNA fragment containing SIRT3 into pGBKT7 (Clontech). SIRT3 was cloned into pBIND (Promega), pEGFP-C1 (Clontech), pGEX-2T vector (GE). Human full-length NMNAT2 cDNA and the cDNAs for its truncated mutants (NMNAT2 N and NMNAT2 C1–2) were PCR-amplified and subcloned in-frame into pCMV-5a (Sigma). NMNAT2 was cloned into pACT (Promega), pDsRed1-N1 (Clontech), pCDNA-3.1(−) myc/his (A) vector (Invitrogen). All plasmids were verified by restriction enzyme analysis and DNA sequencing. The hSIRT3 adenovirus was from Vector BioLabs (Philadelphia, PA, USA).

At 24–30 h after seeding, NSCLC cells were used for adenovirus infection or immunostaining as described below. The adenoviruses were used at a multiplicity of infection (MOI) of 10. Cos7 and HeLa cells were grown in Dulbecco’s modified Eagle’s medium supplemented with penicillin-streptomycin and 10% fetal bovine serum (complete growth medium). Cells were transfected with appropriate plasmids using Superfect transfection reagent (Qiagen) according to the manufacturer’s protocol. For the RNA interference experiments, NSCLC cells were transfected with 200 nM On-Target-plus small interfering RNA (siRNA) specific for hSIRT3 using Dharma-FECT transfection reagents according to the manufacturer’s protocol (11).

The bait plasmid pGBKT7-SIRT3 was used to screen a human lung cDNA library in pGADT7 according to the manufacturer’s instructions (Clontech). Transformants were placed on synthetic medium lacking tryptophan, leucine, adenine and histidine but containing 1 mM 3-aminotriazole. Approximately 4 million transformants were screened. The screened positive clones were also verified by one-on-one transformations and selection on agar plates lacking tryptophan and leucine, or adenine, histidine, tryptophan and leucine, respectively and were also processed by β-galactosidase assay.

For transfection-based co-immunoprecipitation assays, cells were transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen), lysed in 0.5 ml lysis buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 0.25% NP-40, 1 mM DTT and protease inhibitor tablets from Roche) and immunoprecipitated with Protein G Plus/Protein A Agarose Suspension beads (Calbiochem) for 3 h at 4°C. The beads were washed 4 times with the lysis buffer and eluted in SDS sample buffer. The eluted proteins were separated by SDS-PAGE, followed by western blotting with antibody.

To test the hypothesis of NMNAT2 interacting with SIRT3 in vivo using the CheckMate™ Mammalian Two-Hybrid system (Promega), plasmids pACT-NMNAT2 and plasmids pBIND-SIRT3 were constructed that were used for cotransfections of cells cultured in 6-well plates. Two micrograms of pACT-NMNAT2 plasmid and 2 μg of pBIND-SIRT3 plasmid were used for cotransfections by Lipofectamine 2000. The plasmids pACT-NMNAT2+pBIND, pACT+pBIND-SIRT3, pACT, pBIND and blank were used for transfections, respectively, as a negative control. The plasmids pBIND-Id and the pACT-MyoD Control Vector were cotransfectioned as a positive control. At 48 h after transfection, cells were washed 3 times with PBS and then lysed with passive buffer. Firely luciferase assays were performed using the Dual-Glo Luciferase assay system (Promega) following the manufacturer’s instructions. The firefly results were corrected for transfection efficiency using the renilla luciferase. Significance was determined using the paired Student’s t-test on the mean of three different experiments.

The GST- and His-tagged fusion proteins were expressed and purified by glutathione-Sepharose 4B beads (GE) and Ni-NTA agarose (GE), respectively. The expression plasmid for NMNAT2 was used for in vitro transcription and translation in the TNT system (Promega). The NMNAT2 or the purified His-tagged fusion protein was incubated with GST fusion protein bound to glutathione-Sepharose beads in 0.5 ml of the binding buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.3 mM DTT, 0.1% NP-40) at 4°C. The beads were precipitated, washed 4 times with the binding buffer, eluted by boiling in SDS sample buffer and analyzed by SDS-PAGE. Western blotting was performed with anti-His (Santa Cruz). A quantitative measurement of the band intensity was performed using the GE Typhoon Trio (GE, USA).

Cells were grown on glass coverslips in culture plates. Cells were co-transfected with plasmids, 2 μg of pEGFP-C1-SIRT3 (green fluorescent protein, GFP) and 2 μg of pDS-RED1-N1- NMNAT2 by Lipofectamine™ 2000 for 48 h and then cells were treated with 4% paraformaldehyde (PFA) for 10 min, washed 3 times with PBS, stained with PBS including 0.1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) for 30 min at 30°C. Confocal images were acquired using Zeiss 510 META confocal microscope.

Western blot analyses were prepared as described (12). NSCLC cell cultures were lysed and placed in RIPA buffer (Cell signaling) with 1 mM PMSF on ice for 30 min. Cell lysates were centrifuged at 14,000 g for 10 min and cell extracts were mixed with a 1:4 volume of SDS-PAGE loading buffer (10% β-mercaptoethanol, 10% glycerol, 4% SDS, 0.01% bromophenol blue and 62.5 mM Tris-HCl, pH 6.8) and heated to 65°C for 15 min. Samples were loaded on a 10% resolving SDS-polyacrylamide gel and transferred to polyvinyldifluoridine membranes. Membranes were incubated overnight at 4°C with rabbit polyclonal anti-SIRT3 (1:500; Abcam), NMNAT2 antibody (Santa Cruz, 1:1,000 dilution), β-actin (Santa Curz, 1:1,000), GST (Santa Cruz, 1:1,000), Flag (Santa Cruz, 1:1,000), His (Santa Cruz, 1:1,000), SIRT3 (Cell Signaling, 1:1,000), Myc (Santa Cruz, 1:1,000). All affinity-purified and species-specific fluorophore-conjugated secondary antibodies were obtained from Santa Cruz and used at dilutions between 1:500 and 1:800. Immunoreactivity was detected with luminol reagent (GE).

Unlabeled Flag-NMNAT2 was in vitro translated using the TNT coupled transcription-translation rabbit reticulocyte lysate kit (Promega) and immunoprecipitated using anti-Flag M2 affinity beads. Beads with bound protein were washed 4–5 times with radioimmunoprecipitation assay buffer followed by a phosphate-buffered saline (PBS) wash. The final wash was performed in 1X HAT buffer (50 mM Tris, pH 8.0, 10% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol). A typical acetylation reaction mixture contained 1 μg active p300/CBP-associated factor (PCAF) enzyme (Upstate Biotechnology), 0.3 mM acetyl coenzyme A (Sigma) and 10 mM sodium butyrate in 1X HAT buffer. Reaction mixtures were incubated at 30°C for 2 h on a rotator. For the deacetylation assay, acetylated Flag-NMNAT2 that bound to the beads was washed as described above and resuspended in 1X HDAC buffer (50 mM Tris, pH 8.0, 4 mM MgCl₂, 0.2 mM dithiothreitol). The acetylated Flag-NMNAT2 substrate was either incubated only in 1X HDAC buffer (control) or with equal amounts of Myc-SIRT3 with or without NAD (1 mM) in 1X HDAC buffer. Myc-SIRT3 catalytic was produced in a prokaryotic expression system as described (15). Reaction mixtures were incubated for 2–3 h at 37°C on a rotator. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by western blotting (11).

Steady state cell respiration in cells was measured in non-buffered DMEM containing 5.5 mM glucose for cells with XF^e24 analyzer (Seahorse Bioscience) according to the manual.

Steady state cellular ATP levels were measured by using ATP bioluminescence assay kit CLS II in accordance with the protocol (Roche). The optimal detection range is between 10⁻⁷ and 10⁻¹⁰ M. The pH of the sample should be in the range of 7.6–8.0. ATP standard was diluted with redist water by serial dilution in the range of 10⁻⁵ and 10⁻¹⁰ M. Luciferase reagent was added to the samples/standards by automated injection and the measurement was started after a 1-sec delay and integrated for 1–10 sec. The blank (no ATP) was subtracted from the raw data and ATP concentrations calculated from a log-log plot of the standard curve data.

NAD assay was performed as previously described (16–18). Cells were extracted in 0.5 N HClO₄, neutralized with 3 M KOH/125 mM Gly-Gly buffer (pH 7.4) and centrifuged at 10,000 × g for 5 min. Supernatants were mixed with a reaction medium containing 0.1 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), 0.9 mM phenazine methosulfate, 13 U/ml alcohol dehydrogenase, 100 mM nicotinamide and 5.7% ethanol in 61 mM Gly-Gly buffer (pH 7.4). The A560 nm was determined immediately and after 10 min and results were calibrated with NAD standards.

To avoid potential carry-over effects of plasmids transfection-induced cell cycle defects in the previous cycle on the following mitotic entry during the next cycle, we transfected plasmids into cells during the interval between two thymidine blocks, so that we were able to evaluate direct impact of interaction of NMNAT2 with SIRT3 on mitotic entry (13). Cells were synchronized by double thymidine block. Briefly, cells were plated at 40% confluency and arrested with 2 mM thymidine. After 19-h incubation, cells were washed 4 times with fresh medium and treated with siRNA (NMNAT2 and control). After incubation with DNA-lipid mixture for 3 h, cells were washed twice and incubated in fresh medium for an additional 5 h. Subsequently, cells were cultured in medium containing 2 mM thymidine and 2 μg/ml puromycin for the second arrest and drug selection. After 16-h incubation, cells were released into the cell cycle by incubation in fresh medium. Cells were collected or fixed at indicated time-points and subjected to specific analyses.

Following release from the second thymidine arrest at indicated time-points, cells grown in 12-well plate were pulse labeled with BrdU (50 μM) for 30 min. After three washes of PBS, cells were fixed with 1 ml of Carnoy’s fixative (3 parts methano 1:1 part glacial acetic acid) at −20°C for 20 min and followed by three washes of PBS. Subsequently, DNA was denatured by incubation of 2M HCl at 37°C for 60 min, followed by three washes in borate buffer (0.1 M borate buffer, pH 8.5). After incubation with the blocking buffer, cells were stained with anti-BrdU antibody (BD Biosciences, 1:100) overnight at 4°C. After washes in PBS, cells were incubated with Texas Red-conjugated anti-mouse goat IgG for 30 min at RT. After washes, cells were mounted and BrdU positive cells were manually scored under immunofluorescence microscope.

Mitotic events were scored by time-lapse videomicroscopy and DNA staining. Cells were synchronized as described above. Real-time images were captured every 10 min with Openlab software. Mitotic events of cells were scored by their morphological change (from flat to round-up). For each experiment, ≥800 cells were videotaped, tracked and analyzed. Alternatively, nocodazole (100 ng/ml) was added into the medium after release, cells were collected, fixed and stained with DNA dye (Hoechst 33258). Mitotic cells were scored by nuclear morphology and DNA condensation.

Cell growth for 48 h was determined by the colorimetric tetrazolium derived XTT (sodium 3′-(1-(phenylaminocarbonyl)-3, 4-tetrazolium)-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) assay (Roche Applied Science, Mannheim, Germany) and DNA synthesis of cells was assessed by the BrdU (bromodeoxyuridine) incorporation assay (Roche Applied Science). For the cell growth and proliferation assay, at 48 h after culture, the cells of each group were re-seeded in SWNHs-coated 96-well plates at a density of 0.3–1×10⁴ cells per well. After 48 h, XTT and incorporated BrdU were measured colorimetrically using a microtiter plate reader (Bio-Rad) at a wavelength of 450 nm (14).

Cell viability was determined using a CCK-8 cell viability assay kit (Dojindo Laboratories, Japan). The cells (5×10³ cells/well) were pretreated with various methods as indicated and then incubated for 16 h in a 96-well plate. Cell viability assay kit solution (10 μl) was added to each well of the plate. After incubation for 1 h at 37°C in the dark, absorbances were measured at 450 nm using a multi-well plate reader (15).

Apoptotic cells were identified by fluorescence-activated cell sorting (FACS) using Annexin V-Fluos (Biolegend) following the protocol of the manufacturer.

Continuous normally distributed variables are presented as the mean ± standard deviation (SD). For statistical comparison of quantitative data between groups, analysis of variance (ANOVA) or t-test was performed. To determine differences between groups not normally distributed, medians were compared using Kruskal-Wallis ANOVA. χ² test was used when necessary for qualitative data. The degree of association between variables was assessed using Spearman’s non-parametric correlation. All statistical analyses were carried out using SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA). Probability values of ≤0.05 or less were considered to be statistically significant.

To identify novel SIRT3-interacting proteins, we performed a yeast two-hybrid screening of a human lung cDNA library using full-length SIRT3 as bait. Screening of 4 million transformants resulted in the isolation of several positive clones, which were identified to encode the full-length cDNA of NMNAT2 gene. To further identify the interaction of NMNAT2 with SIRT3 in yeast, we next examined the ability of SIRT3 protein to bind to NMNAT2 in yeast cells (Fig. 1A). These data demonstrated that SIRT3 interacts with NMNAT2 in yeast cells.

To identify the interaction between SIRT3 and NMNAT2, we next examined the ability of SIRT3 protein to bind to NMNAT2 in mammalian cells. Cells were co-transfected with Flag-tagged SIRT3, Myc -tagged NMNAT2, or Myc control vector. Immunoprecipitation (IP) of cell lysates with an anti-Flag monoclonal antibody was followed by immunoblotting (IB) with anti-Myc. Results showed specific interaction between the Flag-SIRT3 and Myc-NMNAT2 (Fig. 1B). To test the hypothesis of SIRT3 interacting with NMNAT2 in vivo, the CheckMate™ Mammalian Two-Hybrid system (Promega) was used. The plasmids pACT-SIRT3 and pBIND-NMNAT2 were used for cotransfections by Lipofectamine 2000. The plasmids pACT-SIRT3+pBIND, pACT+pBIND-NMNAT2, pACT, pBIND and blank were used for transfections, respectively, as a negative control. The plasmids pBIND-Id and the pACT-MyoD Control Vector were cotransfected as a positive control. After transfection for 48 h, cells were lysed. Luciferase assays were performed using the Dual-Glo Luciferase assay system (Promega) following the manufacturer’s instructions. The results were corrected for transfection efficiency using renilla luciferase. Significance was determined using the paired Student’s t-test on the mean of three different experiments. The luciferase levels of pACT-SIRT3 and pBIND-NMNAT2 were ≥2.64 times higher than negative control (P<0.05) (Fig. 1C). The expression of SIRT3 and NMNAT2 in lysate of those cells was detected by WB with anti-SIRT3 and anti-NMNAT2 monoclonal antibody (Fig. 1D). To demonstrate the interaction of SIRT3 and NMNAT2 in vitro, GST pull-down assays were performed in which in vitro translated His-SIRT3 was incubated with full-length GST-NMNAT2 or Flag. As shown in Fig. 1E, SIRT3 interacted with GST-NMNAT2 but not with Flag alone (Fig. 1E). To test the colocalization of SIRT3 and NMNAT2 in cells, cells were grown on glass coverslips in culture plates then co-transfected with plasmids pEGFP-C1-SIRT3 and pDS-RED1-N1-NMNAT2. After 48 h, cells were stained with PFA and DAPI, confocal images were acquired using Zeiss 510 META confocal microscope. NMNAT2 (red, Fig. 1F) and SIRT3 (green, Fig. 1G) protein, all localized to the cytoplasma. The nuclear of cells (blue, Fig. 1H) were stained by DAPI. The overlaid images indicated that SIRT3 overlapped partly with NMNAT2 (Fig. 1I) in the cytoplasma. These results indicate that SIRT3 interacted with NMNAT2 in vivo and in vitro.

To identify the region of SIRT3 required for its interaction with NMNAT2, Co-IP experiments were performed in which three deletion mutants. SIRT3 is cleaved by caspase-3 at two sites (D28 and D107, Fig. 2A). As shown in Fig. 2A, both full-length and the carboxy-terminal fragments of SIRT3 C1 protein interacted with Myc-NMNAT2, but the amino-terminal fragment of SIRT3 (SIRT3N, residues 1–28) did not interact with NMNAT2 (Fig. 2A). As shown in Fig. 2B, the Flag-SIRT3 C1 (29–307) bound specifically to NMNAT2, but the Flag-N (1–28) and FLAG-SIRT3 C2 (107–307) did not, suggesting that the 28–107 region is required for the interaction with NMNAT2. Among C-terminal cleavage fragments, SIRT3-C1 appeared to bear the highest affinity to Myc-NMNAT2, indicating that the region between residues 28–107 may be important for SIRT3-NMNAT2 interaction (Fig. 2B). We also tested the NMNAT2-SIRT3 interaction in vitro using purified SIRT3 proteins which were in agreement with our Co-IP results.

To test whether SIRT3 deacelylated NMNAT2, in an acetylation buffer Flag-NMNAT2 was incubated with PCAF. Acetylation of the protein was determined by western blotting with antiacetyllysine antibody (Fig. 3A). Flag-NMNAT2 was acetylated in vitro with PACF and it was precipitated with Flag M2 beads. Acetylated Flag-NMNAT2 was then incubated with beads containing SIRT3 in a deacetylation buffer with or without NAD. SIRT3 was immunoprecipitated from stable A549 cells. This indicated that SIRT3 deacetylated of NMNAT2 in vitro is dependent on the NAD level (Fig. 3B and C). Stable cells expressing SIRT3 were induced to overexpress with Flag-NMNAT2 and treated with NAM (10 mM for 24 h) and/or TSA (5 μM for 6 h) as indicated. Flag-NMNAT2 was immunoprecipitated and the level of acetylation was analyzed by probing with anti-acetyllysine antibody. It indicated that in vivo SIRT3 deacetylated NMNAT2 dependent on the TSA and NAM levels, especially related to TSA (Fig. 3D and E). Together, these data demonstrated that SIRT3 targets the enzyme NMNAT2, which catalyzes the formation of NAD (+) from nicotinamide mononucleotide (NMN) and ATP.

To study the role interaction between SIRT3 and NMNAT2 on mitochondrial function, intact cellular basal oxygen consumption rates (OCR) of A549 cells were measured by Seahorse XF24 analyzer. The OCR of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 increased significantly compared to control (P<0.01), whereas, the OCR of A549 cells decreased significantly with SIRT3 interference (P<0.01) (Fig. 4A). Steady-state cellular ATP levels of A549 cells were also measured. The steady-state cellular ATP level of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 increased significantly compared to the A549 cells. The OCR of A549 cells decreased significantly (P<0.01) (Fig. 4B) with downregulation of SIRT3. There was significant expression of SIRT3 in NSCLC cell line A549. Adenovirus infection caused significant increase of SIRT3 in NSCLC cell lines. Transfection with SIRT1 siRNA caused reduction of protein after transfection (Fig. 4C). This suggested that SIRT1 siRNA selectively silences SIRT3 expression. NAD level of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 increased significantly compared to A549 cells. Downregulation of SIRT3, the OCR of A549 cells decreased significantly (P<0.01) (Fig. 5). The result showed that the interaction between NMNAT2 and SIRT3 promotes mitochondrial function levels of A549 cells.

To investigate the role of interaction between NMNAT2 and SIRT3 on mitotic entry, cell growth and proliferation, A549 cells were transfected with plasmids and synchronized at the G1/S transition as described in Materials and methods. Cells were pulse labeled with BrdU (50 μM) for 30 min at indicated time-points after release from the second thymidine block. BrdU positive cells were detected by immunostaining and scored manually. More than 500 cells were counted in each of three independent experiments (Fig. 6A). Cell cycle progression of >1,000 cells was recorded by time-lapse videomicroscopy. The number of mitotic cells was scored by examination of individual cells (Fig. 6B). The results demonstrated that the interaction of NMNAT2 with SIRT3 promoted mitotic entry (Fig. 6). By XTT assays, we investigated the effect of the interaction between NMNAT2 and SIRT3 on cell growth and found that the growth of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was more significant than in A549 cells (P<0.001). The growth of cells were significantly inhibited by interfering SIRT3 (P<0.001) (Fig. 7A). Cell viability was evaluated by CCK-8 assay. The result showed that the proliferation of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was more significantly than in A549 cells (P<0.001). The growth of cells were significantly inhibited by interfering SIRT3 (P<0.001) (Fig. 7B). Our results indicated that the interaction of NMNAT2 with SIRT3 did not affect DNA synthesis, but low expression of SIRT3 significantly inhibited mitotic entry, growth and proliferation of A549 cells.

Our result showed that the apoptosis of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was less than A549 cells (P>0.05). The apoptosis of cells were significantly promoted by interfering SIRT3 (P<0.001). This indicated that the interaction of NMNAT2 with SIRTs inhibited apoptosis of A549 cells (Fig. 8). Low expression of SIRT3 significantly promoted A549 cell apoptosis (the results from H1299 and PC-9 cells were similar to those from A549 cells).