Int. J. Mol. Sci. 2013, 14(3), 5633-5649; doi:10.3390/ijms14035633

Article
Global Gene Expression Profiling Reveals Functional Importance of Sirt2 in Endothelial Cells under Oxidative Stress
Junni Liu 1,2, Xiao Wu 1, Xi Wang 1,2, Yun Zhang 1, Peili Bu 2,*, Qunye Zhang 1,* and Fan Jiang 1,*
1
Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Public Health, Shandong University, Jinan 250012, Shandong, China; E-Mails: liujunni2012sdu@163.com (J.L.); wuxiao1022@126.com (X.Wu.); wang_xi@126.com (X.Wa.); zhangyun@sdu.edu.cn (Y.Z.)
2
Department of Cardiology, Qilu Hospital, Shandong University, Jinan 250012, Shandong, China
*
Authors to whom correspondence should be addressed; E-Mails: bupeili@medmail.com.cn (P.B.); maogou1974@gmail.com (Q.Z.); fjiang@sdu.edu.cn (F.J.); Tel.: +86-531-8216-9267 (F.J.); Fax: +86-531-8616-9356 (F.J.).
Received: 19 December 2012; in revised form: 22 February 2013 / Accepted: 28 February 2013 /
Published: 11 March 2013

Abstract

: The NAD+-dependent deacetylases Sirt1 and Sirt2 mediate cellular stress responses and are highly expressed in vascular endothelial cells. In contrast to the well-documented protective actions of Sirt1, the role of endothelial Sirt2 remains unknown. Using cDNA microarray and PCR validation, we examined global gene expression changes in response to Sirt2 knock down in primary human umbilical vein endothelial cells under oxidative stress. We found that Sirt2 knock down changed expression of 340 genes, which are mainly involved in cellular processes including actin binding, cellular amino acid metabolic process, transmembrane receptor protein serine/threonine kinase signaling, ferrous iron transport, protein transport and localization, cell morphogenesis, and functions associated with endosome membrane and the trans-Golgi network. These genes and associated functions were largely non-overlapping with those altered by Sirt1 knock down. Moreover, we showed that pharmacological inhibition of Sirt2 attenuated oxidant-induced cell toxicity in endothelial cells. These suggest that Sirt2 is functionally important in endothelial cells under oxidative stress, and may have a primarily distinct role as compared to Sirt1. Our results may provide a basis for future studies aiming to dissect the specific signaling pathway(s) that mediates specific Sirt2 functions in endothelial cells.
Keywords:
Sirt1; Sirt2; endothelial cell; oxidative stress; functional genomics; microarray

1. Introduction

Mammalian Sirt proteins (Sirt1 to Sirt7) are orthologues of the yeast SIR2 gene product, an NAD+-dependent class III histone deacetylase [14]. All Sirt proteins contain a conserved NAD+-dependent catalytic core domain of ~275 amino acids [3,4]. Among the seven Sirt proteins identified, Sirt1, 2, and 3 have the highest homology to yeast Sir2 and all exhibit specific protein deacetylase activity [5,6]. In addition to their important roles in aging and metabolic regulation, different studies have suggested that Sirt is also involved in modulating cardiovascular physiology and disease [1,7]. In the heart, for example, Sirt1 and Sirt3 have been implicated in promoting cardiomyocyte survival and preventing cardiac remodeling in response to different stress stimuli [8]. In blood vessels, activation of Sirt functions, especially those of Sirt1, is associated with multiple beneficial effects such as preventing vascular cell senescence, suppressing inflammation, decreasing cellular oxidative stress, and promoting vascular regeneration [1,7]. Moreover, Sirt may also exert cardiovascular protective actions by improving global glucose and lipid metabolism [8].

Endothelial cells have a pivotal role in maintaining the homeostasis of blood vessels. Endothelial dysfunction is recognized as a major cellular basis of the development of many cardiovascular diseases such as hypertension, atherosclerosis and heart failure [9]. Several lines of evidence have indicated that Sirt1 has an important role in modulating endothelial cell functions. In particular, Sirt1 physically interacts with and deacetylates endothelial nitric oxide synthase (eNOS), leading to enhanced eNOS activation [10]. Both in vitro and in vivo experiments revealed that activation of Sirt1 function led to increases in nitric oxide production and endothelium-dependent vasorelaxation, decreased inflammatory reactions in endothelial cells, and suppressed endothelial cell apoptosis and senescence [1,1114].

Several Sirt members have pivotal roles in modulating cellular stress responses [2,6]. Under oxidative stress, Sirt1 exhibited broad cytoprotective effects in endothelial cells [7,1519]. The molecular mechanisms by which Sirt1 produces these cytoprotective actions are not totally understood, while current evidence indicates that activation of the FoxO family members by Sirt1 is likely to be a major signaling route [2,6]. In contrast to Sirt1, the biological functions of Sirt2 in endothelial cells remain unknown [1]. Results from previous studies in non-endothelial cells indicate that the effects of Sirt2 on cell viability under stress are variable and appear to be cell type- and context-dependent [2027]. Currently, research efforts have been made in the development of selective Sirt2 inhibitors, which may be used as novel chemotherapy agents [28]. Hence, it is important to determine whether and how Sirt2 is involved in modulating endothelial cell homeostasis under stress conditions. Like Sirt1, Sirt2 expression is also responsive to oxidative stress [24]. Moreover, Sirt2 and Sirt1 share a number of common substrates, including FoxO1, FoxO3, nuclear factor (NF)-κB, histone H3 and p300 [4,8,24,2932]. These results prompted us to hypothesize that Sirt2 may also have critical functions in endothelial cells under oxidative stress. Therefore, in the present study we aim to examine the importance of endothelial Sirt2 on a systematic biology basis, by characterizing global gene expression changes after Sirt2 knock down in primary human umbilical vein endothelial cells (HUVECs) using mRNA microarray, an approach that has been used to delineate Sirt1 functions at the whole genome level [33,34].

2. Results and Discussion

To induce oxidative stress in cultured endothelial cells, we treated the cells with H2O2 at 300 μM for 6 h. Induction of a cellular stress response under this condition was demonstrated by the upregulation of detoxification enzymes glutathione peroxidase (GPx) and superoxide dismutases (SOD) as measured by qPCR (Figure 1A). We also confirmed that this treatment protocol did not induce obvious cytotoxic effects as assessed by a MTS cell viability assay (Figure 1B). A previous study demonstrated that Sirt2 expression was upregulated upon oxidant stimulation in adipocytes [24]. To clarify whether this is also the case in endothelial cells, we measured Sirt2 expression with qPCR in H2O2 challenged cells. We found that as compared to untreated cells, H2O2 increased Sirt2 expression by ~2 fold (data not shown). To demonstrate the intracellular localization of Sirt2 and in endothelial cells, we performed immunofluorescence staining. As shown in Figure 1C, the majority of Sirt2 showed a cytosolic localization, which was in contrast to Sirt1, which was mainly nuclear.

To clarify the functions of Sirt2 in endothelial cells at the genome level, we performed microarray experiments comparing the global gene expression profiles between control and Sirt2i cells. The efficiency of siRNA-mediated gene knock down was confirmed by qPCR and Western blot (Figure 2). We showed that under oxidative stress conditions, knock down of Sirt2 significantly changed the expression level of 340 genes, with 152 being upregulated and 188 downregulated (Figure 3A and Table 1). GO analysis of the Sirt2-sensitive genes showed that these genes were mainly involved in cellular processes related to actin binding, cellular amino acid metabolic process, transmembrane receptor protein serine/threonine kinase signaling pathways, ferrous iron transport, protein transport and localization, cell morphogenesis involved in differentiation, and functions associated with endosome membrane and the trans-Golgi network.

To confirm that the altered gene expression after Sirt2 knock down was not caused by non-specific off target effects, we run a parallel experiment by knocking down Sirt1 using the same protocol. Sirt1 gene knock down induced significant alterations of expression of 162 genes (87 upregulated and 75 downregulated) (Figure 3B and Table 1). Among the upregulated genes with Sirt1i, only 31 (36%) overlapped with those changed in Sirt2i cells (20% of those changed in Sirt2i cells). Similarly, among the downregulated genes, only 4 (5%) overlapped with those changed in Sirt2i cells (2% of those changed in Sirt2i cells) (Figure 3C). GO analysis of the Sirt1-sensitive genes showed that these genes were mainly involved in cellular processes related to actin binding, ion binding, endoplasmic reticulum, cellular macromolecule biosynthetic process, cytoskeletal protein binding, and Golgi apparatus, of which the majority was distinct from those related to Sirt2. Further analysis of the differentially expressed genes in relation to disease processes with IPA software revealed that Sirt2-sensitive genes were enriched in categories including infectious disease, connective tissue disorders, developmental disorder, skeletal and muscular disorders, and cardiovascular disease. In comparison, Sirt1-sensitive genes were mainly enriched in categories including cardiovascular disease, inflammatory response, cancer, organismal injury and abnormalities, and connective tissue disorders. We also compared the two sets of cellular pathways significantly over-represented by Sirt1- or Sirt2-sensitive genes respectively, and found that the pathways affected by Sirt1 were primarily distinct from those affected by Sirt2 (Figure 4A). Moreover, IPA-Tox analysis revealed that Sirt1i and Sirt2i exhibited a discrete pattern of gene enrichment in categories of biological mechanisms that were related to toxicity responses (Figure 4B).

To validate our microarray data of Sirt2 effects on global gene expression, we carried out qPCR assays on selected genes including CALD1, CASP7, CNN2, RRAGC, ULBP2. We showed that Sirt2i induced upregulation CALD1, CASP7, CNN2 and downregulation of RRAGC, ULBP2 (Figure 5A). These changes were in accordance with the trend as detected by microarray (see Table 1). In contrast, expressions of these genes were not altered in Sirt1i cells (Figure 5B). To further clarify whether Sirt2 was functionally important in endothelial cells under stress, we treated HUVEC cells with a higher concentration (600 μM) of H2O2 for 2 h in the absence and presence of a selective Sirt2 inhibitor AGK2 (from Merck, Darmstadt, Germany) [35]. We found that pre-treatment with AGK2 (10 μM) attenuated H2O2-induced cell toxicity (Figure 6A), suggesting that under oxidative stress, activation of the Sirt2 pathway might have a detrimental effect on cell viability. In contrast, we showed that pre-treatment with the selective Sirt1 inhibitor EX-527 (10 μM) (from Merck) increased H2O2-induced cell toxicity (Figure 6B).

In the present study, we explored the functional importance of Sirt2 in endothelial cells under oxidative stress by measuring global gene expression changes in cells in which Sirt2 was knocked down. We found that Sirt2 gene knock down significantly altered the expression profile of 340 genes, which were involved in different cellular processes (see Table 1). We also confirmed the microarray data with qPCR for selected genes. Gene clustering analysis suggests that Sirt2-sensitive genes in endothelial cells may be involved in regulation of protein transport and localization, cellular amino acid metabolic process, and functions associated with endosome membrane and the trans-Golgi network. These functional annotations are in agreement with findings from cellular function studies showing that Sirt2 may have a pivotal role in modulating cell autophagy, an intracellular mechanism responsible for clearance of damaged proteins and organelles involving activation and mobilization of the endogenous membranous system [36]. Interestingly, a recent study demonstrated that overexpression of Sirt2 inhibited lysosome-mediated autophagic turnover and increased the sensitivity of cells to proteasomal stress-induced cytotoxicity [37]. Conversely, accumulation of ubiquitinated proteins and cytotoxicity in stressed cells were attenuated by Sirt2 knock down. These results indicate that a complex interaction between Sirt2 and autophagic process may be present. In line with these findings, we observed that pharmacological inhibition of autophagy in endothelial cells augmented H2O2-induced cell death [38]. Moreover, we found that inhibition of Sirt2 decreased H2O2-induced endothelial cytotoxicity (see below). Taken together, we propose that regulation of cellular autophagic processes might be a mechanistic link between Sirt2 and oxidative stress-induced injury in endothelial cells. In addition to the above-mentioned pathways, results from our gene function clustering analysis indicate that Sirt2-regularted genes may also be involved in actin binding, transmembrane receptor protein serine/threonine kinase signaling pathways, ferrous iron transport, and cell morphogenesis involved in differentiation.

Similar to Sirt1, Sirt2 has strong deacetylase activity, and may affect gene expression by modulating functions of multiple transcription factors and co-activators such as FoxO, NF-κB, p300, and histone [46,23,24,2932]. However, the present gene profiling study showed that the potential intracellular pathways regulated by Sirt2 in stressed endothelial cells were primarily different from those regulated by Sirt1. Consistently, Sirt1- and Sirt2-sensitive genes were involved in distinct categories of diseases, for example inflammatory response, cancer, and organismal injury for Sirt2, and infectious disease, developmental disorder, skeletal and muscular disorders for Sirt1. As observed in neural cells [39], our data suggest that Sirt2 is likely to have a distinct functional role from Sirt1 in endothelial cells under stress conditions. Moreover, these data support that the observed gene expression changes in response to Sirt2 knock down are unlikely to be a result of non-specific off target effects of RNA interference.

The precise cellular functions of Sirt2 in endothelial cells remain largely unknown. A previous study has shown that Sirt2 may be implicated in mediating angiotensin II-induced endothelial cell migration via modulating α-tubulin acetylation and microtubule reorganization [40]. Consistent with this observation, we identified (and confirmed with qPCR) that Sirt2 knock down altered the expression of several genes involved in cytoskeletal organization, cell contraction and migration, such as CALD1 (caldesmon) and CNN2 (calponin) [41,42]. Moreover, we demonstrated that Sirt2 also affected expression of genes involved in modulating cell viability. This is exemplified by CASP7 (caspase 7), which is a master regulator of cell apoptosis, and RRAGC (Ras-related GTP binding C), which is involved in activation of the mTOR pathway [43].

To clarify the general role of Sirt2 in endothelial cells under oxidative stress, we challenged the cells with a high concentration of H2O2 and demonstrated that pharmacological inhibition of Sirt2 activity attenuated H2O2-induced cytotoxicity. This result is consistent with previous experiments in neural and cardiac cells showing that activation of Sirt2 promotes cell death, whereas knock down or inhibition of Sirt2 enhances cellular stress-tolerance [25,35]. Moreover, we confirmed that inhibitions of Sirt2 and Sirt1 had divergent effects on endothelial cell viability under H2O2-induced oxidative stress, an observation that was consistent with our microarray data revealing that there was only a small intersection between Sirt2- and Sirt1-sensitive genes in H2O2-challenged endothelial cells. Our experiments supported previous findings that Sirt1 exhibited profound cytoprotective effects in vascular endothelial cells in response to oxidative stress [7,15,16].

3. Experimental Section

3.1. Cell Culture

HUVECs were purchased from the American Type Culture Collection and maintained in ECM (from ScienCell Research Labortories, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum, 1% ECG (endothelial cell growth supplement, ScienCell), and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL). Cells were cultured at 37 °C with 5% CO2. Confluent cells were subcultured with 0.25% trypsin-EDTA, and cells of passage 3 to 5 were used for experimentation. Cell viability was assessed with the tetrazolium-based (MTS) assay using CellTiter 96 Aqueous kit (from Promega, Madison, WI, USA) according to the manufacturer’s direction.

3.2. RNA Interference

Small interfering RNA (siRNA) molecules targeting Sirt1 and Sirt2 were synthesized by GenePharma (Shanghai, China). For each target, 3 different siRNA sequences were tested with quantitative polymerase chain reaction (qPCR), and the one with highest efficacy was selected for following experiments. For siRNA transfection, cells were subcultured 24 h before treatment. Cells were incubated with siRNA (final concentration 30 nM) mixed with Lipofectamin RNAiMAX Reagent (Life Technologies, Carlsbad, CA, USA) for 6 h in antibiotic-free medium, and then changed to normal medium for additional 18 h.

3.3. Microarray Experiments and Data Processing

Cells were transfected with a control siRNA, Sirt2-specific siRNA (Sirt2i) or Sirt1-specific siRNA (Sirt1i). Three biological replicates were included for each group (hence a total of 9 arrays were analyzed). To induce oxidative stress, all transfected cells were treated with H2O2 at 300 μM for 6 h. Total RNA was isolated using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA quality was tested with Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) and further purified with RNeasy Micro kit (Qiagen, Hilden, Germany). Microarray analysis was performed using Affymetrix Human Genome U219 Array, using standard labeling, hybridization and scanning protocols (ShanghaiBio Corporation, China). The raw data were processed and analyzed with GeneSpring GX software. Genes with a fold change of >1.5 and with a p value of <0.05 as compared to control were selected as differentially expressed genes. Gene Ontology (GO) functional annotation of the differentially regulated genes was carried out using DAVID Bioinformatics Resources 6.7 [44]. Further gene function clustering analysis was performed with IPA software (Ingenuity Systems, Redwood City, CA, USA).

3.4. Real-Time qPCR

Total RNA (500 ng) was reverse transcribed to cDNA using Prime Script RT reagent Kit (TaKaRa Biotechnology, Dalian, China). Real-time qPCR was performed with TaqMan gene expression assays primer-probe sets (Applied Biosystems, Carlsbad, CA, USA) or using a Sybr green-based master mix kit (SsoFast EvaGreen from Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. GAPDH or 18S was used as the housekeeping gene.

3.5. Fluorescent Immunocytochemistry

Cells grown on Lab-Tek II chamber slides (Nunc, Roskilde, Denmark) were fixed with cold methanol for 30 min, washed in PBS and blocked with 5% bovine serum albumin. Cells were incubated overnight with polyclonal anti-Sirt1 (1:200) (from Abcam, Cambridge, UK) or anti-Sirt2 (1:100) (from Millipore, Billerica, MA, USA). Immunofluorescent labeling was performed with DyLight594-conjugated donkey anti-rabbit IgG (1:400) (Jackson ImmunoResearch, West Grove, PA, USA). Cell nuclei were counter stained with DAPI. Images were captured using a Zeiss laser scanning confocal microscope (Zeiss LSM710, Oberkochen, Germany). Negative control experiments were performed using corresponding non-immune IgGs.

3.6. Western Blot

Total protein was resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membrane was blocked with 5% non-fat milk at room temperature for 1 h and then incubated with primary antibodies at 4 °C overnight. The blots were developed with ECL Prime reagents from GE Life Sciences (Piscataway, NJ, USA).

3.7. Data and Statistics

Microarray data were tested with Benjamini and Hochberg False Discovery Rate multiple testing correction. Other data were presented as mean ± SEM and tested with unpaired Student’s t-test or one-way ANOVA as appropriate, with a value of p < 0.05 being regarded as statistically significant. SPSS18.0 was used for statistical analysis.

4. Conclusions

In conclusion, to our knowledge this is the first genome-wide characterization of the gene expression profile in response to Sirt2 knockdown in endothelial cells. Sirt2-sensitive genes are involved in multiple cellular functions. Pharmacological inhibition of Sirt2 attenuated oxidant-induced endothelial cell death. These data suggest that Sirt2 is functionally important in endothelial cells under oxidative stress. Our results may provide a basis for future studies aiming to dissect the specific signaling pathway(s) that mediates specific Sirt2 functions in endothelial cells. Nevertheless, a limitation of the present study was that the microarray data did not provide direct evidence about the specific gene products that were involved in mediating the observed effects of Sirt2. Given the number of genes that are responsive to the changed Sirt2 level, it is likely that multiple mechanisms may be implicated in each specific biological function of Sirt2.

Acknowledgments

This research was partially supported by grants from the National 973 Basic Research Program of China (2010CB732605 for F.J.; 2012CB722406 for P.B.), National Natural Science Foundation of China (81070164 for F.J.; 81070076 for P.B.), and Shandong University graduate student independent innovation fund (21300070613085 for J.L.).

Abbreviations

eNOSnitric oxide synthase
NF-κBnuclear factor-κB
HUVEChuman umbilical vein endothelial cell
siRNAsmall interfering RNA
qPCRquantitative polymerase chain reaction
GOGene Ontology
GPxglutathione peroxidase
SODsuperoxide dismutase
  • Conflict of InterestThe authors declare no conflict of interest.

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Figure 1. (A) An oxidative stress response induced by H2O2 (300 μM for 6 h) in cultured human umbilical vein endothelial cells (HUVECs), as revealed by the upregulation of glutathione peroxidase (GPx) and superoxide dismutases (SOD) as measured by qPCR; (B) H2O2 treatment at 300 μM did not result in obvious cytotoxicity in the present experimental system. Cell viability was assessed with a MTS-based assay. Data are mean ± SEM, n = 3–4; (C) pseudo-colored immunofluorescence images showing the intracellular localization of Sirt1 and Sirt2 in untreated HUVEC. Nuclei were counter stained with DAPI (blue). Bar = 20 μm. MERG, merged image.

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Figure 1. (A) An oxidative stress response induced by H2O2 (300 μM for 6 h) in cultured human umbilical vein endothelial cells (HUVECs), as revealed by the upregulation of glutathione peroxidase (GPx) and superoxide dismutases (SOD) as measured by qPCR; (B) H2O2 treatment at 300 μM did not result in obvious cytotoxicity in the present experimental system. Cell viability was assessed with a MTS-based assay. Data are mean ± SEM, n = 3–4; (C) pseudo-colored immunofluorescence images showing the intracellular localization of Sirt1 and Sirt2 in untreated HUVEC. Nuclei were counter stained with DAPI (blue). Bar = 20 μm. MERG, merged image.
Ijms 14 05633f1 1024
Ijms 14 05633f2 200
Figure 2. (A) qPCR and (B) Western blot results showing gene silencing efficiency of siRNA sequences targeting Sirt1 or Sirt2 in H2O2-treated HUVECs. A non-specific siRNA was used as control. Data are mean ± SEM. * p < 0.05, student’s t-test, n = 4–5. Western blots were representative images from two independent experiments.

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Figure 2. (A) qPCR and (B) Western blot results showing gene silencing efficiency of siRNA sequences targeting Sirt1 or Sirt2 in H2O2-treated HUVECs. A non-specific siRNA was used as control. Data are mean ± SEM. * p < 0.05, student’s t-test, n = 4–5. Western blots were representative images from two independent experiments.
Ijms 14 05633f2 1024
Ijms 14 05633f3a 200Ijms 14 05633f3b 200
Figure 3. Heat map diagrams illustrating the significantly changed (p < 0.05 with a fold change value >1.5 as compared to control cells) genes in HUVECs with gene silencing of (A) Sirt2 and (B) Sirt1, determined by Affymetrix Human Genome U219 Array (n = 3 biological replicates each); (C) Venn graphs showing the number of genes up- and downregulated by Sirt1 or Sirt2 gene silencing. A high-resolution version for Figure 3A,B is available online.

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Figure 3. Heat map diagrams illustrating the significantly changed (p < 0.05 with a fold change value >1.5 as compared to control cells) genes in HUVECs with gene silencing of (A) Sirt2 and (B) Sirt1, determined by Affymetrix Human Genome U219 Array (n = 3 biological replicates each); (C) Venn graphs showing the number of genes up- and downregulated by Sirt1 or Sirt2 gene silencing. A high-resolution version for Figure 3A,B is available online.
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Figure 4. Comparison of the potential categories of (A) intracellular pathways and (B) biological mechanisms related to toxicity responses that were significantly over-represented by Sirt1- or Sirt2-regulated genes respectively in stressed endothelial cells. The red line indicates the threshold of statistical significance. Functional gene enrichment analysis was performed with IPA software.

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Figure 4. Comparison of the potential categories of (A) intracellular pathways and (B) biological mechanisms related to toxicity responses that were significantly over-represented by Sirt1- or Sirt2-regulated genes respectively in stressed endothelial cells. The red line indicates the threshold of statistical significance. Functional gene enrichment analysis was performed with IPA software.
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Figure 5. Validation of microarray results with qPCR. The expression levels of CALD1, CASP7, CNN2, RRAGC, ULBP2 were measured in (A) Sirt2i cells and (B) Sirt1i cells in the presence of oxidant stress (H2O2 300 μM for 6 h). Gene expression levels were expressed as fold of control. Data are mean ± SEM. *p < 0.05 vs. Con, Student’s t-test, n = 3–4.

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Figure 5. Validation of microarray results with qPCR. The expression levels of CALD1, CASP7, CNN2, RRAGC, ULBP2 were measured in (A) Sirt2i cells and (B) Sirt1i cells in the presence of oxidant stress (H2O2 300 μM for 6 h). Gene expression levels were expressed as fold of control. Data are mean ± SEM. *p < 0.05 vs. Con, Student’s t-test, n = 3–4.
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Figure 6. Effects of (A) selective Sirt2 inhibitor AGK2 (10 μM) and (B) selective Sirt1 inhibitor EX-527 (10 μM) on H2O2-induced cell toxicity in HUVECs measured with MTS assay. Cells were treated with H2O2 (600 μM) for 2 h in the presence and absence of AGK2 or EX-527 pre-treatment. Data are mean ± SEM. *p < 0.05, one-way ANOVA, n = 4–6. NS: non-significant.

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Figure 6. Effects of (A) selective Sirt2 inhibitor AGK2 (10 μM) and (B) selective Sirt1 inhibitor EX-527 (10 μM) on H2O2-induced cell toxicity in HUVECs measured with MTS assay. Cells were treated with H2O2 (600 μM) for 2 h in the presence and absence of AGK2 or EX-527 pre-treatment. Data are mean ± SEM. *p < 0.05, one-way ANOVA, n = 4–6. NS: non-significant.
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Table 1. List of differentially expressed genes after Sirt1 or Sirt2 gene silencing (read the entire table column-wise).

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Table 1. List of differentially expressed genes after Sirt1 or Sirt2 gene silencing (read the entire table column-wise).
Sirt1
11716338_a_atUINSIG1
11716395_a_atUGPR56
11718915_a_atURGS19
11719218_atUSOCS3
11719513_a_atUADAM15
11719745_s_atUARHGAP27
11720832_x_atUSOX18
11722324_a_atUZNF84
11722353_s_atULDB2
11724390_x_atUZDHHC16
11724394_atUC2orf55
11724395_a_atUZNF83
11724396_x_atUZNF83
11727984_atUODZ3
11728347_atUABI3
11729918_atUADAMTS18
11729919_a_atUADAMTS18
11730211_x_atUPFKL
11731622_x_atUZNF91
11733299_a_atUCLDN5
11736013_atUGLE1
11736029_a_atUITGA6
11736458_x_atUKCNK6
11737089_a_atUTLL1
11737870_s_atUFAM78A
11739146_a_atUMKNK2
11739491_a_atUMGAT4A
11739492_a_atUMGAT4A
11739493_atUMGAT4A
11739650_atUDLL4
11740601_a_atUAPH1B
11722277_s_atUZBED4
11722388_atUMTF1
11722391_atUMTF1
11722531_a_atUCALD1
11722532_s_atUCALD1
11722533_x_atUCALD1
11723092_atUFNIP2
11723414_a_atUCNN2
11723416_x_atUCNN2
11724389_a_atUZDHHC16
11725054_a_atUHNMT
11726328_x_atUGBP1
11726796_a_atUNEXN
11726797_x_atUNEXN
11726824_a_atUZNF565
11727545_atUPANK3
11727784_x_atUTPM4
11728028_a_atUPWWP2A
11728226_a_atUCASP7
11728276_s_atUPLA2G12A
11728488_a_atUNRXN3
11728744_atUC4orf46
11729665_a_atUSTK38L
11730613_atUMBOAT7
11731665_a_atUPDGFB
11732339_atUBCL11A
11733043_a_atUSLC7A2
11733084_a_atUPALLD
11734281_a_atUZNF17
11734865_a_atUPSMC3IP
11736812_atUPDCD1LG2
11736959_a_atUTNFSF15
11724786_s_atDC14orf129
11725510_a_atDSLC7A6
11725569_atDKLHL15
11725596_a_atDZDHHC23
11725733_a_atDNFYA
11725850_atDUSP48
11726455_x_atDMYO5B
11726551_s_atDRAET1L
11726552_x_atDULBP2
11727222_atDEVI5
11727286_a_atDZNF323
11727361_a_atDMYLK
11727406_a_atDTEK
11727485_atDTPR
11727854_s_atDNUP50
11727905_a_atDIL13RA1
11727984_atDODZ3
11728195_s_atDTRO
11728288_a_atDKRT15
11728353_atDMSMP
11728960_a_atDKRT80
11729449_s_atDTINF2
11729550_a_atDGPR126
11729840_s_atDZCCHC2
11730033_a_atDRPS6KL1
11730195_atDSEC14L2
11730615_a_atDPLCD4
11730616_atDPLCD4
11730623_atDDSEL
11732160_a_atDNUDCD1
11732713_atDFST
11733051_a_atDSCAMP1
11740602_s_atUAPH1B
11740624_a_atUAZI1
11742830_a_atUPHF3
11744239_a_atUCCDC159
11744430_a_atUKIF9
11744505_x_atUTP53I11
11744948_x_atUSEMA3F
11745450_a_atUAUH
11745927_x_atUADAM15
11746516_a_atUADAM15
11747060_a_atUMGAT4A
11747977_a_atUZNF180
11748731_a_atUZNF616
11749511_a_atUZNF595
11751946_a_atUARHGAP21
11752002_a_atUPLEKHG1
11752675_a_atUOGFR
11754022_s_atUGAP43
11754446_x_atUZNF761
11754754_s_atUADAMTS18
11755219_a_atUTHBD
11755232_s_atUZNF468
11755474_a_atUADAM15
11757861_a_atUTSHZ1
11760814_x_atUKANK3
11715679_s_atDPRNP
11715889_a_atDATP5I
11715890_x_atDATP5I
11716027_atDSELT
11716028_x_atDSELT
11716404_s_atDEIF4EBP2
11718102_atDCD59
11718141_atDDKK1
11718769_a_atDMAPKSP1
11719164_a_atDCLCN5
11719267_s_atDSERINC1
11719394_a_atDFBXO32
11719395_atDEXTL2
11719396_a_atDEXTL2
11719479_atDALOX5AP
11719712_atDPPM1B
11719816_s_atDBET1
11720240_atDTMSB15A
11720514_atDC9orf150
11721024_a_atDIL11RA
11721112_a_atDACP6
11739010_a_atUMYH9
11739086_x_atUMESDC2
11739088_atUMESDC2
11739451_a_atUFRMD6
11739672_x_atUZNF253
11740133_a_atUCALD1
11740743_a_atUTPM2
11740744_x_atUTPM2
11741168_a_atUMSRB3
11741188_a_atUSLC30A6
11742483_a_atUC1orf110
11743015_a_atUDIP2C
11743020_atUZSCAN29
11743253_x_atUCALD1
11743458_a_atUFAM49A
11743696_atUCLEC14A
11743705_atUETAA1
11744034_a_atUVASP
11744323_s_atUPWWP2A
11745608_a_atUWDR1
11745924_atULOC220930
11746173_a_atUDDHD1
11746476_x_atUCALD1
11746548_s_atUCNN2
11747300_a_atUCDK14
11747469_x_atUKDM2B
11747711_a_atULDB1
11748208_a_atUZNF317
11748391_x_atUZDHHC16
11748400_s_atULOC643634
11748401_x_atUTPM4
11748403_x_atUCNN2
11748527_a_atUARHGAP24
11749172_x_atUNEXN
11749732_a_atULONP2
11749734_s_atUJRKL
11749921_a_atUSDC4
11750198_a_atUCASP7
11750623_a_atUFILIP1L
11751244_s_atUCNN2
11751245_x_atUCNN2
11751993_a_atUJAG1
11752164_x_atUKDM2B
11752276_a_atUDIDO1
11752361_s_atUNEXN
11752499_a_atUCALD1
11733054_a_atDSCAMP1
11733929_a_atDARMC8
11734059_a_atDPSTK
11734150_x_atDPLAGL1
11735224_a_atDKLRG1
11735991_atDLARS
11736192_atDRRM2B
11736343_x_atDOPA3
11736345_x_atDOPA3
11736528_a_atDSMC2
11736785_atDHOXA2
11737052_x_atDPLAGL1
11737816_x_atDFAM119B
11739064_s_atDGNG12
11739245_a_atDANKFY1
11739596_a_atDKIAA1429
11739640_atDDIP2A
11739942_s_atDSEPSECS
11740096_a_atDTMCC1
11740176_atDARSK
11740213_a_atDTBL1X
11741152_x_atDPLAGL1
11742720_atDLRRC58
11742722_atDLRRC58
11742962_a_atDIP6K2
11743404_atDZMAT2
11743573_atDTMEM184C
11743574_x_atDTMEM184C
11743648_a_atDDCAF6
11743649_a_atDDCAF6
11743763_atDGTF3C3
11744083_atDANKIB1
11744415_s_atDMFSD6
11744788_x_atDTMEM68
11745010_a_atDDCBLD2
11745230_a_atDC3orf23
11745231_a_atDC3orf23
11745700_s_atDULBP2
11746163_a_atDLARP4B
11746536_a_atDWSB2
11747146_s_atDTMBIM6
11748416_a_atDDCAF6
11749027_x_atDHERPUD2
11750354_a_atDTMEM184C
11750993_x_atDMAP3K7
11751165_a_atDRBMS2
11722843_a_atDENAH
11723533_x_atDBRMS1L
11723534_atDBRMS1L
11723580_atDLOC221710
11724238_atDCYB5R4
11726140_s_atDSIRT1
11726750_a_atDGTF3C4
11727022_atDTMEM64
11727370_atDTSN
11727935_atDC4orf49
11729128_atDCPA4
11729710_a_atDMARS2
11731195_atDSEMA3D
11731263_a_atDZNF365
11734371_a_atDSCML1
11734955_a_atDSCML1
11736470_atDSLC35D1
11738893_s_atDTPM1
11739119_s_atDCNPY3
11742308_s_atDTPM1
11742483_a_atDC1orf110
11742743_a_atDCNN3
11743092_atDTHEM4
11743197_atDTLR4
11743334_a_atDMRPL35
11743973_a_atDMRPL38
11743974_atDMRPL38
11745482_s_atDPRNP
11746622_a_atDPHC1
11746928_a_atDENC1
11747834_a_atDSPATA5
11748315_s_atDPRNP
11750985_a_atDEXTL2
11751191_a_atDLIPG
11752333_a_atDITGAV
11754940_s_atDTSN
11754976_x_atDCNPY3
11755458_a_atDHDDC2
11755848_a_atDC17orf51
11756471_a_atDMFSD2A
11756882_a_atDRTTN
11757738_s_atDFAT1
11758013_s_atDC8orf4
11758101_s_atDEIF4EBP2
11758326_s_atDTHEM4
11758872_atDCDC37L1
11752930_a_atUGBP1
11754084_x_atUMYL9
11754644_x_atUCNN2
11754887_a_atUMSRB3
11754911_x_atUNEXN
11755122_a_atUPALLD
11755734_x_atUCCDC107
11757637_a_atUMUS81
11758013_s_atUC8orf4
11759169_a_atUC1orf222
11759711_a_atURBM25
11760202_atUIGFBP7
11760611_x_atUSETD6
11760918_a_atUMRRF
11763500_a_atUZNF93
11715207_atDWDFY4
11715265_atDFIG4
11715477_atDTFRC
11715545_atDTMED10
11715550_atDDAG1
11715651_s_atDFSTL1
11715761_a_atDTBC1D14
11716015_a_atDCMTM3
11716208_s_atDGLUD1
11716288_s_atDESYT1
11716391_a_atDBACE1
11716620_a_atDTNPO1
11716626_atDKIF3B
11716787_a_atDB3GNT1
11716788_atDB3GNT1
11718184_a_atDFCHSD2
11718287_atDUBL3
11718288_atDUBL3
11718406_s_atDTMBIM6
11718439_atDNSUN4
11718734_a_atDPOGK
11718900_a_atDTGFBR3
11718901_atDTGFBR3
11719088_atDMMGT1
11719353_s_atDGCC2
11719397_a_atDRRAGC
11719398_s_atDRRAGC
11719409_a_atDHIPK2
11719628_a_atDHDHD1A
11719786_atDSMCR7L
11719912_a_atDKDM1B
11751297_s_atDSUB1
11751305_a_atDB3GNT1
11751353_a_atDRRAGC
11751354_a_atDHDHD1A
11753308_s_atDULBP2
11753549_a_atDCMTM3
11754031_s_atDCKS1B
11754827_x_atDFBXO17
11755251_x_atDFADS1
11756152_s_atDPCNP
11756156_s_atDTFRC
11756181_x_atDYWHAZ
11756205_x_atDDCAF6
11756254_a_atDGGT5
11756259_s_atDNFATC1
11756285_s_atDIGF2BP3
11756497_a_atDVWCE
11756603_a_atDC9orf6
11756861_s_atDULBP2
11757430_s_atDTMED5
11757523_s_atDWDR35
11757542_s_atDSSR1
11757787_x_atDFTL
11757799_s_atDVAMP7
11757810_s_atDTMED10
11757880_s_atDDAG1
11757989_s_atDANKRD46
11758200_x_atDCKS1B
11758212_s_atDKIAA0494
11758452_s_atDCENPQ
11758454_s_atDFAM116A
11758750_x_atDYWHAZ
11758751_atDYWHAZ
11758809_atDRRAGC
11758820_atDDNAJC10
11758873_a_atDHPSE
11758995_atDLOX
11758999_s_atDUBE2H
11759720_x_atDLOC400590
11763276_a_atDPWP1
11763395_a_atDZC3HAV1L
11764275_s_atDSLC11A2
Co-regulated
11715698_a_atUNOLC1
11717961_atUMED11
11718908_s_atUCHST2
11758929_atDTFDP2
11759503_atDFAM103A1
11759776_atDGTDC1
11759943_atDFAM13A
Sirt2
11715270_s_atUKLF7
11715586_atUMAPRE1
11715713_a_atUVOPP1
11715803_a_atUANXA6
11716299_a_atUITGAV
11716344_a_atUZCCHC3
11716413_x_atUMT1E
11716582_a_atUG3BP2
11716771_s_atUSIK1
11717055_a_atUCORO1C
11717056_a_atUCORO1C
11717952_atUZDHHC18
11717968_atUTMEM216
11719647_a_atUCASP7
11719648_a_atUCASP7
11719833_atUMPZL2
11720028_x_atULDLR
11720063_a_atUGLIPR2
11720223_atUGFPT1
11720823_atUJAG1
11720849_a_atURAB23
11721218_a_atUMSRB3
11721525_s_atULOC440354
11721684_a_atUZW10
11721778_a_atUACBD5
11722193_a_atUC12orf75
11722218_a_atUWBP4
11722220_a_atUWBP4
11720046_x_atDDNAJC10
11720111_atDSNTB2
11720112_atDSNTB2
11720146_a_atDDAPK1
11720273_atDSFT2D3
11720570_a_atDPHF15
11720599_s_atDSUB1
11720602_atDSYT11
11720798_atDRAB8B
11720799_s_atDRAB8B
11720800_a_atDRAB8B
11720893_s_atDSOS1
11721524_s_atDZNF706
11721585_a_atDTMCC1
11721622_a_atDKATNAL1
11721834_a_atDGET4
11721993_atDSLC6A6
11722111_atDHLX
11722273_s_atDKIAA1826
11722338_atDPEX7
11722377_atDPNPO
11722425_s_atDNEDD4L
11722460_atDPCDH12
11722475_a_atDARID3B
11722662_a_atDHPSE
11722969_s_atDTRPV4
11722977_atDHOXB5
11723228_s_atDSGCB
11723230_a_atDRNF138
11723586_a_atDGRB14
11723639_s_atDC11orf58
11724011_atDSIKE1
11724171_a_atDFCHO2
11718909_x_atUCHST2
11721860_s_atUSTX12
11721862_a_atUSTX12
11722853_a_atUHABP4
11724388_atUZNF721
11724959_s_atUCDK14
11727522_a_atUZNF267
11727523_x_atUZNF267
11727782_a_atUTPM4
11727783_s_atUTPM4
11728155_s_atUFUT4
11731868_a_atUBICD2
11734554_a_atUBCL7B
11738988_a_atUGANAB
11738989_a_atUGANAB
11738990_x_atUGANAB
11743449_a_atUBICD2
11744786_x_atUOGFR
11748926_a_atUGANAB
11752163_a_atUKDM2B
11752676_x_atUOGFR
11753089_a_atUOGFR
11753090_x_atUOGFR
11754869_s_atUZNF267
11755270_a_atUGANAB
11757525_s_atUBICD2
11759004_atUSLC33A1
11759757_a_atUSLC33A1
11741758_x_atDTRPV4
11754398_atDLOC644538
11758147_s_atDMAPK13
11758902_atDZNF641
Int. J. Mol. Sci. EISSN 1422-0067 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert