Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: Plasma and gene expression levels of SIRT2 are associated with viral parameters in chronic untreated HIV infection. Proteomic array applied to plasma samples from untreated chronically infected individuals with different degrees of HIV control ( n = 40). (A) Heatmap showing the relative plasma levels of the most differentially detected soluble factors between untreated HIV-infected individuals with high viral loads (HIV-high, with more than 50,000 viral copies/mL, n = 20) and those with low viral loads (HIV-low, viral loads < 10,000 viral copies/mL, n = 20). Red indicates high protein abundance in plasma, and green indicates reduced protein levels. (B) Volcano plot representing the relative expression of the 185 molecules measured, identifying SIRT2 as the most significant and differentially detected soluble factor between HIV-high and HIV-low (Log 2 FC = 1.039; log 10 P value = 2.478). The log 2 fold change is shown on the x axis and the −log 10 of the P value on the y axis. (C) Scatterplot showing the relative plasma protein levels of SIRT2 in both study groups (HIV-high [ n = 20, orange dots] and HIV-low [ n = 20, green dots]). (D) SIRT2 gene expression in PBMCs from chronic untreated HIV-infected individuals grouped as HIV-high ( n = 16, orange dots), HIV-low ( n = 30, green dots), and elite controllers (i.e., untreated HIV-infected individuals with undetectable HIV viral load in plasma, n = 12, blue dots). Study groups are shown on the x axis, and relative SIRT2 gene expression corrected for CD4 counts is shown on the y axis. (E and F) Correlation between relative SIRT2 gene expression and plasma viral load (E) and HIV proviral DNA levels (F) in PBMC for all three groups. HIV-high patients ( n = 16, orange dots), HIV-low patients ( n = 30, green dots), and elite controllers ( n = 12, blue dots) are indicated in the plot. Relative SIRT2 gene expression corrected for CD4 counts is shown on the x axis, and viral load (HIV RNA copies/mL) and proviral levels (HIV DNA copies/10 6 PBMCs) are shown on the y axis. Plasma proteome data were analyzed using the t test. SIRT2 plasma levels between HIV-high and HIV-low were analyzed using the Mann-Whitney test. SIRT2 gene expression levels between HIV-high, HIV-low, and EC were analyzed using ANOVA test for multiple comparisons corrected by original FDR method of Benjamini and Hochberg. The Spearman's rank test was applied for the correlation analysis. Statistical significance was set at P < 0.05.

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: Clinical Proteomics, Gene Expression, Infection, Control, Quantitative Proteomics, Expressing, MANN-WHITNEY

Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: Top candidates identified by plasma proteomic analysis

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: Clinical Proteomics

Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: Plasma levels of SIRT2 are associated with neurological factors in chronic untreated HIV infection. (A) Correlation matrix showing the significant relationship between SIRT2 and proteins measured in the antibody array considering all HIV-infected groups (HIV-high and HIV-low). The color scale shows positive correlations in blue and negative correlations in red. (B) The functional analysis performed using the STRING webpage represents the interaction between SIRT2 and the remaining correlated factors. Several functions derived from Gene Ontology, Uniprot, Reactome, and PubMed were identified as follows: Regulation of Cell Death category (GO, 0010941; FDR = 0.0030) in blue, Innate Immunity (KW0399; FDR = 3.57 × 10E7) in red, Complement Cascade (HSA-166658; FDR = 3.44 × 10E6) in yellow, and epigenetic mechanisms involved in neurological disorders (PMID25774124; FDR = 0.00025) in green. (C to E). Correlation plots showing the associations between sirtuin-2 and plasma levels of SNCA (C), MAPT (D), and BDNF (E) measured in the antibody array across all chronic untreated HIV-infected individuals. HIV-high ( n = 20, orange dots) and HIV-low ( n = 20, green dots) groups are indicated in the plot. The x axis shows relative plasma SIRT2 levels, and the y axis shows the relative plasma levels for SNCA, BDNF, and MAPT, respectively. The Spearman's rank test was applied for the correlation analysis. Statistical significance was set at P < 0.05.

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: Clinical Proteomics, Infection, Ab Array, Functional Assay, Derivative Assay

Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: SIRT2 levels in the CNS of HIV-infected individuals. (A to C) Scatterplot showing the relative plasma protein levels of SNCA (A), MAPT (B), and BDNF (C) in both study groups (HIV-high [ n = 20, orange dots] and HIV-low [ n = 20, green dots]). (D) Plot representing normalized protein expression (NPX) levels of SIRT2 in the plasma (left) and CSF (right) in HIV-high ( n = 10, red bar), HIV-low ( n = 10, green bar), and seronegative individuals ( n = 5, black bar). The mean and standard deviation are shown. (E and F) Correlation between relative SIRT2 levels and relative CSF levels of MAPT (E) and NFL (F) in HIV-high ( n = 10, red dots), HIV-low ( n = 10, green dots), and seronegative individuals ( n = 5, black dots). Relative MAPT and NFL levels are shown on the y axis, and relative SIRT2 levels are shown on the x axis. (G) Plot representing SIRT2 gene expression in postmortem brain tissues measured by microarray in the GSE28160 study performed in seronegative ( n = 6), cART-treated HAD (HIV-associated disorders, n = 6), and cART-untreated HAD individuals ( n = 8). Values are expressed as mean and standard deviation. (H) Correlation between gene expression levels of SIRT2 ( x axis) and MAPT ( y axis) in postmortem brain tissue samples in HIV-infected individuals (cART-treated HAD [ n = 6] and cART-untreated HAD individuals [ n = 8]). For comparisons between groups, ANOVA test for multiple comparisons corrected by original FDR method of Benjamini and Hochberg was applied. The Spearman's rank test was applied for the correlation analysis. Statistical significance was set at P < 0.05.

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: Infection, Clinical Proteomics, Expressing, Standard Deviation, Gene Expression, Microarray

Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: Plasma SIRT2 levels are associated with neurological dysfunction in treated HIV infection. (A) Schematic representation of the following two study arms included in the ARBRE study: early-cART ( n = 9) and later-cART ( n = 10) HIV-infected individuals who started cART at different time points after estimated HIV acquisition and underwent longitudinal neurological evaluation (neuropsychological tests and neuroimaging) and quantification of SIRT2 in plasma. Early-cART patients initiated treatment within a maximum of 3 months since the estimated date of infection, and later-treated patients initiated cART at least 6 months after the estimated date of infection. Study visits were at baseline (BSL), 1 month (4 weeks), and 1 year after initiation of cART. (B to D) Plots representing results of the NPZ12 and global gray matter and medial orbitofrontal cortex volume (peak coordinate at MNI, x = 6, y = 44, z = −29) in early- and later-cART individuals at baseline and after 1 year on treatment (early-cART, n = 9, green group; later-cART, n = 9, red group). The values on the y axis represent the NPZ12 score, global gray matter (cm 3 ) and medial orbitofrontal cortex volume (a.u.). The box plot shows the median and minimum to maximum values for each group. (E to H) Absolute plasma NFL, GFAP, UCHL1, and MAPT levels at baseline (BSL) and after 1 year on treatment in early-cART ( n = 9, green group) and later-cART ( n = 10, red group) individuals expressed in picograms per milliliter. The upper limit of the bar is the median value of protein plasma levels. (I) Absolute MAPT (tau protein) plasma levels represented longitudinally and expressed in picograms per milliliter. (J) Correlation between longitudinal plasma MAPT (tau protein) levels ( x axis, pg/mL) and the results for medial orbitofrontal cortex volumetry ( y axis) expressed as the difference between 1 year and baseline in the ARBRE study including early- ( n = 9, green dots) and later-cART individuals ( n = 5, red dots). (K and L) Plots representing the absolute plasma SIRT2 in early-cART ( n = 9, green line) and later-cART ( n = 10, red line) individuals cross-sectionally (K) and longitudinally (L) at BSL (baseline) and 1 year time points. Weeks of treatment are shown on the x axis, and absolute plasma levels of SIRT2 (ng/mL) are shown on the y axis. (M) Correlation between longitudinal plasma SIRT2 ( x axis, ng/mL) and the results for medial orbitofrontal cortex volumetry expressed as the difference between 1 year on cART and baseline in the ARBRE study including early- ( n = 9, green dots) and later-cART individuals ( n = 9, red dots). Differences between the groups were analyzed using the Mann-Whitney U test, changes over time were assessed using the paired t test, and the correlation analysis was based on the Spearman's rank test. Statistical significance was set at P < 0.05.

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: Clinical Proteomics, Infection, MANN-WHITNEY

Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: Clinical information of neurologically evaluated HIV cohort (ARBRE study)

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: Transmission Assay, Infection, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: Longitudinal neurological evaluation and SIRT2 plasma levels in ARBRE study

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: Effect of in vitro SIRT2 inhibition on HIV replication. (A to C) Inhibition of HIV replication in the presence of SIRT2 inhibitor (AK-1), tested in HIV-infected PHA blasts (seven independent experiments) (A and B) with HIV NL4-3 strain, at day 3 and 1 week postinfection, respectively, and HIV-infected monocyte-derived macrophages (MDMs) (six independent experiments) (C) infected with HIV BaL strain at day 4 postinfection. (D to F) Cell viability in the presence of SIRT2 inhibitor (AK-1), tested in HIV-infected PHA blasts (seven independent experiments) (D and E) with HIV NL4-3 strain, at day 3 and 1 week, respectively, and monocyte-derived macrophages (MDMs) (six independent experiments) (F) infected with HIV BaL strain at day 4 postinfection. Experimental conditions are shown on the x axis; quantification of viability (% live cells) is shown on the y axis. (G) Glial cells infected with the HIV NLAD8 virus strain (MOI, 0.01) in the presence of different doses of AK-1 inhibitor. Experimental conditions are shown on the x axis; quantification of absolute p24 supernatant (pg/mL) is shown on the y axis. The data presented correspond to the mean of two duplicates performed in a single experiment. (H) HIV-infected microglial cells (six independent experiments) with the NLAD8 virus strain in the presence of different doses of AK-1 inhibitor. Experimental conditions are shown on the x axis; quantification of p24 levels (pg/mL) is shown on the y axis. For results from HIV-infected PHA blasts, MDMs, and primary glial cells, ANOVA test for multiple comparisons corrected by original FDR method of Benjamini and Hochberg was used to analyzed differences between conditions. For all comparisons, P < 0.05 was considered significant. The plots show the median of all experiments for each condition.

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: In Vitro, Inhibition, Infection, Derivative Assay, Virus

Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: Effect of in vitro SIRT2 inhibition on HIV reactivation. (A) Histogram plot showing green fluorescent protein (GFP) cellular counts of three different conditions of the experiment, including nonstimulated cells (black), PMA-stimulated cells (orange), and cells stimulated with PMA in the presence of AK-1 (blue) from one representative experiment. (B and C) HIV reactivation measured by the percentage of GFP expression measured by flow cytometry in J-LAT A2 cells is shown on the y axis; different conditions of the experiment, including nonstimulated cells, PMA-stimulated cells, and cells stimulated with PMA in the presence of AK-1 are shown on the x axis. (D) Gating strategy of HIV virus reactivation in the J-LAT A2 cell line experiment. For multiple comparisons, ANOVA test corrected by original FDR method of Benjamini and Hochberg was used to analyze differences between conditions. For all comparisons, P < 0.05 was considered significant. The plots show the median of all experiments for each condition.

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: In Vitro, Inhibition, Expressing, Flow Cytometry, Virus

Journal: Free radical biology & medicine

Article Title: NQO1 regulates mitotic progression and response to mitotic stress through modulating SIRT2 activity

doi: 10.1016/j.freeradbiomed.2018.08.009

Figure Lengend Snippet: NQO1 and SIRT2 colocalize to the mitotic spindle during mitosis. (A, B) Confocal microscopy images show the locations of endogenous SIRT2 (red) and NQO1 (green) during both metaphase (A) and telophase (B) in MCF-7 human breast cancer cells. (C, D) Confocal microscopy images show the locations of endogenous SIRT2 (red) and NQO1 (green) during both metaphase (C) and telophase (D) in BxPC-3 human pancreatic cancer cells. In all panels above, DNA was stained with DAPI (blue). On the right, graphs represent signal intensity scans along the lines drawn for each panel. Scale bar, 20 µm. (E, F, G) Higher magnification images (60x) under same experimental conditions as shown in A, B.

Article Snippet: Recombinant human SIRT2 and GST tagged NQO1 (GST-NQO1) proteins were purchased from Abcam and Creative BioMart, respectively.

Techniques: Confocal Microscopy, Staining

Journal: Free radical biology & medicine

Article Title: NQO1 regulates mitotic progression and response to mitotic stress through modulating SIRT2 activity

doi: 10.1016/j.freeradbiomed.2018.08.009

Figure Lengend Snippet: NQO1 interacts directly with SIRT2. (A) MCF-7 cells were transiently transfected with Flag-NQO1 and HA-SIRT2. 48 h after transfection, cell lysates were subjected to immunoprecipitation (IP) using either a Flag (left) or an HA (right) antibody followed by western blotting using antibodies against HA and Flag, respectively. Specificity was confirmed by using species-matched control IgG as a negative control. (B) Cell lysates from MCF-7 cells were subjected to immunoprecipitation (IP) using a SIRT2 antibody followed by western blotting using an anti-NQO1 antibody. * denotes Ig heavy and light chain. (C) Cell lysates from MCF-7 cells as described in (A) either unsynchronized or synchronized in mitosis after treatment with nocodazole (Noc, 200 nM for 12 h) were subjected to immunoprecipitation using an antibody against HA followed by western blotting using a Flag antibody. Specificity was confirmed by using species-matched control IgG as a negative control. Overexpression of both Flag-NQO1 and HA-SIRT2 is confirmed by western blotting (lower). (D) Reciprocal co-immunoprecipitation of endogenous SIRT2 and NQO1 in either unsynchronized or synchronized in mitosis Hela cells. For cell synchronization, HeLa cells were serum starved for 48 h and then released into regular media for 16 h. Cell lysates were subjected to immunoprecipitation (IP) using either a SIRT2 (left) or an NQO1 (right) antibody followed by western blotting using antibodies against SIRT2 and NQO1. Specificity was confirmed by using species-matched control IgG as a negative control. (E) Either unsynchronized or synchronized in mitosis (syn, as described in C) MCF-7 cells were fractionated into cytoplasmic extracts (cyt) and nuclear extracts (nuc). Levels of both SIRT2 and NQO1 were detected by western blotting. Successful cellular fractionation was confirmed by western blotting using antibodies against markers such as Lamin A/C (nuc) and actin (cyt). (F) Recombinant human SIRT2 and GST-NQO1 were used to check protein-protein interaction in vitro. Coomassie staining shows the molecular weight of the two proteins. GST-NQO1 was immunoprecipitated followed by SIRT2 immunodetection of (upper). In addition, SIRT2 was immunoprecipitated first, followed by immunodetection of GST-NQO1 (lower). Beads alone were used in the pull-down assays as negative controls.

Article Snippet: Recombinant human SIRT2 and GST tagged NQO1 (GST-NQO1) proteins were purchased from Abcam and Creative BioMart, respectively.

Techniques: Transfection, Immunoprecipitation, Western Blot, Negative Control, Over Expression, Cell Fractionation, Recombinant, In Vitro, Staining, Molecular Weight, Immunodetection

Journal: Free radical biology & medicine

Article Title: NQO1 regulates mitotic progression and response to mitotic stress through modulating SIRT2 activity

doi: 10.1016/j.freeradbiomed.2018.08.009

Figure Lengend Snippet: NQO1 positively regulates SIRT2 activity. (A) MCF-7 cells were transfected with either control (ctr) siRNA or NQO1 siRNA. 48 h after transfection, lysates were analyzed by western blotting using antibodies against Lys-40 acetylated tubulin (Ac-Tubulin), NQO1, SIRT2 and tubulin/actin. Quantification of acetylated tubulin levels are presented, **p<0.01. (B) MDA-MB-231 cells (NQO1 null) were transfected with either empty vector (ctr), Flag-NQO1 or Flag-NQO1P187S. 48h after transfection, lysates were analyzed by western blotting using antibodies against Lys-40 acetylated tubulin (Ac-Tubulin), Flag, SIRT2 and tubulin/actin. Quantification of acetylated tubulin levels are presented, **p<0.01. (C) MCF-7 cells were treated with Dicoumarol (100 uM) for different times or with different concentrations as indicated for 24h. Lysates were analyzed by western blotting using antibodies against Lys-40 acetylated tubulin (Ac-Tubulin), SIRT2 and tubulin. Quantification of acetylated tubulin levels are presented, *p<0.05. (D, E, F) MCF-7 cells were transfected with either control (ctr) siRNA or NQO1 siRNA. Cells were lysed in extraction buffer, and NADtotal (NAD and NADH) (D) as well as NADH alone (E) were calculated. The ratio of NAD/NADH (F) is calculated based on the formula (NADtotal-NADH)/NADH. Data represent Mean ± SEM of three independent experiments, *P<0.05. (G) MCF-7 cells were transfected with either control (ctr) siRNA or NQO1 siRNA. 48 h after transfection, SIRT2 activity in these lysates was determined by using a fluorometric assay based on the deacetylation of a unique target peptide included in the kit (FLUOR DE LYS® SIRT2 Deacetylase Fluorometric Assay Kit). Data represent Mean ± SEM of three independent experiments, *P<0.05. (H) MCF-7 cells were transfected with either control (ctr) siRNA or NQO1 siRNA. NQO1 knockdown cells were next transfected with NQO1 cDNA or treated with NAD+ (0.5 mM). Cell extracts were analyzed by western blotting using antibodies against Acetylated-Tubulin (Ac-tubulin), NQO1, SIRT2 and actin/GAPDH. * denotes exogenously expressed Flag-NQO1, whereas ** denotes the expression of endogenous NQO1. (I) MCF-7 cells were transfected with either control (ctr) siRNA or SIRT2 siRNA in the presence or absence of exogenously expressed NQO1 or NQO1P187S. Cell extracts were analyzed by western blotting using antibodies against acetylated histone H4 lysine16 (Ac-H4K16), SIRT2, Flag (NQO1) and actin. Note that NQO1P187S cannot be detected as this mutation affects enzymatic activity by extremely decreasing stability due to ubiquitination and proteasomal degradation [44]. (J) Wild-type (Sirt2+/+) and Sirt2 knockout (Sirt2-/-) MEFs were transfected with either control (ctr) siRNA, Nqo1 siRNA or Flag-NQO1 cDNA. Cell extracts were analyzed by western blotting using antibodies against acetylated histone H4 lysine16 (Ac-H4K16), SIRT2, Flag (NQO1) and actin.

Article Snippet: Recombinant human SIRT2 and GST tagged NQO1 (GST-NQO1) proteins were purchased from Abcam and Creative BioMart, respectively.

Techniques: Activity Assay, Transfection, Western Blot, Plasmid Preparation, Histone Deacetylase Assay, Expressing, Mutagenesis, Knock-Out

Journal: Free radical biology & medicine

Article Title: NQO1 regulates mitotic progression and response to mitotic stress through modulating SIRT2 activity

doi: 10.1016/j.freeradbiomed.2018.08.009

Figure Lengend Snippet: NQO1-SIRT2 axis regulates APC/C complex during mitosis. (A) Cell extracts from control shRNA (sh ctr) and NQO1 shRNA (sh NQO1) MCF-7 cells were used to immunoprecipitate endogenous Cdc27. Interaction with several components of the APC/C complex was determined by western blotting using the indicated antibodies. Specificity was confirmed by using species-matched control IgG as a negative control. Input levels of the interacting proteins are shown (right). (B) MCF-7 cells overexpressing HA-Cdh1 were transfected with either control (ctr) siRNA or NQO1 siRNA followed by immunoprecipitation using an anti-acetylated lysine antibody (Ac-K). Acetylated levels of Cdh1 were checked by western blotting using an anti-HA antibody. Specificity was confirmed by using species-matched control IgG as a negative control. In parallel, immunoprecipitation with an antibody against Cdc27 was performed to check interaction between Cdc27 and Cdh1. Input levels of both Cdc27 and NQO1 are shown. (C) MCF-7 cells were transfected with either control (ctr) siRNA or NQO1 siRNA followed by immunoprecipitation using an anti-acetylated lysine antibody (Ac-K). Acetylated levels of endogenous Cdh1 were checked by western blotting using an anti-Cdh1 antibody. Specificity was confirmed by using species-matched control IgG as a negative control. In parallel, immunoprecipitation with an antibody against Cdc27 was performed to check interaction between Cdc27 and Cdh1. Input levels of Cdh1, Cdc27 and NQO1 are shown. (D) Western blot analysis in whole cell lysates MCF-7 cells transfected with either control (ctr) siRNA or NQO1 siRNA. Antibodies against well–established mitotic proteins including Aurora A, Cyclin B1 and Cdc20 were used. Mitotic cells following nocodazole treatment were collected by shake-off, replated and harvested at indicated time points. Protein levels of SIRT2, NQO1 and actin are shown. (E) Quantification of Aurora A protein levels in (D) using the ImageJ software is presented. (F) For in vivo ubiquitination assay, Myc-Aurora A and HA-Ubiquitin were co-transfected into control shRNA (sh ctr) and NQO1 shRNA (sh NQO1) MCF-7 cells. To block proteasomal degradation, cells were treated with MG132 (10 µM, 4 h). Aurora A was immunoprecipitated using an anti-Myc antibody followed by western blotting against HA to detect ubiquitinated levels of Aurora A. Levels of NQO1 and actin are shown. (G) A similar in vivo ubiquitination assay was performed in MCF-7 cells transfected with either a control (ctr) vector or a Flag-NQO1 vector. Aurora A was immunoprecipitated using an anti-Myc antibody followed by western blotting against HA to detect ubiquitinated levels of Aurora A. Levels of Flag-NQO1 and actin are shown.

Article Snippet: Recombinant human SIRT2 and GST tagged NQO1 (GST-NQO1) proteins were purchased from Abcam and Creative BioMart, respectively.

Techniques: shRNA, Western Blot, Negative Control, Transfection, Immunoprecipitation, Software, In Vivo, Ubiquitin Assay, Blocking Assay, Plasmid Preparation

Journal: Free radical biology & medicine

Article Title: NQO1 regulates mitotic progression and response to mitotic stress through modulating SIRT2 activity

doi: 10.1016/j.freeradbiomed.2018.08.009

Figure Lengend Snippet: NQO1 inhibition sensitizes cancer cells to mitotic stress. (A) Mitotic index in nocodazole (400 nM) treated control and NQO1 siRNA MCF-7 cells as well as untreated cells. Mitotic index values are compared at different time points. ** P<0.01. (B) Mitotic index in nocodazole treated control, SIRT2 overexpressing (SIRT2) and SIRT2 overexpressing cells followed by NQO1 siRNA (SIRT2 + NQO1 siRNA) MCF-7 cells. Mitotic index values are compared at different time points between SIRT2 and SIRT2 + NQO1 siRNA cells. n.s not-significant, * P<0.05, ** P<0.01. (C) Chronic mortality assay in control (shctr) and NQO1 knocked down MCF-7 cells (shNQO1–1, shNQO1–4) following treatment with nocodazole for the indicated times. ** P<0.01. (D) Mitotic index in paclitaxel (20 nM) treated control, NQO1 siRNA, SIRT2 overexpressing (SIRT2) and SIRT2 overexpressing cells followed by NQO1 siRNA (SIRT2 + NQO1 siRNA) MCF-7 cells. Mitotic index values are compared at different time points between control and NQO1 siRNA cells as well as SIRT2 and SIRT2 + NQO1 siRNA cells. n.s not-significant, ** P<0.01. (E) BxPC3 cells were treated with increasing concentrations of paclitaxel and dicoumarol as indicated. Cell viability was assessed using the membrane-permeable dye Calcein AM. A heat map was made based on the fraction of viable cells (numbers) as compared to untreated cells. n.s not-significant. ** P<0.01. (F) Chronic mortality assay in control (shctr) and NQO1 knocked down MCF-7 cells (shNQO1–1, shNQO1–4) following treatment with paclitaxel for the indicated times. * P<0.05, ** P<0.01. (G, H) Kaplan-Meier analysis of the prognostic value of NQO1 expression in (G) all breast cancer patients and (H) ER-negative patients. (I) Proposed model regarding the effect of NQO1-SIRT2 axis on regulating mitotic progression through APC/C complex. (J) The interplay between the spindle assembly checkpoint (SAC) and APC/C is crucial for the response of cancer cells to mitotic stress. (i) High SAC activity drives mitotic arrest (though inhibiting APC/C) whereas high APC/C activity drives mitotic exit (through inhibiting SAC). (ii) Under mitotic stress, SAC activity remains high which is required for efficient APC/C inhibition, and mitotic arrest. APC/C activity is not fully inhibited which may eventually contribute to SAC inhibition, escape from mitotic arrest, mitotic slippage and resistance to anti-mitotic drugs. (iii) Given that NQO1-SIRT2 is a positive regulator of APC/C, NQO1 inhibition tips the balance towards APC/C inhibition and enhanced mitotic arrest which may confer sensitivity to microtubule poisons.

Article Snippet: Recombinant human SIRT2 and GST tagged NQO1 (GST-NQO1) proteins were purchased from Abcam and Creative BioMart, respectively.

Techniques: Inhibition, Expressing, Activity Assay