Ndy1 overexpression renders MEFs resistant to oxidative stress. (A) Cell viability as determined by the MTT assay. MEFs transduced with an Ndy1.myc or Ndy1-ΔJmjC.myc retroviral construct or with the empty retroviral vector (EV) were treated with different concentrations of H₂O₂ for 12 h. Cell viability was measured using the MTT assay. Data are mean percentages of live cells ± SEM. Asterisks indicate statistically significant differences (P < 0.05). (B) Apoptosis as determined by the TUNEL assay. MEFs transduced with an Ndy1.myc or Ndy1-ΔJmjC.myc retroviral construct or with the EV were treated with H₂O₂ (0.5 mM). The rates of apoptosis in H₂O₂-treated and untreated cells harvested 1 h later were determined by TUNEL staining and flow cytometry. (Top) Data are mean percentages of apoptotic cells ± SEM and are derived from five independent experiments. (Bottom) Representative plots of FACS analysis. (C) Cell cycle analysis. The DNA contents of MEFs harvested before and 12 h after treatment with H₂O₂ were determined. (Top) The percentages of cells in the G₁ (left), S (middle), and G₂/M (right) phases of the cell cycle were measured before and 12 h after treatment with H₂O₂ (0.5 mM). Data are the mean percentage of cells in a given phase ± the SEM and are derived from six independent experiments. (Bottom) Representative plots of FACS analysis before (upper row) and after (lower row) treatment with H₂O₂. Asterisks indicate statistically significant differences (P < 0.05).

Ndy1 knockdown enhances sensitivity to oxidative stress. (A) Cell viability as determined by the MTT assay. MEFs transfected with Ndy1 siRNA (siNdy1) or scrambled siRNA (siControl) were treated with different concentrations of H₂O₂ for 12 h. Cell viability was measured using the MTT assay. Data are mean percentages of live cells ± SEM. Asterisks indicate statistically significant differences (P < 0.05). (B) Apoptosis as determined by the TUNEL assay. MEFs transfected with siNdy1 or siControl were treated with H₂O₂ (0.5 mM), subjected to TUNEL staining 1 h later, and analyzed by FACS. (Left) Data are mean percentages of apoptotic cells ± SEM and are based on five independent experiments. (Right) Representative plots of FACS analyses. (C) Cell cycle analysis. The DNA contents of MEFs harvested before and 12 h after treatment with H₂O₂ were determined. (Top) The percentages of cells in the G₁ (left), S (middle), and G₂/M (right) phases of the cell cycle were measured before and 12 h after treatment with H₂O₂. Data are the mean percentage of cells in a given phase ± the SEM and are derived from six independent experiments. Asterisks indicate statistically significant differences (P < 0.05). (Bottom) Representative plots of FACS analysis before (upper row) and after (lower row) treatment with H₂O₂.

Ndy1 expression modifies the signaling response to H₂O₂-induced oxidative stress. (A) MEFs transduced with the pBabe-puro-based retroviral construct Ndy1.myc or with the empty retroviral vector (EV) were treated with H₂O₂ (0.5 mM). Lysates of H₂O₂-treated and untreated cells harvested 2 h later were analyzed by Western blotting for the phosphorylation of redox-sensitive signaling proteins. The data are representative of three independent experiments. (B) MEFs transfected with Ndy1 siRNA (siNdy1) or scrambled siRNA (siControl) were treated with H₂O₂ (0.5 mM). Lysates of H₂O₂-treated and untreated cells harvested 2 h later were analyzed by Western blotting for the phosphorylation of redox-sensitive signaling proteins. The data are representative of three independent experiments. Notably, no effect on the total levels of these proteins was observed.

Ndy1 expression inhibits deoxyguanosine oxidation and DNA damage. (A) Deoxyguanosine oxidation. MEFs transduced with an Ndy1.myc or Ndy1-ΔJmjC.myc retroviral construct or with the empty retroviral vector (EV) were treated with H₂O₂ (0.5 mM). H₂O₂-treated and untreated cells were analyzed by immunofluorescence 30 min later for 8-oxo-dG accumulation. The ratio of red (8-oxo-dG) to blue (DAPI) staining was quantified for each sample. The ratio was given the arbitrary value of 1 in untreated EV cells, which were used as the basis for all comparisons. (Left) Representative microphotography of 8-oxo-dG and DAPI staining of H₂O₂-treated and untreated cells. Bar, 50 μm. (Right) Cumulative data are presented as the means of the relative 8-oxo-dG levels ± SEM and are based on three independent experiments. (B) DNA damage. MEFs transduced with an Ndy1.myc or Ndy1-ΔJmjC.myc retroviral construct or with the EV were treated with H₂O₂ (0.5 mM). H₂O₂-treated and untreated cells were subjected to single gel electrophoresis 45 min later. Following electrophoresis, samples were stained with Sybr green and scored by fluorescence microscopy. (Top) Representative microphotographs of Sybr green-stained samples. Bar, 100 μm. (Bottom) DNA damage, defined by the intensity and length of the comet tails, was quantified using Comet Assay IV image analysis software. Data are expressed as percentages of cells with a given level of DNA damage reflected in the intensity (left) or length (right) of the tail. Data are based on three independent experiments. (C) Deoxyguanosine oxidation. MEFs transfected with Ndy1 siRNA (siNdy1) or with scrambled siRNA (siControl), were treated with H₂O₂ (0.5 mM). H₂O₂-treated and untreated cells were analyzed 30 min later for 8-oxo-dG accumulation. The ratio of red (8-oxo-dG) to blue (DAPI) staining was quantified for each sample. The ratio in untreated cells transfected with siControl, which were used as the basis for all comparisons, was given the arbitrary value of 1. Cumulative data are presented as the means of the relative 8-oxo-dG levels ± SEM and are based on two independent experiments. (D) DNA damage. MEFs transfected with siNdy1 or with siControl were treated with H₂O₂ (0.25 mM), subjected to single-cell gel electrophoresis, stained with Sybr green, and scored by fluorescence microscopy. DNA damage, defined by the intensity and length of the comet tails, was quantified using Comet Assay IV image analysis software. Data are expressed as percentages of cells with a given level of DNA damage reflected in the intensity (left) and length (right) of the tail. Data are based on two independent experiments.

Ndy1 expression regulates the intracellular levels of H₂O₂ in H₂O₂-treated and untreated cells. (A) ROS levels in cells overexpressing Ndy1. MEFs transduced with an Ndy1.myc or Ndy1-ΔJmjC.myc retroviral construct or with the empty vector (EV) were treated with H₂O₂ (0.5 mM). H₂O₂-treated and untreated cells were stained with ROS indicators (DCFDA, DHE, and MitoROS) and analyzed by FACS. (Top left) Cumulative data from six experiments are presented as mean DCFDA fluorescence ± SEM. EV cell fluorescence was given the arbitrary value of 100. (Top right) Representative overlay plots of DCFDA-stained cells analyzed by FACS. (Bottom) DHE (left) and MitoROS (right) staining. Untreated cells are shown. (B) ROS levels in cells in which Ndy1 was knocked down. MEFs transfected with Ndy1 siRNA (siNdy1) or with scrambled siRNA (siControl) were treated with H₂O₂ (0.5 mM). H₂O₂-treated and untreated cells were stained with ROS indicators (DCFDA, DHE, and MitoROS) and analyzed by FACS. (Left) Representative overlay plots of DCFDA-stained cells analyzed by FACS. Similar data were obtained in four independent experiments. (Right) DHE (top) and MitoROS (bottom) staining. Untreated cells are shown. There was no difference between samples before and after treatment with H₂O₂. (C) (Left) Total antioxidant activity in cells overexpressing Ndy1 and in cells in which Ndy1 was knocked down. Extracts from MEFs transduced with the Ndy1.myc or Ndy1-ΔJmjC.myc retroviral construct or with the EV were analyzed for their capacities to detoxify H₂O₂ compared to Trolox. (Right) The antioxidant capacities of lysates of MEFs transfected with siNdy1 or with siControl were measured as in the left panel. In both panels, cumulative data were derived from three independent experiments performed in triplicate.

Ndy1 regulates the expression of antioxidant genes. (A) Real-time RT-PCR array of antioxidant genes. Results are expressed as mean changes (n-fold) in mRNA levels in cells transduced with the Ndy1.myc or Ndy1-ΔJmjC.myc retroviral construct relative to empty vector (EV)-transduced cells and in cells transfected with siRNA for Ndy1 (siNdy1) relative to scrambled siRNA (siControl). Notably, no differences between EV and siControl cells were observed. The genes that were further analyzed are highlighted in red. (B) Real-time RT-PCR and Western blot analysis verified the data obtained from the array. (Left) Results are expressed as mean changes (n-fold) in mRNA levels in cells transduced with Ndy1.myc or Ndy1-ΔCXXC.myc relative to EV-transduced cells and in cells transfected with siNdy1 relative to siControl-transfected cells. Cumulative data from two independent experiments performed in triplicate are shown. The expression of all genes tested was given the arbitrary value of 1 in EV-transduced cells, which were used as the basis for all comparisons. No differences were observed between EV and siControl cells. (Right) Western blots of lysates of Ndy1- or EV-transduced cells and siNdy1- or siControl-transfected cells were probed with antibodies to Nqo1, Prdx4, or tubulin (loading control). (C) ChIP analysis. (Top) Schematic diagrams of the topology of the primer sets used for the Nqo1 and Prdx4 genes. (Center and bottom) ChIP analyses of MEFs transduced with the Ndy1.myc or Ndy1-ΔCXXC.myc retroviral construct or with the EV were performed using monoclonal antibodies against the myc tag (center) and histone H3 dimethylated K36 (bottom). Immunoprecipitated DNA fragments were quantified using real-time PCR and the indicated sets of primers. Data from three independent experiments are expressed as mean enrichment of a given DNA fragment in ChIPs of experimental cells relative to control cells, where the level of immunoprecipitated DNA was given the arbitrary value of 1. Ndy1 binding and histone H3K36me2 levels were also examined by ChIP in the negative-control Ndy1-insensitive gene Prdx2 (see Fig. S11 in the supplemental material).

Downregulation of the Ndy1-regulated antioxidant genes in Ndy1-overexpressing MEFs to the levels of control cells restores cellular sensitivity to H₂O₂. (A) Titration of target gene knockdown. siRNAs targeting the Ndy1-regulated genes were titrated in Ndy1-overexpressing cells to knock down these genes to the levels in empty vector (EV) cells. siRNAs were transfected separately or in combination, and their effects on the expression of each gene relative to siControl-transfected and EV cells were recorded. The levels of all genes in the siControl-transfected cells were given the arbitrary value of 1. Data from three independent experiments are expressed as mean RNA levels relative to those in siControl cells ± SEM. (B) H₂O₂ levels in Ndy1-expressing cells transfected with siRNAs of Ndy1 target genes. MEFs transduced with Ndy1 were transfected with each of the four siRNAs alone or in combination, stained with carboxy-DCFDA, and analyzed by FACS. Representative FACS analysis plots from three independent experiments are shown. (C) Survival of Ndy1-expressing cells transfected with siRNAs of Ndy1 target genes. MEFs transduced with Ndy1 transfected with each of the four siRNAs, alone or in combination, were treated with H₂O₂ for 12 h. Relative numbers of live cells were measured using the MTT assay. Data are mean percentages of live cells ± SEM. Asterisks indicate statistically significant differences (P < 0.05).