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Double-strand break repair deficiency in NONO knockout murine embryonic fibroblasts and compensation by spontaneous upregulation of the PSPC1 paralog.

Li S, Li Z, Shu FJ, Xiong H, Phillips AC, Dynan WS - Nucleic Acids Res. (2014)

Bottom Line: We tested whether NONO and PSPC1 might also affect repair indirectly by influencing mRNA levels for other DSB repair genes.Of 12 genes tested, none were downregulated, and several were upregulated.Thus, NONO or related proteins are critical for DSB repair, NONO and PSPC1 are functional homologs with partially interchangeable functions and a compensatory response involving PSPC1 blunts the effect of NONO deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Emory University, Atlanta, GA 30322, USA Department of Biochemistry, Emory University, Atlanta, GA 30322, USA Institute of Molecular Medicine and Genetics, Georgia Regents University, Augusta, GA 30912, USA.

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Effect of NONO deficiency on cell growth and radiosensitivity. (A) Growth curves for six MEF populations derived from different embryos of indicated genotypes. Cells were seeded at 3 × 104 cells per well in 6-well plates and individual wells were harvested and counted daily. Each point represents the mean of triplicate cultures. Error bars denote standard deviation. Genotypes are indicated. (B) Cell cycle distribution of WT-2 and gt-2 populations determined by flow cytometry using propidium iodide staining. Data reflect mean and standard deviations from three independent experiments. (C) Clonogenic survival assays performed as described in the Materials and Methods section using indicated MEF populations. Each point represents pooled data from two independent experiments, each performed in triplicate. Data were normalized to the surviving fraction in non-irradiated control cells in each experiment. Mean and standard deviations are shown. Doses of 137Cs gamma rays are indicated. (*P < 0.05 and **P < 0.01).
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Figure 2: Effect of NONO deficiency on cell growth and radiosensitivity. (A) Growth curves for six MEF populations derived from different embryos of indicated genotypes. Cells were seeded at 3 × 104 cells per well in 6-well plates and individual wells were harvested and counted daily. Each point represents the mean of triplicate cultures. Error bars denote standard deviation. Genotypes are indicated. (B) Cell cycle distribution of WT-2 and gt-2 populations determined by flow cytometry using propidium iodide staining. Data reflect mean and standard deviations from three independent experiments. (C) Clonogenic survival assays performed as described in the Materials and Methods section using indicated MEF populations. Each point represents pooled data from two independent experiments, each performed in triplicate. Data were normalized to the surviving fraction in non-irradiated control cells in each experiment. Mean and standard deviations are shown. Doses of 137Cs gamma rays are indicated. (*P < 0.05 and **P < 0.01).

Mentions: We compared the growth of wild-type and NONO gene trap MEFs, using cells isolated separately from three different embryos of each genotype. Although there were small differences between independently derived cell populations, there was no consistent effect of genotype on growth rate (Figure 2A). We chose two isolates that had approximately equal growth rates under 3% oxygen conditions (WT-2 and gt-2) and tested their growth in atmospheric (21%) oxygen. Although all cells grew more slowly in 21% oxygen, both genotypes were affected approximately equally (Supplementary Figure S1). We also compared the cell cycle distribution in WT-2 and gt-2 MEFs. The distributions were similar (Figure 2B and Supplementary Figure S1).


Double-strand break repair deficiency in NONO knockout murine embryonic fibroblasts and compensation by spontaneous upregulation of the PSPC1 paralog.

Li S, Li Z, Shu FJ, Xiong H, Phillips AC, Dynan WS - Nucleic Acids Res. (2014)

Effect of NONO deficiency on cell growth and radiosensitivity. (A) Growth curves for six MEF populations derived from different embryos of indicated genotypes. Cells were seeded at 3 × 104 cells per well in 6-well plates and individual wells were harvested and counted daily. Each point represents the mean of triplicate cultures. Error bars denote standard deviation. Genotypes are indicated. (B) Cell cycle distribution of WT-2 and gt-2 populations determined by flow cytometry using propidium iodide staining. Data reflect mean and standard deviations from three independent experiments. (C) Clonogenic survival assays performed as described in the Materials and Methods section using indicated MEF populations. Each point represents pooled data from two independent experiments, each performed in triplicate. Data were normalized to the surviving fraction in non-irradiated control cells in each experiment. Mean and standard deviations are shown. Doses of 137Cs gamma rays are indicated. (*P < 0.05 and **P < 0.01).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Effect of NONO deficiency on cell growth and radiosensitivity. (A) Growth curves for six MEF populations derived from different embryos of indicated genotypes. Cells were seeded at 3 × 104 cells per well in 6-well plates and individual wells were harvested and counted daily. Each point represents the mean of triplicate cultures. Error bars denote standard deviation. Genotypes are indicated. (B) Cell cycle distribution of WT-2 and gt-2 populations determined by flow cytometry using propidium iodide staining. Data reflect mean and standard deviations from three independent experiments. (C) Clonogenic survival assays performed as described in the Materials and Methods section using indicated MEF populations. Each point represents pooled data from two independent experiments, each performed in triplicate. Data were normalized to the surviving fraction in non-irradiated control cells in each experiment. Mean and standard deviations are shown. Doses of 137Cs gamma rays are indicated. (*P < 0.05 and **P < 0.01).
Mentions: We compared the growth of wild-type and NONO gene trap MEFs, using cells isolated separately from three different embryos of each genotype. Although there were small differences between independently derived cell populations, there was no consistent effect of genotype on growth rate (Figure 2A). We chose two isolates that had approximately equal growth rates under 3% oxygen conditions (WT-2 and gt-2) and tested their growth in atmospheric (21%) oxygen. Although all cells grew more slowly in 21% oxygen, both genotypes were affected approximately equally (Supplementary Figure S1). We also compared the cell cycle distribution in WT-2 and gt-2 MEFs. The distributions were similar (Figure 2B and Supplementary Figure S1).

Bottom Line: We tested whether NONO and PSPC1 might also affect repair indirectly by influencing mRNA levels for other DSB repair genes.Of 12 genes tested, none were downregulated, and several were upregulated.Thus, NONO or related proteins are critical for DSB repair, NONO and PSPC1 are functional homologs with partially interchangeable functions and a compensatory response involving PSPC1 blunts the effect of NONO deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Emory University, Atlanta, GA 30322, USA Department of Biochemistry, Emory University, Atlanta, GA 30322, USA Institute of Molecular Medicine and Genetics, Georgia Regents University, Augusta, GA 30912, USA.

Show MeSH
Related in: MedlinePlus