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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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Green cell microcolony formation assay. (A) Verification of miRNA-mediated PSPC1 knockdown. Vectors encoding control or PSPC1 miRNA were introduced by electroporation into NONO-deficient (gt/0) MEFs. A linked EmGFP gene serves as a marker for miRNA expression. Cells were fixed and analyzed by indirect immunofluorescence using anti-PSPC1 primary and red secondary antibodies. Cells were counterstained for DNA with DAPI. Each row depicts blue (DAPI), green (EmGFP) and red (anti-PSPC1) channels for the same field. Scale bar, 10 μm. (B) Representative images of multi-cell and 1-cell microcolonies after 5 days. Scale bar, 10 μm. (C) Clonogenic survival. Colonies of ≥2 cells were expressed as a proportion of total colonies. Data are normalized to fraction of colonies with ≥2 cells in non-irradiated control populations, which did not vary significantly with genotype. For each genotype and radiation dose, 200 green microcolonies were scored. Left panel, microcolony formation in the absence of DNA-PK inhibitor; right panel, microcolony formation in the presence of 2-μM NU7441.
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Figure 4: Green cell microcolony formation assay. (A) Verification of miRNA-mediated PSPC1 knockdown. Vectors encoding control or PSPC1 miRNA were introduced by electroporation into NONO-deficient (gt/0) MEFs. A linked EmGFP gene serves as a marker for miRNA expression. Cells were fixed and analyzed by indirect immunofluorescence using anti-PSPC1 primary and red secondary antibodies. Cells were counterstained for DNA with DAPI. Each row depicts blue (DAPI), green (EmGFP) and red (anti-PSPC1) channels for the same field. Scale bar, 10 μm. (B) Representative images of multi-cell and 1-cell microcolonies after 5 days. Scale bar, 10 μm. (C) Clonogenic survival. Colonies of ≥2 cells were expressed as a proportion of total colonies. Data are normalized to fraction of colonies with ≥2 cells in non-irradiated control populations, which did not vary significantly with genotype. For each genotype and radiation dose, 200 green microcolonies were scored. Left panel, microcolony formation in the absence of DNA-PK inhibitor; right panel, microcolony formation in the presence of 2-μM NU7441.

Mentions: To investigate whether the spontaneous upregulation of PSPC1 expression in NONO-deficient MEFs blunts the DNA damage sensitivity phenotype, we used an MEF-specific electroporation protocol to introduce PSPC1 or control miRNA vectors. We estimated the transfection efficiency to be 30–40%, based on co-expression of an Emerald Green Fluorescent Protein (EmGFP) marker encoded by the miRNA vector. Cells expressing EmGFP showed essentially complete loss of PSPC1 expression as determined by immunofluorescence (Figure 4A).


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)

Green cell microcolony formation assay. (A) Verification of miRNA-mediated PSPC1 knockdown. Vectors encoding control or PSPC1 miRNA were introduced by electroporation into NONO-deficient (gt/0) MEFs. A linked EmGFP gene serves as a marker for miRNA expression. Cells were fixed and analyzed by indirect immunofluorescence using anti-PSPC1 primary and red secondary antibodies. Cells were counterstained for DNA with DAPI. Each row depicts blue (DAPI), green (EmGFP) and red (anti-PSPC1) channels for the same field. Scale bar, 10 μm. (B) Representative images of multi-cell and 1-cell microcolonies after 5 days. Scale bar, 10 μm. (C) Clonogenic survival. Colonies of ≥2 cells were expressed as a proportion of total colonies. Data are normalized to fraction of colonies with ≥2 cells in non-irradiated control populations, which did not vary significantly with genotype. For each genotype and radiation dose, 200 green microcolonies were scored. Left panel, microcolony formation in the absence of DNA-PK inhibitor; right panel, microcolony formation in the presence of 2-μM NU7441.
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Figure 4: Green cell microcolony formation assay. (A) Verification of miRNA-mediated PSPC1 knockdown. Vectors encoding control or PSPC1 miRNA were introduced by electroporation into NONO-deficient (gt/0) MEFs. A linked EmGFP gene serves as a marker for miRNA expression. Cells were fixed and analyzed by indirect immunofluorescence using anti-PSPC1 primary and red secondary antibodies. Cells were counterstained for DNA with DAPI. Each row depicts blue (DAPI), green (EmGFP) and red (anti-PSPC1) channels for the same field. Scale bar, 10 μm. (B) Representative images of multi-cell and 1-cell microcolonies after 5 days. Scale bar, 10 μm. (C) Clonogenic survival. Colonies of ≥2 cells were expressed as a proportion of total colonies. Data are normalized to fraction of colonies with ≥2 cells in non-irradiated control populations, which did not vary significantly with genotype. For each genotype and radiation dose, 200 green microcolonies were scored. Left panel, microcolony formation in the absence of DNA-PK inhibitor; right panel, microcolony formation in the presence of 2-μM NU7441.
Mentions: To investigate whether the spontaneous upregulation of PSPC1 expression in NONO-deficient MEFs blunts the DNA damage sensitivity phenotype, we used an MEF-specific electroporation protocol to introduce PSPC1 or control miRNA vectors. We estimated the transfection efficiency to be 30–40%, based on co-expression of an Emerald Green Fluorescent Protein (EmGFP) marker encoded by the miRNA vector. Cells expressing EmGFP showed essentially complete loss of PSPC1 expression as determined by immunofluorescence (Figure 4A).

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