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. 2010 Jan 15;38(9):2891–2903. doi: 10.1093/nar/gkp1250

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© The Author(s) 2010. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 7. — Knockdown of Nudt16 mRNA suppressed ITPA-deficient phenotypes in immortalized Itpa^−/– MEFs. (A) Expression of Nudt16 mRNA. To knock down the expression of Nudt16, two different siRNAs (80, 82 or 80 + 82) against Nudt16 mRNA or control siRNA were introduced into immortalized Itpa^−/– MEFs. Forty-eight hours after the introduction, total RNA was prepared and quantitative real-time RT–PCR was performed. Levels of Nudt16 mRNA were normalized to those of Gapdh mRNA and their relative values are shown. Data are shown in a bar graph (mean ± SD, n = 3). (B) Expression of NUDT16 protein. Western blotting was performed for crude cell extracts (40 µg) prepared from immortalized Itpa^−/– MEFs treated with two Nudt16 siRNAs (80 + 82) or control siRNA, using anti-NUDT16. GAPDH was detected as an internal control. (C) Knockdown of Nudt16 mRNA suppressed proliferation of immortalized Itpa^−/– MEFs. Expression of Nudt16 mRNA was blocked using a mix of two different Nudt16 siRNAs and cell proliferation was examined. Circles, Itpa^+/+; triangles, Itpa^−/–; open marks, control siRNA; closed marks, Nudt16 siRNAs. Result of repeated measures ANOVA, two-tailed, P < 0.0001; Bonferroni/Dunn post hoc test, **P < 0.01 (versus other three measures). Error bars represent the SD (n = 4). (D) Knockdown of Nudt16 mRNA significantly increased the accumulation of inosine in RNA of immortalized Itpa^−/– MEFs. Level of inosine in RNA was determined by LC–MS/MS analysis of RNA prepared from immortalized Itpa^−/– MEFs treated with control or Nudt16 siRNAs. Result of unpaired Student’s t-test (two-tailed), **P < 0.01. Data are shown as a bar graph with the mean ± SD (n = 3). (E) Knockdown of Nudt16 mRNA significantly increased the accumulation of dI in nuclear DNA of immortalized Itpa^−/– MEFs. Level of dI in nuclear DNA was determined by LC–MS/MS analysis of nuclear DNA prepared from immortalized Itpa^−/– MEFs treated with control or Nudt16 siRNAs. Result of unpaired Student’s t-test (two-tailed), **P < 0.01. Data are shown as a bar graph with the mean ± SD (n = 3). (F) Increased immunoreactivity against ssDNA in immortalized Itpa^−/– MEFs after Nudt16 knockdown. Immunofluorescence microscopy with anti-ssDNA antibody revealed significantly increased immunoreactivity after Nudt16 knockdown. Result of unpaired Student’s t-test (two-tailed), **P < 0.01. Data are shown as a bar graph with the mean ± SD (n = 4). (G) Knockdown of Nudt16 mRNA increased chromosomal abnormalities in immortalized Itpa^−/– MEFs. The frequency of chromosomal abnormalities was increased significantly in immortalized Itpa^−/– MEFs after Nudt16 knockdown. Result of unpaired Student’s t-test (two-tailed), P < 0.05 (versus control siRNA). Data are shown as pie charts with the mean ± SD (n = 4).