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. 2014 Jan 6;204(1):129–146. doi: 10.1083/jcb.201305084

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© 2014 Zambrano et al.

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Figure 7. — T3 induces oxidative DNA damage. (A) Relative oxidized glutathione (Rel. GSSG) levels in TP53KO MEFs incubated with or without T3 for 3 h. (B) Cellular ROS levels measured with H₂DCFDA in TP53KO, ATMKO, and wild-type MEFs treated or not treated with T3 for 3 h (P = 0.0022, n = 3). Treatment for 2 h with 1 mM H₂O₂ was used as a positive control. (C) Percentage of TP53KO MEFs positive for MitoSOX after incubation with T3 (P = 0.0001, n = 3). 0.1 mM paraquat (1 h) was used as a positive control. (D) Percentages of cells positive for oxidized MitoSOX in Thra/Thrb KO MEFs transduced with THRA or THRB and incubated with T3 during 3 h (P < 0.0001, n = 3). (E) Immunofluorescence of TP53BP1 and 8-OH-dG in MEFs treated for 24 h with and without T3. Merge and negative control images are also shown. (bottom) Confocal microscopy images showing colocalization of TP53BP1 and 8-OH-dG in foci. Bars, 10 µm. (F) Quantification of TP53BP1/8-OH-dG foci. (G) High resolution respirometry of intact TP53KO, wild-type, and ATMKO MEFs incubated with or without T3. Cellular oxygen flow (pmol/s × 10⁻⁶ cells) was measured under routine conditions (Cr; P < 0.0001, n = 3), inhibition by oligomycin (CrO; P = 0.0011, n = 3), uncoupling to maximum flux (CrU; P < 0.0001, n = 3), and nonmitochondrial respiration (ROX). Results are presented as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. WT, wild type.