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. Author manuscript; available in PMC: 2011 Dec 16.
Published in final edited form as: Oncogene. 2010 Sep 6;29(50):6603–6608. doi: 10.1038/onc.2010.406

Figure 4.

Figure 4

Tempol affects Brca1 transcription. (A) Tempol (0.1 mM for 16 h) decreases free NADH reflected by a decreased cellular lactate/pyruvate (L/P) ratio in Fadu cells. The lactate and pyruvate concentrations of Fadu cells were measured by colorimetric assays and used to calculate the free cellular NADH/NAD ratio (Williamson et al., 1967). Since free NAD is in greater excess than free NADH, the conversion of NAD to NADH mainly affects NADH level. Therefore, the L/P ratio indicates the free NADH level (Williamson et al., 1967). Hypoxia attenuated the NADH decrease induced by Tempol treatment. (B) CtBP1 level and localization stay unchanged during Tempol treatment of Fadu cells. Scale bar=2 µm. (C) Tempol treatment decreased CtBP1 recruitment to E2F4 (E2F4-CtBP1) at the Brca1 promoter. Sequential ChIP using an E2F4 antibody (Santa Cruz; E2F4 single ChIP: E2F4) followed by anti-CtBP1 (double ChIP: E2F4-CtBP1) was performed in Fadu cells treated with Tempol; p<0.02 vs. non-treated Fadu cells (Control). Primers surrounding the proximal promoter region of Brca1 were used to PCR-amplify the ChIP sample. E2F4 single ChIP of non-treated Fadu cells was used for normalization. (D) Tempol increases Brca1 expression in Fadu cells measured by qRT-PCR; * p<0.05 vs. non-treated Fadu cells (Control). Furthermore, hypoxia attenuated the stimulatory effect of Brca1 by Tempol; # p<0.02 vs. Tempol-treated Fadu cells (Tempol). (E) Tempol increases Brca1 foci formation from 13.5 ± 1.3 to 40.2 ± 1.4 per 100 cells (p<0.01). Scale bar = 2 µm.