Figure 6
Figure 6. 17-AAG inhibits FANCD2 activation but not H2AX phosphorylation. (A) Effects of 17-AAG on MMC-induced FANCD2 monoubiquitination. HeLa cells were treated with 40 ng/mL MMC alone (MMC) or with 250 nM 17-AAG (MMC + 17-AAG) for the indicated times. Cell lysates were immunoblotted with anti-FANCD2 and anti–tubulin-β antibodies. Protein bands corresponding to FANCD2 (D2) and a monoubiquitinated form of FANCD2 (Ub-D2) were quantified, and ratios of the 2 isoforms (Ub-D2/D2) were determined for each sample. Data represent means ± SE from 3 independent experiments (bottom graph). (B) Effects of 17-AAG on MMC-induced H2AX phosphorylation. HeLa cells were treated with 40 ng/mL MMC alone (MMC) or with 250 nM 17-AAG (MMC + 17-AAG) for the indicated times. Cell lysates were immunoblotted with anti-γ-H2AX and anti-H2AX. Protein bands were quantified and fold inductions of γ-H2AX normalized against H2AX were determined for each sample. Data represent means ± SE from 3 independent experiments (bottom graph). (C) Effects of 17-AAG on FANCD2 monoubiquitination and H2AX phosphorylation in control and HU- and MMC-treated HeLa cells. HeLa cells were treated with 1 mM HU or 40 ng/mL MMC in the absence (−) or presence (+) of 250 nM 17-AAG for 8 hours. Cell lysates were immunoblotted with the indicated antibodies. Ratios of FANCD2 isoforms (Ub-D2/D2) and fold inductions of γ-H2AX normalized against H2AX were determined for each sample as described. Data represent means ± SD from 3 independent experiments. (D-E) Effects of 17-AAG on formation of FANCD2 and γ-H2AX nuclear foci. HeLa cells were treated with 1 mM HU or 40 ng/mL MMC in the absence (−) or presence (+) of 250 nM 17-AAG for 8 hours. Cells were stained with anti-FANCD2 (D) or anti-γ-H2AX (E) antibodies. Cell nuclei were visualized with DAPI staining. In each sample, at least 200 nuclei were examined at original magnification ×200. Nuclei containing more than 10 bright foci were scored as FANCD2- and γ-H2AX foci-positive cells. Data represent means ± SD from 3 independent experiments (bottom graphs). Images were obtained on an Olympus AX70 microscope equipped with UPlanApo 20×/0.70 NA and WH 10×/22 objectives (Olympus, Tokyo, Japan) using a PXL charge-coupled device camera (model CH1; Photometrics, Osnabrück, Germany).

17-AAG inhibits FANCD2 activation but not H2AX phosphorylation. (A) Effects of 17-AAG on MMC-induced FANCD2 monoubiquitination. HeLa cells were treated with 40 ng/mL MMC alone (MMC) or with 250 nM 17-AAG (MMC + 17-AAG) for the indicated times. Cell lysates were immunoblotted with anti-FANCD2 and anti–tubulin-β antibodies. Protein bands corresponding to FANCD2 (D2) and a monoubiquitinated form of FANCD2 (Ub-D2) were quantified, and ratios of the 2 isoforms (Ub-D2/D2) were determined for each sample. Data represent means ± SE from 3 independent experiments (bottom graph). (B) Effects of 17-AAG on MMC-induced H2AX phosphorylation. HeLa cells were treated with 40 ng/mL MMC alone (MMC) or with 250 nM 17-AAG (MMC + 17-AAG) for the indicated times. Cell lysates were immunoblotted with anti-γ-H2AX and anti-H2AX. Protein bands were quantified and fold inductions of γ-H2AX normalized against H2AX were determined for each sample. Data represent means ± SE from 3 independent experiments (bottom graph). (C) Effects of 17-AAG on FANCD2 monoubiquitination and H2AX phosphorylation in control and HU- and MMC-treated HeLa cells. HeLa cells were treated with 1 mM HU or 40 ng/mL MMC in the absence (−) or presence (+) of 250 nM 17-AAG for 8 hours. Cell lysates were immunoblotted with the indicated antibodies. Ratios of FANCD2 isoforms (Ub-D2/D2) and fold inductions of γ-H2AX normalized against H2AX were determined for each sample as described. Data represent means ± SD from 3 independent experiments. (D-E) Effects of 17-AAG on formation of FANCD2 and γ-H2AX nuclear foci. HeLa cells were treated with 1 mM HU or 40 ng/mL MMC in the absence (−) or presence (+) of 250 nM 17-AAG for 8 hours. Cells were stained with anti-FANCD2 (D) or anti-γ-H2AX (E) antibodies. Cell nuclei were visualized with DAPI staining. In each sample, at least 200 nuclei were examined at original magnification ×200. Nuclei containing more than 10 bright foci were scored as FANCD2- and γ-H2AX foci-positive cells. Data represent means ± SD from 3 independent experiments (bottom graphs). Images were obtained on an Olympus AX70 microscope equipped with UPlanApo 20×/0.70 NA and WH 10×/22 objectives (Olympus, Tokyo, Japan) using a PXL charge-coupled device camera (model CH1; Photometrics, Osnabrück, Germany).

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