Additional file 5: of Geminin prevents DNA damage in vagal neural crest cells to ensure normal enteric neurogenesis

Figure S4. Efficient ablation of the Gem locus when combined with the inducible Sox10iCreER T2 line. Whole-mount gut preparations of control (A) and Sox10CreER(i8.5)|Gem (B) E12.5 embryos, immunostained for GFP to visualise the distribution of ENCCs within the gut. Red arrows indicate the position of the most caudally located ENCCs in the gut preparations. (C–D), Relative quantitation of Gem transcript levels in the FACS-purified ENCCs of Sox10CreER(i8.5)|Gem and Sox10CreER(i10)|Gem embryos normalised to the levels of b-actin. Unpaired t-test with Welch’s correction, **P value < 0.01, ***P value < 0.001. Scale bar: (A, B) 400 μm. (TIF 1130 kb

Differential regulation of Cdt1 in response to the topoisomerase inhibitors Doxorubicin and Etoposide.

<p>HeLa and HepG2 cells were treated for 6 h with (A) Doxorubicin (0.2, 2 and 10 µM) (Doxo) or (B) Etoposide (20 and 80 µM) (Etopo), in the presence or absence of the proteasome inhibitor MG-132 (+MG-132). Total protein extracts were prepared and subjected to western blot analysis using antibodies against Cdt1, PARP, Geminin and Tubulin. (C) HeLa and HepG2 cells were synchronized in M phase with nocodazole, and subsequently were incubated with Etoposide (20 and 80 µM) (lanes 2–3, 7–8) or Doxorubicin (0.2 and 2 µM) (lanes 4–5, 9–10). Protein extracts were subjected to Western blot analysis using antibodies against Cdt1 and Tubulin.</p

5-Fluoruracil (5-FU) does alter Cdt1 protein expression levels in HepG2 but not in HeLa cells.

<p>HeLa and HepG2 cells were incubated for 6 h with 5-FU (0.1, 10 and 100 µg/ml) in the absence (lanes 1–4 and 9–10) or in the presence (lanes 5–8 and 12–14) of MG-132 (20 µM). Protein extracts were analyzed by Western blotting using antibodies against Cdt1, PARP, Geminin and Tubulin.</p

Treatment with 5-Fluoruracil (5-FU) doesn't alter Cdt1 protein expression levels in HeLa or HepG2 cells.

<p>Asynchronous HeLa (A) and HepG2 cells (C) were incubated with 5-FU (0.1 and 100 µg/ml) in the presence of BrdU (20 µM, for 1 h). Cells were subjected to immunofluorescence using antibodies against Cdt1, Cyclin A and BrdU. DNA was visualized with DAPI or Hoechst 3258. The percentage of HeLa (B) and HepG2 (D) cells expressing Cdt1, Cyclin A and BrdU in presence of 5-FU, 0.1 µg/ml (grey columns), 100 µg/ml (black columns) and control cells (white columns) is shown; Data are the mean values of the quantifications from at least 3 different experiments from each condition and represent mean ± SD. **p<0.01, ***p<0.001. (E) HeLa and HepG2 cells were synchronized with nocodazole, released to enter G1 phase, and incubated with 5-FU (10 and 100 µg/ml) for 6 hours. Total cell lysates were extracted and subjected to Western blot analysis using antibodies against Cdt1 and Tubulin. Scale bars: A, C, 50 µm.</p

Cdt1 is targeted for proteolysis in response to DNA damage caused by Cisplatin and MMS.

<p>HeLa and HepG2 cells were cultured in the presence of Cisplatin (10, 50 and 100 µg/ml) for 6 h (lanes 1–4 and 9–12) or (B) MMS (150 and 600 µM) for 3 h (lanes 1–3 and 7–9) and in the presence of MG-132 (20 µM) (+MG-132) (lanes 5–8 and 13–16 (A) and lanes 4–7 and 10–12 (B)). Cellular protein extracts were prepared and western blot analysis was performed using antibodies against Cdt1, PARP, Geminin and Tubulin as a loading control. (C) HeLa cells cultured in absence or in presence of Cisplatin (50 µg/ml) or MMS (150 µM) were subjected to immunofluorescence analysis using antibodies against Cdt1 and Cyclin A, whereas DNA was stained with DAPI. (D) Percentage of HeLa cells expressing Cdt1 or CyclinA after Cisplatin or MMS treatment. Scale bars: C, 50 µm.</p

Treatment with Tamoxifen does not affect Cdt1 protein expression levels.

<p>HeLa and HepG2 cells were treated with Tamoxifen (0.2, 2 and 10 µM) for 6 h, in absence (lanes 1–4, 9–11) or in presence (lanes 5–8, 12–14) of MG-132. Cells were harvested, protein extracts were prepared and subjected to Western blot analysis using antibodies against Cdt1 and Tubulin as a loading control.</p

UV irradiation of HeLa cells promotes rapid Cdt1 degradation.

<p>(A) HeLa cells were irradiated with 20 and 50 J/m² UV and cells were analyzed after 0.5, 1, 3 and 6 hours. In addition, cells were cultured in the presence of the proteasome inhibitor MG-132 for 2 hr and then irradiated with 50 J/m² UV. Total protein extracts were prepared and subjected to western blot analysis using antibodies against Cdt1. Cdc2 was used as a loading control. (B) HeLa cells were irradiated with 2, 5 and 10 J/m² UV and incubated for 1 hour. Cells were fixed and stained with anti-Cdt1 (green) and anti-CPDs (red) antibodies. DNA was counterstained with DAPI (blue). Scale bars: B, 50 µm.</p

PCNA is involved in the Cdt1 proteolysis pathway.

<p>HeLa cells were transfected with 100 nM siRNAs for PCNA (PCNA RNAi) and Luciferase (Lucifer. RNAi) for 72 h. Subsequently, cells were either uncultured or cultured in the presence of MMS (600 µM) (lanes 1–3) for 3 h before cell lysis. Total cell lysates were prepared and analyzed by Western blot using antibodies against PCNA, Cdt1, and Tubulin.</p