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Time course of H2A.X phosphorylation after SAHA and Actinomycin D treatment. LA1-55n cells were treated (+) or not (-) with 0.1 nM of actinomycin D (ActD) in the presence (+) or in the absence (-) of 1 μ M SAHA. Indicated protein expression was determined by Western blot analysis at the indicated times after the treatment. (DOCX 710 kb

E2F1 enhanced ROS production is essential for the apoptotic function.

<p>(A) PC12 ER-E2F1 cells were serum deprived and treated with OHT for the indicated hours. ROS levels were analysed by using the oxidation-sensitive fluorescent probe H2DCFDA as described in Material and Methods. Results are presented as Mean ± SEM, for n = 6 (B) Stable ER-E2F1 PC12 cells were treated with (+) or without OHT in the presence (+) or in the absence (−) of 1 mM NAC, or in the presence (+) or in the absence of 40 mM LiCl or serum for 4 hours. After cell collection and lysis, caspase-3 activity of cell extracts was analysed by using the <i>p</i>NA colorimetric assay as indicated in Material and Methods. Results are presented as Mean ± SEM, for n = 3. (C) ER-E2F1 cells were transiently transfected with YFP-Bax, treated for 3 hours with (+) or without (−) OHT in the presence (+) or absence (−) of 1 mM NAC, and the punctuated mitochondrial clusters of YFP-Bax were quantified by immunostaining analysis. Results are presented as Mean ± SEM, for n = 3. In all of the figures, data are compared as indicated individually. Student’s <i>t</i>-test values of *p<0,05, **p<0,01 and ***p<0,0001 were considered statistically significant.</p

Heatmap of oxidative and antioxidative genes regulated by E2F1.

<p>mRNA from ER-E2F1 SH-SY5Y was isolated from OHT treated or not treated cells and quantified using the RT² qPCR array platform as described in Material and Methods. All OHT regulated genes were compared against controls, using Hypoxanthine phosphoribosyltransferase 1 gene as the reference. The relative magnitude of expression is indicated on a spectrum ranging from minimum (green) to the maximum detected (red). All genes tested in the array are listed on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051544#pone.0051544.s004" target="_blank">Table S1</a>.</p

mRNA levels changes after OHT addition in SH-SY5Y cells.

<p>RT²Profiler human oxidative stress and antioxidant defense PCR Arrays (Bioscience) were performed according to the manufacture’s protocols. Expression levels were compared between with and without OHT addition. Hypoxanthine phosphoribosyltransferase 1 gene was used as control for each gene expression calculation, and the extent of change in the expression of each gene was calculated by the ΔC_t method. Only genes whose expression was downregulated at least 2 fold are shown in the Table. We have also removed all genes whose expression change significantly between duplicates. When ΔC_t was over 12 and therefore expression was thought to be extremely low, the gene was omitted from analysis.</p

Apoptotic function of E2F1 is correlated with its ability to induce ROS accumulation.

<p>(A) Indicated SH-SY5Y and SK-N-JD neuroblastoma ER-E2F1 stable cell lines were treated (+) or not treated (−) with OHT, in the presence (+) or absence (−) of serum for 4 hours. Cell extracts were collected and lysed, and caspase-3 activities (left panels) and ROS levels (right panels) were analysed as described in Material and Methods. (B) Indicated SH-SY5Y and SK-N-JD neuroblastoma ER-E2F1 stable cell lines were treated (OHT) or not treated (control) with OHT for 4 hours. Cells were immunostained for E2F1 protein (red) and DAPI (blue). (C) Indicated SH-SY5Y and SK-N-JD neuroblastoma ER-E2F1 stable cell lines were transfected for 16 hours with a E2F luciferase reported construct and treated with (+) or without (−) OHT. Luciferase was measured and normalized for <i>Renilla</i> luciferase readings in the same extracts and the values were indicated. Results are presented as Mean ± SEM, for n = 4. In all figures, Student’s <i>t</i>-test values of *p<0,05, **p<0,01 and ***p<0,0001 were considered statistically significant.</p

LiCl inhibits ROS production and Bax activation induced by E2F1.

<p>(A) PC12 ER-E2F1 cells were serum deprived and treated with OHT (+) or not (−) with OHT, in the presence (+) or in the absence (−) of 40 mM LiCl for 4 hours. ROS levels were analysed by using the oxidation-sensitive fluorescent probe H2DCFDA as described in Material and Methods. Results are presented as Mean ± SEM, for n = 4. (B) PC12 ER-E2F1 cells were transiently transfected with an YFP-Bax plasmid, serum deprived and treated with OHT (+) or not (−) with OHT, in the presence or absence of 40 mM LiCl for 3 hours. Quantification of the punctuated mitochondrial clusters of YFP-Bax achieved in the immunofluorescence assay is described in Material and Methods. Results are presented as Mean ± SEM, for n = 3. In all of the figures, data are compared as indicated individually. Student’s <i>t</i>-test values of *p<0,05 and **p<0,01were considered statistically significant.</p

E2F1 does not regulate the ROS levels through Nox, but is capable to modulate NF-κB activity.

<p>(A) PC12 ER-E2F1 cells were serum deprived and treated with (+) or without (−) OHT in the presence (+) or absence (−) of DPI for 4 hours. After cell collection and lysis, caspase-3 activity of cell extracts was analysed by using the <i>p</i>NA colorimetric assay as indicated in Material and Methods. (B) Stable ER-E2F1 PC12 cells were treated with (+) or without OHT, in the presence (+) or in the absence (−) of 1 mM NAC, or DPI for 4 hours. ROS levels were analysed by using the oxidation-sensitive fluorescent probe H2DCFDA as described in Material and Methods. (C) Serum-deprived cells were treated (+) or not treated (−) with OHT for the indicated hours. Expression of the referred proteins was determined by Western Blot analysis. (D) Stable ER-E2F1 cells were transfected for 16 hours with a NF-κB reported construct and treated with (+) or without (−) OHT in the presence (+) or absence (−) of 1 mM NAC for 4 hours. Luciferase was measured and normalized with protein content. Results are presented as Mean ± SEM, for n = 3. In all of the figures, data are compared as indicated individually. Student’s <i>t</i>-test values of *p<0,05, **p<0,01 and ***p<0,0001 were considered statistically significant.</p

Effect of over-expression of Bcl-xL and cFLIP in E2F1-induced apoptosis.

<p>Stable ER-E2F1 PC12 cells were transfected or not with Bcl-xL (A) or cFLIP (B) expression plasmids and treated (+) or not treated (−) with OHT. At 48 hours, apoptotic cells were identified by TUNEL and counted against 100 nuclei identified by Hoescht staining in randomly taken photographs. Results are presented as Mean ± SEM, for n = 3. Statistically significant differences were obtained comparing with cells untransfected. Student’s <i>t</i>-test value of **p<0,01 was considered statistically significant. Expression of the transfected proteins were measured by Western Blot analysis and shown on the right of the figures. HA antibody was used for Bcl-xL detection and FLAG for FLIP. Arrow indicates FLIP position and asterisk a non specific protein.</p

Expression analysis of reported E2F1-target genes in ER-E2F1 PC12 cells.

<p>(A) Serum-deprived cells were treated (+) or not treated (−) with OHT for the indicated hours. Expression of the indicated proteins was determined by Western Blot analysis. (B) Total mRNA was extracted and the levels of indicated transcripts were analyzed by RT-PCR. (C) ER-E2F1 PC12 cells were serum deprived and treated with OHT for the indicated hours, or with 400 µM of H₂O₂ for 6 hours. Mitochondrial fractions and cytoplasmic (cyto) fractions were obtained and analyzed for the indicated protein contents by Western blot analysis.</p

Additional file 1: Figure S1. of Effect of low doses of actinomycin D on neuroblastoma cell lines

Cell cycle distribution after actinomycin D treatment. Indicated cell lines were treated with 10 nM of actinomycin D in the presence or in the absence of 20 μM of Oph-QVD for 24 h or 48 h. Cell cycle distribution was detected by the propidium iodide staining method and indicated in the histograms. (DOCX 866 kb