Pen conformation and p53 protein expression. 3.five Chromatin Immunoprecipitation assay To confirm that the capacity of p53 protein to bind the promoter of miR-34a target gene is not compromised by mutation at site 175 we performed ChIP assay. The 7 / 15 Osteosarcoma Cell Response to Etoposide DNA Damage Fig. 2. Growth-inhibition assay. U2-OS and U2-OS175 cells showed a similar viability Trend with larger sensitivity to etoposide at 72 h than MG63 and Saos-2 cells. No variations between U2- OS and U2-OS/e had been observed. Information have been presented as mean SE from three independent experiments. Student’s test indicated significantly decrease IC50 mean values at 72 h of remedy in U2-OS, U2-OS/e and in U2-OS175 than in p53-deficient Saos-2 and MG63. doi:ten.1371/journal.pone.ARS-853 chemical information 0114757.g002 evaluation showed binding between p53 plus the promoter of miR-34a in U2-OS and U2-OS175 cells, but not within the p53-deficient cell lines, MG63 and Saos-2 suggesting that increase of miR-34a expression in response to etoposide was dependent on p53 recruitment and not impaired by expression of dominant negative p53. Fig. 3. RT-PCR analysis of miR-34a. Increased expression of miR-34a was seen in U2-OS, U2-OS/e and in U2-OS175 in response to etoposide remedy at 24 h and 48 h respectively. No relevant alterations had been evident in p53-deficient MG63 and Saos-2, also displaying decrease basal miR-34a levels. Data have been presented as mean SE 
from 3 independent experiments. doi:10.1371/journal.pone.0114757.g003 8 / 15 Osteosarcoma Cell Response to Etoposide DNA Damage Fig. 4. miR-34a gene genomic organization and methylation precise PCR. The position of p53 binding web site and primers for wild-type and methylation sequences on CpG region are indicated. Immediately after bisulphite therapy, U2-OS, U2-OS/e and U2-OS175 showed full unmethylation of miR34a promoter; in contrast MG63 and Saos-2 cells presented methylation of each alleles. doi:ten.1371/journal.pone.0114757.g004 three.6 Cell cycle distribution and co-immunoprecipitation Following 48 h exposure to IC50 etoposide, BrDU incorporation showed a diverse cell cycle distribution in OS cell lines. U2-OS cells presented cell accumulation in G1 phase, and G2/M accompanied by a relevant cell reduce in S phase. While a reduced G1 accumulation in U2-OS175 cells was anticipated, offered the expression of dominant adverse p53, slight modifications in cell cycle distribution had been observed after etoposide remedy. No substantial differences had been observed in PubMed ID:http://jpet.aspetjournals.org/content/123/3/180 between U2OS and U2-OS/e cells. p53-deficient MG63 responded to etoposide having a marked Fig. 5. Chromatin Immunoprecipitation assay. Interaction between p53 and miR-34a promoter was present in both U2-OS and U2-OS175. INPUT5positive handle; IgG5negative control. doi:ten.1371/journal.pone.0114757.g005 9 / 15 Osteosarcoma Cell Response to Etoposide DNA Damage Fig. 6. Cell cycle analysis and apoptosis. Following 48 h of etoposide remedy, BrDU incorporation showed cell accumulation in G1 phase in U2-OS and U2-OS175 cells and in G2/M phase in MG63 and Saos-2 when in comparison to untreated cells. By Annexin V-FITC assay, no important boost of CTX-0294885 (hydrochloride) biological activity apoptotic cells was observed in OS cell lines soon after 24 h and 48 h of treatment. Information had been presented as mean SE from 3 independent experiments. C5Untreated cells. doi:ten.1371/journal.pone.0114757.g006 accumulation of G2/M, concomitant with pronounced cell reduce in G1 and S phase. Similarly, in Saos-2 cells, etoposide-induced cell accumulation in G2/M accompanied by a powerful decr.Pen conformation and p53 protein expression. 3.five Chromatin Immunoprecipitation assay To confirm that the capacity of p53 protein to bind the promoter of miR-34a target gene is just not compromised by mutation at web-site 175 we performed ChIP assay. The 7 / 15 Osteosarcoma Cell Response to Etoposide DNA Harm Fig. 2. Growth-inhibition assay. U2-OS and U2-OS175 cells showed a comparable viability Trend with greater sensitivity to etoposide at 72 h than MG63 and Saos-2 cells. No differences in between U2- OS and U2-OS/e were observed. Information have been presented as imply SE from 3 independent experiments. Student’s test indicated significantly reduce IC50 mean values at 72 h of remedy in U2-OS, U2-OS/e and in U2-OS175 than in p53-deficient Saos-2 and MG63. doi:ten.1371/journal.pone.0114757.g002 evaluation showed binding between p53 as well as the promoter of miR-34a in U2-OS and U2-OS175 cells, but not within the p53-deficient cell lines, MG63 and Saos-2 suggesting that raise of miR-34a expression in response to etoposide was dependent on p53 recruitment and not impaired by expression of dominant negative p53. Fig. three. RT-PCR evaluation of miR-34a. Improved expression of miR-34a was seen in U2-OS, U2-OS/e and in U2-OS175 in response to etoposide remedy at 24 h and 48 h respectively. No relevant changes were evident in p53-deficient MG63 and Saos-2, also displaying reduce basal miR-34a levels. Information had been presented as 
imply SE from three independent experiments. doi:ten.1371/journal.pone.0114757.g003 eight / 15 Osteosarcoma Cell Response to Etoposide DNA Harm Fig. four. miR-34a gene genomic organization and methylation precise PCR. The position of p53 binding web site and primers for wild-type and methylation sequences on CpG area are indicated. Following bisulphite treatment, U2-OS, U2-OS/e and U2-OS175 showed complete unmethylation of miR34a promoter; in contrast MG63 and Saos-2 cells presented methylation of both alleles. doi:ten.1371/journal.pone.0114757.g004 three.6 Cell cycle distribution and co-immunoprecipitation After 48 h exposure to IC50 etoposide, BrDU incorporation showed a various cell cycle distribution in OS cell lines. U2-OS cells presented cell accumulation in G1 phase, and G2/M accompanied by a relevant cell lower in S phase. Though a reduced G1 accumulation in U2-OS175 cells was expected, provided the expression of dominant negative p53, slight modifications in cell cycle distribution had been seen just after etoposide treatment. No significant differences were observed among U2OS and U2-OS/e cells. p53-deficient MG63 responded to etoposide using a marked Fig. five. Chromatin Immunoprecipitation assay. Interaction in between p53 and miR-34a promoter was present in each U2-OS and U2-OS175. INPUT5positive manage; IgG5negative handle. doi:10.1371/journal.pone.0114757.g005 9 / 15 Osteosarcoma Cell Response to Etoposide DNA Damage Fig. six. Cell cycle analysis and apoptosis. Immediately after 48 h of etoposide therapy, BrDU incorporation showed cell accumulation in G1 phase in U2-OS and U2-OS175 cells and in G2/M phase in MG63 and Saos-2 when compared to untreated cells. By Annexin V-FITC assay, no important raise of apoptotic cells was observed in OS cell lines following 24 h and 48 h of remedy. Information were presented as mean SE from 3 independent experiments. C5Untreated cells. doi:10.1371/journal.pone.0114757.g006 accumulation of G2/M, concomitant with pronounced cell reduce in G1 and S phase. Similarly, in Saos-2 cells, etoposide-induced cell accumulation in G2/M accompanied by a robust decr.