Pen conformation and p53 protein expression. 3.5 Chromatin 
Immunoprecipitation assay To verify that the capacity of p53 protein to bind the promoter of miR-34a target gene is just not compromised by mutation at web page 175 we performed ChIP assay. The 7 / 15 Osteosarcoma Cell Response to Etoposide DNA Damage Fig. two. Growth-inhibition assay. U2-OS and U2-OS175 cells showed a equivalent viability Trend with higher sensitivity to etoposide at 72 h than MG63 and Saos-2 cells. No differences between U2- OS and U2-OS/e have been observed. Data have been presented as imply SE from three independent experiments. order M2I-1 Student’s test indicated drastically lower 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:10.1371/journal.pone.0114757.g002 analysis showed binding in between p53 plus the promoter of miR-34a in U2-OS and U2-OS175 cells, but not in the p53-deficient cell lines, MG63 and Saos-2 suggesting that enhance of miR-34a expression in response to etoposide was dependent on p53 recruitment and not impaired by expression of dominant damaging p53. Fig. 3. PQR620 chemical information RT-PCR analysis of miR-34a. Improved expression of miR-34a was seen in U2-OS, U2-OS/e and in U2-OS175 in response to etoposide therapy at 24 h and 48 h respectively. No relevant changes had been evident in p53-deficient MG63 and Saos-2, also displaying reduce basal miR-34a levels. Information were presented as mean SE from three 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 certain PCR. The position of p53 binding web site and primers for wild-type and methylation sequences on CpG area are indicated. Just after bisulphite treatment, U2-OS, U2-OS/e and U2-OS175 showed total unmethylation of miR34a promoter; in contrast MG63 and Saos-2 cells presented methylation of both alleles. doi:ten.1371/journal.pone.0114757.g004 3.six Cell cycle distribution and co-immunoprecipitation Soon after 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 decrease in S phase. Even though a lowered G1 accumulation in U2-OS175 cells was anticipated, offered the expression of dominant unfavorable p53, slight changes in cell cycle distribution were seen following etoposide remedy. 
No significant differences had been observed between U2OS and U2-OS/e cells. p53-deficient MG63 responded to etoposide with a marked Fig. 5. Chromatin Immunoprecipitation assay. Interaction between p53 and miR-34a promoter was present in both U2-OS and U2-OS175. INPUT5positive control; IgG5negative manage. doi:10.1371/journal.pone.0114757.g005 9 / 15 Osteosarcoma Cell Response to Etoposide DNA Harm Fig. six. Cell cycle analysis and apoptosis. 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 in comparison to untreated cells. By Annexin V-FITC assay, no significant improve of apoptotic cells was observed in OS cell lines soon after 24 h and 48 h of treatment. Data have been presented as imply SE from 3 independent experiments. C5Untreated cells. doi:ten.1371/journal.pone.0114757.g006 accumulation of G2/M, concomitant with pronounced cell lower 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. three.5 Chromatin Immunoprecipitation assay To verify that the capability of p53 protein to bind the promoter of miR-34a target gene will not be compromised by mutation at web 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 comparable viability Trend with greater sensitivity to etoposide at 72 h than MG63 and Saos-2 cells. No differences involving U2- OS and U2-OS/e had been observed. Information have been presented as imply SE from three independent experiments. Student’s test indicated drastically reduced 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 among p53 and also the promoter of miR-34a in U2-OS and U2-OS175 cells, but not in the p53-deficient cell lines, MG63 and Saos-2 suggesting that boost of miR-34a expression in response to etoposide was dependent on p53 recruitment and not impaired by expression of dominant adverse p53. Fig. 3. RT-PCR evaluation of miR-34a. Increased expression of miR-34a was noticed in U2-OS, U2-OS/e and in U2-OS175 in response to etoposide treatment at 24 h and 48 h respectively. No relevant modifications have been evident in p53-deficient MG63 and Saos-2, also displaying reduced basal miR-34a levels. Data have been presented as imply SE from three independent experiments. doi:ten.1371/journal.pone.0114757.g003 8 / 15 Osteosarcoma Cell Response to Etoposide DNA Damage Fig. 4. miR-34a gene genomic organization and methylation particular PCR. The position of p53 binding web site and primers for wild-type and methylation sequences on CpG area are indicated. After bisulphite treatment, U2-OS, U2-OS/e and U2-OS175 showed total unmethylation of miR34a promoter; in contrast MG63 and Saos-2 cells presented methylation of both alleles. doi:10.1371/journal.pone.0114757.g004 three.6 Cell cycle distribution and co-immunoprecipitation Soon after 48 h exposure to IC50 etoposide, BrDU incorporation showed a distinctive cell cycle distribution in OS cell lines. U2-OS cells presented cell accumulation in G1 phase, and G2/M accompanied by a relevant cell decrease in S phase. Even though a reduced G1 accumulation in U2-OS175 cells was anticipated, provided the expression of dominant adverse p53, slight alterations in cell cycle distribution have been noticed right after etoposide therapy. No significant differences had been observed amongst U2OS and U2-OS/e cells. p53-deficient MG63 responded to etoposide using a marked Fig. five. Chromatin Immunoprecipitation assay. Interaction amongst p53 and miR-34a promoter was present in both U2-OS and U2-OS175. INPUT5positive manage; IgG5negative control. doi:10.1371/journal.pone.0114757.g005 9 / 15 Osteosarcoma Cell Response to Etoposide DNA Harm Fig. six. Cell cycle analysis and apoptosis. Soon after 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 when compared with untreated cells. By Annexin V-FITC assay, no significant enhance of apoptotic cells was observed in OS cell lines just after 24 h and 48 h of therapy. Data have been presented as mean SE from three 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 sturdy decr.
