Pen conformation and p53 protein expression. 3.five Nutlin3 Chromatin Immunoprecipitation assay To verify that the ability of p53 protein to bind the promoter of miR-34a target gene will not be compromised by mutation at site 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 related viability Trend with larger 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 3 independent NP-031112 experiments. Student’s test indicated substantially 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 between p53 and 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 unfavorable p53. Fig. three. RT-PCR analysis of miR-34a. Improved expression of miR-34a was observed 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 had 
been evident in p53-deficient MG63 and Saos-2, also displaying decrease basal miR-34a levels. Information were presented as imply SE from 3 independent experiments. doi:10.1371/journal.pone.0114757.g003 eight / 15 Osteosarcoma Cell Response to Etoposide DNA Harm Fig. 4. miR-34a gene genomic organization and methylation specific PCR. The position of p53 binding website and primers for wild-type and methylation sequences on CpG region 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 each alleles. doi:10.1371/journal.pone.0114757.g004 three.six Cell cycle distribution and co-immunoprecipitation Immediately after 48 h exposure to IC50 etoposide, BrDU incorporation showed a different 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 damaging p53, slight modifications in cell cycle distribution were seen soon after etoposide treatment. 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 amongst p53 and miR-34a promoter was present in both U2-OS and U2-OS175. INPUT5positive handle; IgG5negative handle. doi:10.1371/journal.pone.0114757.g005 9 / 15 Osteosarcoma Cell Response to Etoposide DNA Harm Fig. 6. Cell cycle evaluation 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 compared to untreated cells. By Annexin V-FITC assay, no substantial increase of apoptotic cells was observed in OS cell lines right after 24 h and 48 h of therapy. Information were 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 strong decr.Pen conformation and p53 protein expression. three.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 site 175 we performed ChIP assay. The 7 / 15 Osteosarcoma Cell Response to Etoposide DNA Harm 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 involving U2- OS and U2-OS/e had been observed. Information had been presented as imply SE from three independent experiments. Student’s test indicated drastically reduced IC50 imply 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 analysis showed binding among p53 along with the promoter of miR-34a in U2-OS and U2-OS175 cells, but not inside 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. 3. RT-PCR analysis of miR-34a. Enhanced 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 adjustments were evident in p53-deficient MG63 and Saos-2, also showing lower basal miR-34a levels. Information had been presented as imply SE from 3 independent experiments. doi:ten.1371/journal.pone.0114757.g003 8 / 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 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 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 unique 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 lowered G1 accumulation in U2-OS175 cells was anticipated, provided the expression of dominant adverse p53, slight adjustments in cell cycle distribution had been noticed right after etoposide remedy. No significant differences have 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 using a marked Fig. 5. Chromatin Immunoprecipitation assay. Interaction amongst p53 and miR-34a promoter was present in each U2-OS and U2-OS175. INPUT5positive handle; IgG5negative manage. doi:ten.1371/journal.pone.0114757.g005 9 / 15 Osteosarcoma Cell Response to Etoposide DNA Harm Fig. six. Cell cycle analysis and apoptosis. Just after 48 h of etoposide treatment, 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 significant improve of apoptotic cells was observed in OS cell lines immediately after 24 h and 48 h of remedy. Data 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 decrease in G1 and S phase. Similarly, in Saos-2 cells, etoposide-induced cell accumulation in G2/M accompanied by a powerful decr.
