Se: 59CAA-ACA-AAA-CAC-ATAAAA-ACA-ACA-39, U-MSP 34a Forward: 59GGG-GAT-GAG-GAT-TAG-GATTTT-39, M-MSP 34a Reverse: 59ACA-AAA-CGC-ATA-AAA-ACG-ACG-39, MMSP 34a Forward: 59GGG-GAT-GAG-GAT-TAG-GAT-TTC-39. PCR goods had been analyzed on 2 agarose gel. four / 15 Osteosarcoma Cell Response to Etoposide DNA Damage two.six Chromatin Immunoprecipitation assay DNA and protein complexes had been reversibly cross-linked in living cells by adding formaldehyde directly to cell culture medium at 1 final concentration to maintain the association of proteins with their target DNA sequence. Chromatin extract was then shared by sonication to DNA fragments with an typical size of 2001000 bps, cleared by centrifugation together with the addiction of sonicated salmon sperm DNA/protein A agarose. Precleared chromatin was buy Amezinium metilsulfate incubated overnight at four C on rotating plate with anti-p53 dilution:1:1000. Precipitation continued together with the addition of salmon sperm DNA/protein A agarose. Precipitates have been washed sequentially below stringent condition to get rid of unspecifically bound chromatin and were eluted. Cross-links had been reversed, proteins had been digested and ChiP DNA purified. DNA sequences linked with precipitated protein have been identified by PCR applying two mL of immunoprecipitated DNA and promoter-specific primers for miR34a promoter sequence containing p53 cis-elements. Immunoprecipitated DNA with non-specific immunoglobulins was regarded as as unfavorable manage. PCR products have been run on two agarose gel and visualized. 2.7 Cell cycle evaluation OS cells have been plated overnight at 1.56105 cells per well in 6-well plates and cell cycle distribution analysis was performed just before and just after 2448 h exposure to etoposide concentration corresponding to IC50. Soon after trypsinization and fixation with 70 ethanol, cells had been stained for total DNA 
content material having a answer containing 20 mg/ml propidium iodide. Cell cycle distribution was then analyzed using a FACScan flow cytometer. Cell fraction percentage was presented as imply from 3 independent experiments. two.eight Apoptosis measurement Apoptotic cell death was analyzed with Annexin V-FITC apoptosis detection kit. The green and red fluorescence of Annexin/propidium iodide -stained live cells and PI-stained fixed cells was analyzed having a 
FACSCalibur flow cytometer and Dan shen suan A CellQuest Application, working with a peak fluorescence gate to exclude cell aggregates. According to protocol, after 24 h and 48 h from transfection, adherent cells had been briefly trypsinized and re-suspended in 500 ml staining option containing FITC-conjugated Annexin V antibody and PI. Right after incubation, cells were analyzed by flow cytometry. Basal apoptosis and necrosis were identically determined on untreated cells making use of precisely the same process. Data were presented as mean SE from 3 independent experiments. five / 15 Osteosarcoma Cell Response to Etoposide DNA Damage 2.9 Co-immunoprecipitation and western blot evaluation Based on normal procedures, 300 mg of OS cell lysate had been immunoprecipitated with antibodies anti-p-p53 and antip53, fractioned by 8 SDSpolyacrylamide gel and transferred to nitrocellulose membranes. Western blot evaluation was performed by using anti-p-p53 and anti-p53 . Expression levels of total CDK4, cyclin D1 and CDK4 bound to cyclin D1 were determined before and following 48 h exposure to etoposide concentration corresponding to IC50. 250 mg of cell lysate have been immunoprecipitated with ten ml of antibodies to CDK4 and cyclin D1 adding Gamma Binding Plus Sepharose. Precipitates have been analy.Se: 59CAA-ACA-AAA-CAC-ATAAAA-ACA-ACA-39, U-MSP 34a Forward: 59GGG-GAT-GAG-GAT-TAG-GATTTT-39, M-MSP 34a Reverse: 59ACA-AAA-CGC-ATA-AAA-ACG-ACG-39, MMSP 34a Forward: 59GGG-GAT-GAG-GAT-TAG-GAT-TTC-39. PCR solutions were analyzed on 2 agarose gel. 4 / 15 Osteosarcoma Cell Response to Etoposide DNA Damage two.6 Chromatin Immunoprecipitation assay DNA and protein complexes had been reversibly cross-linked in living cells by adding formaldehyde straight to cell culture medium at 1 final concentration to sustain the association of proteins with their target DNA sequence. Chromatin extract was then shared by sonication to DNA fragments with an typical size of 2001000 bps, cleared by centrifugation with the addiction of sonicated salmon sperm DNA/protein A agarose. Precleared chromatin was incubated overnight at 4 C on rotating plate with anti-p53 dilution:1:1000. Precipitation continued using the addition of salmon sperm DNA/protein A agarose. Precipitates had been washed sequentially below stringent condition to remove unspecifically bound chromatin and had been eluted. Cross-links had been reversed, proteins were digested and ChiP DNA purified. DNA sequences related with precipitated protein have been identified by PCR applying two mL of immunoprecipitated DNA and promoter-specific primers for miR34a promoter sequence containing p53 cis-elements. Immunoprecipitated DNA with non-specific immunoglobulins was regarded as as negative control. PCR products were run on two agarose gel and visualized. 2.7 Cell cycle analysis OS cells had been plated overnight at 1.56105 cells per well in 6-well plates and cell cycle distribution analysis was performed prior to and following 2448 h exposure to etoposide concentration corresponding to IC50. Following trypsinization and fixation with 70 ethanol, cells were stained for total DNA content material with a resolution containing 20 mg/ml propidium iodide. Cell cycle distribution was then analyzed using a FACScan flow cytometer. Cell fraction percentage was presented as mean from three independent experiments. two.8 Apoptosis measurement Apoptotic cell death was analyzed with Annexin V-FITC apoptosis detection kit. The green and red fluorescence of Annexin/propidium iodide -stained live cells and PI-stained fixed cells was analyzed having a FACSCalibur flow cytometer and CellQuest Computer software, utilizing a peak fluorescence gate to exclude cell aggregates. Based on protocol, after 24 h and 48 h from transfection, adherent cells were briefly trypsinized and re-suspended in 500 ml staining remedy containing FITC-conjugated Annexin V antibody and PI. Immediately after incubation, cells have been analyzed by flow cytometry. Basal apoptosis and necrosis had been identically determined on untreated cells applying the identical process. Data had been presented as mean SE from three independent experiments. 5 / 15 Osteosarcoma Cell Response to Etoposide DNA Damage 2.9 Co-immunoprecipitation and western blot evaluation In accordance with common procedures, 300 mg of OS cell lysate have been immunoprecipitated with antibodies anti-p-p53 and antip53, fractioned by eight SDSpolyacrylamide gel and transferred to nitrocellulose membranes. Western blot analysis was performed by using anti-p-p53 and anti-p53 . Expression levels of total CDK4, cyclin D1 and CDK4 bound to cyclin D1 had been determined ahead of and immediately after 48 h exposure to etoposide concentration corresponding to IC50. 250 mg of cell lysate have been immunoprecipitated with 10 ml of antibodies to CDK4 and cyclin D1 adding Gamma Binding Plus Sepharose. Precipitates were analy.