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. 2016 Jul 29;7(34):54503–54514. doi: 10.18632/oncotarget.10950

Figure 5. The TAp73β-mediated Cisplatin induced cell death is opposed by the miR-193a-5p in human Bone Sarcoma cells.

Figure 5

a. The TAp73β's expression was assessed in the MG63 Osteosarcoma cell line both at mRNA level by qRT-qPCR (left panel) and at protein level by Western Blotting (right panel) forty-eight hours after the cell's transient transfection with the empty vector pcDNA3 or the TAp73β one. Glyceraldehyde-3-phosphate dehydrogenase and β2-microglobulin were used as housekeeping genes for qRT-PCR. Actin was used as a loading control for Immunoblotting. b. MG63 Osteosarcoma cell line was transiently transfected with either the empty vector pcDNA3 or the TAp73β one and was cultured forty-eight hours later in the presence of Cisplatin at the indicated concentrations for four days. The cell viability was determined by WST-1 assay and compared with control. The viability of the non-treated control was assigned as 100%. Error bars show the standard deviation for n = 2 measurements from representative experiments. An unpaired Student's t-test was used to compare the different conditions in the viability assays. c. MG63 cells were transiently transfected with either the pre-miR control or the pre-miR-193a-5p as the same time as pcDNA3 empty vector or the TAp73β containing-one. The cells were then cultured forty-eight hours later in the presence of 3 μM Cisplatin for additional forty-eight hours. The apoptosis was then evaluated by dosage of the caspase 3/7 activity in protein extracts. Error bars show the standard deviation for n = 3 measurements from representative experiments. A two-tailed paired Student's t-test was used to compare the different conditions in the caspase 3/7 activity assays. d. MG63 cells were transiently transfected with either the pre-miR control or the pre-miR-193a-5p as the same time as pcDNA3 empty vector or the TAp73β containing-one. The cells were then cultured forty-eight hours later in the presence of 3 μM Cisplatin at the indicated concentrations for additional forty-eight hours. The cell viability was determined by WST-1 assay and compared with control. The viability of the non-treated control was assigned as 100%.