Figure 4. The TAp73β's targeting miR-193a-5p is implicated in the Cisplatin chemoresistance of human Bone Sarcomas.

a. miR-193a-5p expression was assessed by qRT-PCR in the RDES Ewing Sarcoma cell line forty-eight hours after either the pre-miR control or the pre-miR-193a-5p mimic's transfection. The expression of TAp73β b., p21 c. and MDM2 d. were assessed by qRT-PCR in the same conditions as described in (a). RNU6B, Glyceraldehyde-3-phosphate dehydrogenase and β2-microglobulin were used as housekeeping genes for qRT-PCR and the error bars show the standard deviation for n = 3 measurements from representative experiments. e. RDES Ewing Sarcoma cell line was transiently transfected in the same conditions as in (a) and was treated forty-eight hours later with 3 μM Cisplatin or the same amount of NaCl 0.9% 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. f. Protein's extracts from RDES Ewing Sarcoma cell line in same conditions as in (a) were subjected to Immunoblotting with anti-cleaved-PARP antibodies. Glyceraldehyde-3-phosphate dehydrogenase was used as loading control. g. RDES Ewing Sarcoma cell line was transiently transfected with either pre-miR control or pre-miR-193a-5p mimics and was cultured forty-eight hours later in the presence of Cisplatin at the indicated concentrations for two 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%. A two-way ANOVA test was used to compare the different conditions in the viability assays. Data refer to three different experiments and western blot images are representative of these. Black lines show where the original gel was cropped to obtain the final image