Figure 6
MT2A silencing amplifies osteosarcoma cell response to cytotoxic agents. U2OS cell line was transduced with an integrative lentiviral vector encoding a specific shRNA sequence targeting MT2A (sh-MT2A). (a) MT2A mRNA levels were evaluated by RT-qPCR in U2OS parental and MT2A-silenced cells. (b) MT2A protein levels were evaluated by western blot analysis in U2OS parental and MT2A-silenced cells. (c) U2OS parental and MT2A-silenced cells were incubated with increasing doses of ZnCl2. Free intracellular zinc content was evaluated by fluorimetry. (d) U2OS parental and MT2A-silenced cells were incubated with or without cisplatin, transplatin, doxorubicin or vincristine for 48 h. Cell viability was evaluated using the MTT test. (e) Relative amounts of doxorubicin combined to DNA were evaluated by flow cytometry into parental, silenced and overexpressing U2OS cells. (f) Relative amounts of Hoechst 33342 bound to DNA were evaluated by flow cytometry into parental, silenced and overexpressing U2OS cells. Results are expressed as mean±S.D. *P<0.05 versus untreated cells; #P<0.05 versus parental cells
