Figure 5.

Treatment of human cells. Human osteoclasts transfected with the indicated expression vectors were treated for 48 hours with the indicated concentrations of (a) Clcn7^G215R-, (b) Clcn7^R767W-, and (c) Clcn7^R286W-specific siRNAs. Real-time RT-PCR using primer pairs specific for EGFP, normalized with GAPDH. (d) Osteoclasts were generated from the peripheral blood mononuclear cells of CLCN7^G215R/WT ADO2 patient onto bone slices and treated with the indicated concentration of SCR and Clcn7^G215R-specific siRNA. At the end of experiment, cells were removed by sonication and bone resorption evaluated by the pit assay. (e) Human breast cancer cells MDA-MB-231 (MDA) were transfected with Clcn7^WT-EGFP or Clcn7^R767W-EGFP and treated for 96 hours with the indicated concentrations of Clcn7^R767W-specific siRNA. Real-time RT-PCR using primer pairs specific for EGFP, normalized with GAPDH. (f) Balb/c nu/nu atymic mice were subcutaneously inoculated in both flanks with MDA cells transfected with Clcn7^R767W-EGFP expression vector. When tumors reached the volume of 1 cm³, animals were treated with 4 mg/kg body weight of Clcn7^R767W-specific siRNA. After 96 hours, tumors were excised, RNA extracted, and evaluated for Clcn7^R767W expression by real time RT-PCR, using primer pairs specific for EGFP, normalized with GAPDH. Results are the mean ± SD of (d)one single experiment, (a-c,e) three independent experiments and (f) three mice/group. a–c,f: Student's t-test; e: one-way analyis of variance.