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. 2012 Sep 25;7(9):e46185. doi: 10.1371/journal.pone.0046185

Figure 3. Dasatinib reduces cytotoxicity of mutant SOD1s in NSC-34 cells.

Figure 3

A: Protein levels of phosphorylated c-Abl (Tyr245), c-Abl, phosphorylated c-Src (Tyr416), c-Src, and GAPDH in NSC-34 cells overexpressing human wild-type or mutant SOD1s treated with various concentrations of dasatinib or SU6656 were measured by western blot. Cells were cultured in serum-free culture medium with doxycycline (Dox, 2 µg/ml), and western blot was performed at 24 h after dasatinib or SU6656 addition. B: Cells were grown in 96-well collagen-coated plates (3,500 cells per well) with doxycycline (Dox, 2 µg/ml) in culture medium containing 10% FBS for 16 h. Culture medium was then replaced with 1% FBS-containing medium including the indicated concentrations of dasatinib and 2 µg/ml doxycycline (Dox). MTS assays were performed at 24 h after addition of dasatinib or SU6656. Viability was measured as the level of absorbance at 490 nm. Absorbance at 490 nm was expressed as the mean ± SEM (n = 6). Ratios of relative cell viability based on the MTS assay were calculated to determine the beneficial effect of dasatinib in mutant cells overexpressing SOD1s. Absorbance at 490 nm was standardized relative to the absorbance at each corresponding time point for 0 nM dasatinib. Cell viability assay confirmed that dasatinib significantly reduced the cytotoxicity of mutant SOD1s, whereas SU6656 did not. Statistics were evaluated using 1-way ANOVA with Dunnett's post-hoc test. *P<0.05, **P<0.01. C: Cells were grown in 96-well collagen-coated plates (3,500 cells per well) with doxycycline (Dox, 2 µg/ml) in culture medium containing 10% FBS for 16 h. Culture medium was then replaced with 1% FBS-containing medium with the indicated concentrations of dasatinib and 2 µg/ml doxycycline (Dox). LDH assays were performed at 24 h after dasatinib or SU6656 addition. Cytotoxicity was measured as the level of absorbance at 490 nm. Ratios of relative LDH release were calculated to determine the beneficial effect of dasatinib in mutant cells overexpressing SOD1s. Absorbance at 490 nm was standardized relative to the absorbance at each corresponding time point for 0 nM dasatinib. LDH assay confirmed that dasatinib significantly reduced the cytotoxicity of mutant SOD1s, whereas SU6656 did not. Values represent the mean ± SEM of the ratio of LDH release (n = 4). Statistics were evaluated using 1-way ANOVA with Dunnett's post-hoc test. *P<0.05, **P<0.01.