Research Paper Advance Articles

Figure 6. FGF21 mitigates cytotoxicity in NSC-34 and ALS motor neurons induced oxidative stress. (A) The viability of NSC-34 cells expressing FLAG-tagged WT-SOD1 or SOD1^G93A was determined using the Vialight assay. Viability for WT SOD1-transfected cells was set at 1. ****P < 0.0001, unpaired two-tailed t-test. (B) Caspase activation was measured in the same cells and values were normalized to activity in WT SOD1-transfected cells which was set at 1. ****P < 0.0001, unpaired two-tailed t-test. (C) FGF21 protein in the conditioned media of NSC-34 cells transfected with a FLAG-tagged FGF21 plasmid was detected by western blot (upper panel) and by ELISA (graph). ****P < 0.0001; unpaired two-tailed t-test. (D, E) NSC-34 cells expressing either WT-SOD1 or SOD1^G93A were transfected with FLAG-FGF21 and assessed for viability as in (A) and Caspase-3/7 as in (B). ****P < 0.0001, unpaired two-tailed t-test. (F) NSC-34 cells expressing either WT SOD1 or SOD1^G93A were treated with recombinant FGF21 (100 ng/ml) and assessed for viability as in (A). **P < 0.01, unpaired two-tailed t-test. (G) Cell viability was assessed in NSC-34 cells exposed to methionine-cystine (MetCys)-deprived media or treated with 100 mM H₂O₂. ****P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparisons test. (H) NSC-34 cells transfected with FLAG-FGF21 (or empty vector) were subjected to stressors as described in (G) for 24h and then assayed for viability. **P = 0.007, ***P = 0.0002; unpaired two-tailed t-test. (I) iPSC motor neurons derived from C9-ALS patients or normal donors were exposed to different doses of H₂O₂ as indicated, and cell viability was assessed 24 h after treatment. **P < 0.01; unpaired two-tailed t-test with Welch’s correction. (J) iPSC motor neurons exposed to 12.5 μM of H₂O_2, were treated with different doses (0, 25 and 50 ng/ml) of recombinant FGF21 and cell viability was assessed as in (I). *P < 0.05, **P < 0.01; paired two-tailed t-test among the different doses of rec-FGF21 treatment in C9-ALS groups and unpaired two-tailed test in normal vs. C9-ALS groups. Data points represent biological replicates and bars are the mean ± SD.