Figure 3.

Viability of astrocytes and neurons after oxidative insult by different concentrations of H₂O₂ and the protective effects of Klotho. Neurons and astrocytes were pretreated with PBS or Klotho with different concentrations. After 24 h, the cells were exposed to a 30-min insult with H₂O₂ at different concentrations. Cellular viability was measured by MTT. (A) Astrocytes were challenged with different concentrations of H₂O₂. All concentrations tested were sufficient to reduce their viability compared to the control group. One-way ANOVA followed by Tukey’s multiple comparisons tests [F = 110.5; R squared = 0.8962; P < 0.0001, n = 12]. A concentration of 500 µM was selected. (B) Astrocytes were pretreated with different concentrations of Klotho and challenged with 500 µM H₂O₂. Results suggest that 1 nM Klotho alone was not able to alter cell viability, although 500 µM H₂O₂ decreased cell viability, as expected. The pretreatment with all different Klotho concentrations protected astrocytes from death. One-way ANOVA followed by Tukey’s multiple comparisons tests [F = 11.83; R squared = 0.5221; P < 0.0001, n = 4–12]. (C) Neurons were challenged with different concentrations of H₂O₂. All concentrations tested were sufficient to reduce their viability compared to the control group. One-way ANOVA followed by Tukey's multiple comparisons tests [F = 20.36; R squared = 0.8498; P < 0.0001, n = 4]. (d) Neurons were pretreated with 1 nM Klotho for 24 h and challenged with 500 µM H₂O₂ for 30 min. Compared to the control group, as expected, H₂O₂ altered neuronal viability but Klotho did not. However, pretreatment with Klotho did not avoid cell death caused by oxidative insult. Therefore, compared to the control group, both groups (H₂O₂ and H₂O₂ + Klotho) had a decreased viability. One-way ANOVA followed by Tukey’s multiple comparisons tests [F = 59.01; R squared = 0.9568; P < 0.0001, n = 3]. *P < 0.05; ***P < 0.0001.