Figure 2.

Hydralazine inhibited mitochondrial fragmentation and depolarization induced by oxidative stress. (A) Representative confocal images of HeLa cells showing mitochondrial fragmentation in induced by H₂O₂, an effect which was attenuated in cells pre-treated with hydralazine. Scale bars are 10 µm. (B) HeLa cells subjected to H₂O₂ displayed more mitochondrial fragmentation when compared to baseline cells (no H₂O₂), and this effect was attenuated in cells pre-treated with hydralazine. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison post-test. Data are expressed as mean±EM. ***P=0.0002. Baseline (no H₂O₂), N=3 independent experiments. Vehicle control+H₂O₂, N=5 independent experiments. Hydralazine+H₂O₂, N=6 independent experiments. (C) H₂O₂ induced mitochondrial membrane depolarization in HeLa cells, when compared to baseline cells (no H₂O₂), and this effect was attenuated in cells pre-treated with hydralazine. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison post-test. Data are expressed as mean±SEM. *P<0.05. **P<0.01. Baseline (no H₂O₂), N=3 independent experiments. Vehicle control+H₂O₂, N=5 independent experiments. Hydralazine+H₂O₂, N=6 independent experiments. (D) Representative confocal images of WT and Drp1 KO MEFs. Under baseline conditions (no H₂O₂), pre-treatment with hydralazine reduced mitochondrial fragmentation in WT MEFs but not in Drp1 KO MEFs. In response to H₂O₂, mitochondrial fragmentation was increased in both WT and Drp1 KO MEFs, and hydralazine attenuated mitochondrial fragmentation in WT MEFS, but not in Drp1 KO MEFs. Statistical analysis was performed using one-way ANOVA with Sidak’s multiple comparison post-test. Data are expressed as mean±SEM. N=11 independent experiments. For Drp1 WT: *P=0.0445. **P=0.0031 for vehicle control no H₂O₂ vs. hydralazine no H₂O₂ and ***P<0.0001 for vehicle control no H₂O₂ vs. vehicle control+H₂O₂. For Drp1 KO: ***P<0.0001. Scale bars are 10 µm. (E) H₂O₂ induced cell death in both WT and Drp1 KO MEFs, and hydralazine significantly attenuated cell death in WT MEFs but not in Drp1 KO MEFs. Statistical analysis was performed using one-way ANOVA with Sidak’s multiple comparison post-test. N =9 independent experiments. Data are expressed as mean±SEM. For Drp1 WT: ***P<0.0001. **P<0.0018. For Drp1 KO. ***P<0.0001. (F) H₂O₂ induced mitochondrial membrane depolarization in both WT and Drp1 KO MEFs, and this effect was attenuated in cells pre-treated with hydralazine. CCCP was used as a positive control to induce mitochondrial membrane depolarization. Statistical analysis was performed using one-way ANOVA with Sidak’s multiple comparison post-test. N=7 independent experiments. Data are expressed as mean±SEM. For Drp1 WT: **P=0.0047 vehicle control+H₂O₂ vs. hydralazine+H₂O₂ and **P=0.0081 for vehicle control no H₂O₂ vs. CCCP. For Drp1 KO: *P=0.0337. (G) H₂O₂ induced the formation of mitochondrial ROS in both WT and Drp1 KO MEFs, and this effect was attenuated in cells pre-treated with hydralazine. N-acetyl cysteine (NAC) was used as a positive control for scavenging ROS. Statistical analysis was performed using one-way ANOVA with Sidak’s multiple comparison post-test. N=7 independent experiments. Data are expressed as mean±SEM. For Drp1 WT: ***P<0.0001 vehicle control no H₂O₂ vs. vehicle control+H₂O₂ and ***P<0.0001 for vehicle control+H₂O₂ vs. hydralazine+H₂O₂ and ***P<0.0001 vehicle control+H₂O₂ vs. NAC+H₂O₂. For Drp1 KO: ***P<0.0001 vehicle control no H₂O₂ vs. vehicle control+H₂O₂ and ***P=0.0008 for vehicle control+H₂O₂ vs. hydralazine+H₂O₂ and ***P<0.0001 vehicle control+H₂O₂ vs. NAC+H₂O₂.