Research Paper Volume 13, Issue 18 pp 22188—22207

Cx32 inhibits the autophagic effect of Nur77 in SH-SY5Y cells and rat brain with ischemic stroke

Nur77 attenuated mitochondrial dysfunction after the inhibition of Cx32 following OGD/R injury. Cells were transfected with Cx32 siRNA or Nur77 siRNA for 24 h followed by OGD/R (2 h OGD following by 24 h recovery) before harvested. (A) The level of ROS was detected by DCF-DA. Scale bars, 100 μm. (B) Mitochondria was labeled with GFP (green); cell nuclei were counterstained with DAPI (blue). Photomicrographs were captured under a Nikon Ni-U fluorescence microscope. Scale bars, 50 μm. (C) Δψm was detected by JC-1 dye. Images were shown as the ratio of JC-1 aggregates to JC-1 monomers. Scale bars, 50 μm. (D–E) Representative bands of p-Drp-1, COX4 and TOMM20 protein after OGD/R. Variation in protein loading was determined by blotting with an anti-β-actin antibody. Densitometric scanning of band intensities were calculated as means ± SD (n = 3). **p ##p F) Images were collected by transmission electron microscope. Green square represents normal mitochondria with cylindrical shape. Their cristae was well-defined, the double membranes was intact and the density was homogenous; Red square represents abnormal mitochondria. Their cristae was disordered, double membrane was discontinuous, electron density was decreased and minor axis was increased consistent with mitochondrial swelling. Scale bars, 0.5 μm.

Figure 3. Nur77 attenuated mitochondrial dysfunction after the inhibition of Cx32 following OGD/R injury. Cells were transfected with Cx32 siRNA or Nur77 siRNA for 24 h followed by OGD/R (2 h OGD following by 24 h recovery) before harvested. (A) The level of ROS was detected by DCF-DA. Scale bars, 100 μm. (B) Mitochondria was labeled with GFP (green); cell nuclei were counterstained with DAPI (blue). Photomicrographs were captured under a Nikon Ni-U fluorescence microscope. Scale bars, 50 μm. (C) Δψm was detected by JC-1 dye. Images were shown as the ratio of JC-1 aggregates to JC-1 monomers. Scale bars, 50 μm. (DE) Representative bands of p-Drp-1, COX4 and TOMM20 protein after OGD/R. Variation in protein loading was determined by blotting with an anti-β-actin antibody. Densitometric scanning of band intensities were calculated as means ± SD (n = 3). **p < 0.01 vs. Ctrl group, ##p < 0.01 vs. OGD/R. (F) Images were collected by transmission electron microscope. Green square represents normal mitochondria with cylindrical shape. Their cristae was well-defined, the double membranes was intact and the density was homogenous; Red square represents abnormal mitochondria. Their cristae was disordered, double membrane was discontinuous, electron density was decreased and minor axis was increased consistent with mitochondrial swelling. Scale bars, 0.5 μm.