Fig. 2.

Mdivi-1 treatment or silencing Drp1 expression using siRNA transfection alleviated R28 cell injury induced by OGD/R.

A: A schematic diagram of siRNA application. R28 cells were plated and cultured to 30% confluence for transfection. After 4 h transfection, R28 cells were cultured for an additional 48 h and then subjected to OGD/R modeling; B: After Mdivi-1 treatment or silencing Drp1 expression, the cell death rate was detected using Hoechst 33342 and PI dual staining. Scale bar = 100 μm; C: Quantification of the percentage of PI-positive cells(n = 9); D: After Mdivi-1 treatment or silencing Drp1 expression, the ΔψM was measured by JC-1 staining(2.0 μg/ml). Scale bar = 100 μm; E: Quantification of the relative red/green ratio(n = 9); F: After Mdivi-1 treatment or silencing Drp1 expression, mitochondrial morphology was identified using Tom20 antibodies by immunofluorescence. Scale bar = 2 μm; G: Average mitochondrial lengths were analyzed (n = 9); H: After Mdivi-1 treatment or silencing Drp1 expression, ATP assay kit was used to detect ATP production(n = 9); I: After Mdivi-1 treatment or silencing Drp1 expression, Mito-SOX(2 μM) staining to detect the ROS level. Red fluorescent spots represent mitochondrial ROS production, and Hoechst staining in the nuclei is shown in blue. Scale bar = 100 μm; J: Quantification of the relative Mito-SOX red fluorescent intensity(n = 9). Values are expressed as the mean ± SD. One-way ANOVA was followed by the Dunnett's test. *P < 0.05, **P < 0.01, ***P < 0.001and****P < 0.0001 vs OGD/R.