Figure 6.

SOD2-Induced Neurogenesis Is by Enhancing Mitochondrial Fusion Process

(A) Protein levels of mitochondrial fission (FIS1) and fusion (MFN1 and MFN2) markers upon SOD2 over-expression. (B) Representative images of mitochondria upon WT or mutSOD2 over-expression in cells where Mfn1, Mfn2, or both are knocked down. (C) Quantification of mitochondrial length in the conditions as mentioned in the (B). (D) mRNA levels of early neural markers (Foxg1 and Nestin) in P40H1 cells with conditions wherein SOD2 is over-expressed in cells with Mfn2 knockdown. (E) Representative immunofluorescence images of NESTIN in neural cells with Mfn 1, Mfn 2 individual or simultaneous knockdown and subsequent SOD2 over-expression. Scale bar represents 50 μm. (F) Relative fold change in the percentage of NESTIN-positive cells upon knockdown of Mfn 1, Mfn2, or both, and subsequent SOD2 over-expression. (G) Transcript analysis showing rescue in neural markers Sox1, Nestin, and Zic1 of mESCs differentiated to neural progenitors under Mfn2 shRNA and over-expression of SOD2 conditions. (H) Immunofluorescence images of NESTIN and SOX1 showing rescue in neural differentiation upon over-expression of SOD2 in Mfn2 knockdown background. (I) Transcript analysis of Nestin and (J) immunofluorescence for NESTIN and SOX1 showing rescue in neural differentiation of mESCs upon over-expression of antioxidant activity mutant SOD2 under Mfn2 shRNA condition. Scale bar represents 100 μm. Data representative of mean ± SE, n = 3 independent experiments, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Number of mitochondria counted per condition ∼400. Un-cropped full western blot images are available in data S1. List of primers used for transcript analysis and antibodies used for protein detection are available in Tables S1 and S2, respectively.