Fig. 4

MEF2C regulates mitochondria gene expression and mitochondrial function in motor neuron cell line (NSC-34). (A) MEF2C transcription factor binding sites at mouse mitochondrial DNA detected by TRANSFAC 6.0-based algorithm, Patch 1.0. (B) A scheme illustrating performing MEF2C ChIP assay on NSC-34 cells infected by Mef2c-KD (shMef2c) and control (shControl) viruses, and measuring level of specific DNA in ChIP samples in mitochondria genes by qPCR. (C) MEF2C ChIP-qPCR showed Mef2c occupancy level in Nd2, Nd4 and Nd5 mitochondrial genes in shControl and shMef2c cells. Data generated from 3 samples which are duplicated and normalized to input DNA. Statistics were calculated using Student’s t-test (ND2, *, P = 0.04;  ND4, *, P = 0.047; ND5, *, P = 0.05). (D) A scheme illustrating performing qPCR on NSC-34 cells infected by shControl and shMef2c viruses and harvested after 24 h. (E) qPCR results showed mRNA level of mitochondria genes, Nd2, Nd4, Nd5, decreased by Mef2c-KD. Statistics were calculated using Student’s t-test (ND2, *, P = 0.035; ND4, *, P = 0.05; ND5, **, P = 0.025). (F) A scheme illustrating a series of experiments to evaluate mitochondrial function in in NSC-34 cells infected by Mef2c-KD virus. Immunostaining of (G) MitoSox (red), (H) MitoTracker (red) and (I) cytochrome c (Cyto c) (red) in GFP⁺ NSC-34 cells infected by Mef2c-KD virus. The nuclei were counterstained with DAPI (blue). Scale bars (white): 5 μm. Right: quantification of MitoSox, MitoTracker and cytochrome c levels in GFP⁺ cells. A total of 30 cells/group were counted (10 cells/well) from n = 3 wells/group (shControl and shMef2c). Statistics were calculated using LMM (*, P = 0.012; ***, P < 0.001). (J) Cellular respiration rate of differentiated NSC-34 cells infected by Mef2c-KD virus measured by a Seahorse XFe24 analyzer. Right: quantitative analysis of the basal and maximum respiratory rate, and ATP production amount of GFP⁺ cells. Statistics were calculated using Student’s t-test (n = 4 wells/group: *, P < 0.05 ,). (K) Immunofluorescence staining of cleaved caspase-3 in NSC-34 cells infected by Mef2c-KD virus. Scale bars (white): 5 μm. Right: densitometry analysis for GFP⁺ cells. A total of 30 cells/group were counted (10 cells/well) from n = 3 wells/group (shControl and shMef2c). Statistics were calculated using LMM (***, P < 0.001). (L) Decrease of ATP level by Mef2c-KD at 24, 48 and 72 h after the virus infection in NSC-34 cells. Statistics were calculated using Student’s t-test (n = 4 wells/group: P = 0.097 at 24 h; *, P = 0.025 at 48 h; *, P = 0.023 at 72 h). Error bars represent means ± SEM