Fig. 2

Defective mitochondrial complex I causes cellular dysfunction in SMA MNs. a Complex I activity using 20 µg mitochondria extract isolated from WT and SMA MNs (10DIV) (N = 4). Quantification represents the increase in mean OD₄₅₀ nm/h. b Representative images and quantification of WT and SMA MNs labeled with CellROX (green and rainbow color) for ROS detection, DAPI (blue), and Phalloidin (red). Rainbow color indicates the intensity of CellROX signal. Quantification of mean fluorescence intensity of the CellROX signal in WT and SMA MN (red squares) (n = 40, N = 4). Scale bars are written on individual images. c Quantification of CellROX signal of WT and SMA MNs measured with a microplate reader (N = 5). d Western blot analysis and quantification of carbonylated proteins (DNP = 2,4-dinitrophenyl) in 10DIV WT and SMA MNs (N = 4). Bar graph represents the quantification of the DNP signal. ACTB was used as a loading control. e ATP levels in WT (N = 7) and SMA MNs (N = 8). Data are normalized to solubilized protein amounts. f ATP levels in WT and SMA MNs (N = 6) after 50 mM pyruvate treatment for 1 h. Data are normalized to solubilized protein amounts. Individual data (circle or square) represent biological replicates. g ROS levels of SMA MNs after 50 mM pyruvate or 100 µM menadione treatment for 1 h. Two-tailed unpaired t-tests with Holm-Bonferroni correction for multiple comparisons were used on independent biological replicates (N = 6) to determine statistical significance. *p < 0.05, **p < 0.01. a–f Blue circles represent data from WT and red squares represent SMA MNs. 10DIV MNs were used. Each dot represents the quantification of individual biological replicates. A two-tailed unpaired t-test was used on independent biological replicates to determine statistical significance. Bar graphs depict the mean ± S.D., n.s. p > 0.05, *p < 0.05, **p < 0.01