Fig 1: The m.4349C>T mutation results in decreased levels of mitochondrial proteins in patient’s muscle and mutant cybrid cells. (A) Western blots of mtDNA-encoded subunits of OXPHOS complexes in muscle samples. Control muscle samples, C1 and C2. VDAC1 as a loading control. ND1 and ND5 indicate subunits 1 and 5 of the complex I; CYB, subunit of complex III; CO1, subunit 1 of complex IV; ATP6, subunit 6 of complex V. (B) Western blots of nDNA-encoded subunits (NDUFB8, SDHB, UQCRC2, CO4, and ATP5A) of OXPHOS complexes. (C) Western blots of mtDNA-encoded subunits of OXPHOS complexes in cybrid cells. Two WT cybrid cells line, C1 and C2; Two mutant cybrid cell lines, M1 and M2. (D) Western blots of nDNA-encoded subunits of OXPHOS complexes in cybrid cells. Two WT cybrid cells line, C1 and C2; Two mutant cybrid cell lines, M1 and M2.
Fig 2: Myg1 mediates selective RNA turnover in mitochondria and governs OXPHOS. (A) Silexoseq analysis of isolated mitochondria from control and Myg1 silenced cells was carried out. Representation of the heavy strand of mitochondria; coordinates in the x-axis represent the genomic coordinates (in 5' to 3' direction) in continuum and each of the genes are highlighted in different colors at the backdrop. (B) RNA decay analysis performed by EU labeling in B16 cells treated with control and Myg1 siRNA. (C) Western blot analysis of ND5, CO1, ATP6, CYTB along with Myg1 from the whole cell lysates of B16 cells normalized to tubulin levels in control and Myg1 silenced cells. (D) Flow cytometric analysis of B16 mouse melanoma cells treated with Control and Myg1 siRNA and stained with TMRE to check the mitochondrial potential and MitoTracker green to check the mitochondrial mass. Experiments were performed with three independent biological replicates and represented as mean and individual data points (students t test for mitotracker green staining means not significantly different, and for TMRE staining P value < 0.0001). (E) Oxygen consumption measured using Clark Electrode in control and Myg1 siRNA treated B16 mouse melanoma cells. The traces correspond to the level of oxygen at various time intervals after addition of cells to the Oroboros chamber normalized to the level of oxygen at the time of addition of cells (inset). Oxygen consumption rate (OCR) determined from the slope of the curve is depicted as pmoles of oxygen consumed per minute per 107 of cells, calculated from two independent biological replicates with comparable Myg1 silencing represented as mean and individual data points (Student's t test, P < 0.01).
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