Figure 2
Mitochondrial morphology, UPRmtand autophagy in skeletal muscles of NOS1-/- mice. Tibialis anterior muscles were isolated from wild-type and NOS1-/- mice at P120. (A) In vivo imaging of the mitochondrial network by two-photon confocal microscopy. Muscles were transfected with the mitochondrially targeted red fluorescent protein pDsRed2-Mito. The images are representative of results obtained from at least five different animals per experimental group. Scale bar: 10 μm. (B) TEM images detecting the presence of abnormal, enlarged subsarcolemmal mitochondria (asterisks) or autophagic vacuoles (arrowheads) in NOS1-/- muscles. The inset depicts a multivesicular body in NOS1-/- fibres taken at higher magnification. The images are representative of results obtained from at least three different animals per experimental group. (C-D) Subsarcolemmal mitochondrial ultrastructure analysis by TEM. Data represent the quantification of the mitochondrial area and morphometric analysis of mitochondrial cristae complexity. Each histogram represents the data obtained from at least three different animals per experimental group. * P <0.05 and ** P <0.01 versus the respective wild-type control. (E) Western blot analysis of HSP60 and ClpP expression. Actin was used as the internal standard. The image is representative of results obtained from at least five to seven different animals per experimental group. (F) In vivo imaging of autophagosome formation by two-photon confocal microscopy. Muscles were transfected with YFP-LC3. The images are representative of results obtained from at least five different animals per experimental group. Scale bar: 10 μm. (G) Western blot analysis of LC3 lipidation. Actin was used as the internal standard. The image is representative of results obtained from at least 10 different animals per experimental group.