For total proteins and cytosolic protein, b-actin was used as a loading control

activate PAGFP to show GFP green fluorescence. GFP and TMRE images were simultaneously recorded. GFP was excited at 488 nm and its emitted fluorescence was collected at 490-540. TMRE was excited at 543 and its emitted fluorescence was collected at 560-620. Before photoactivation Dendra is a green fluorescent protein. Photoactivation with 364 nm laser light converts Dendra from a green to a red fluorescent protein. Thus after photoactivation Dendra was excited at 543 and its emitted fluorescence was collected at 560-620. A confocal microscope capable of lineinterleaving images excited with different lasers, was used. TMRE was purchased from Invitrogen. Results Abnormal mitochondrial dynamics in skeletal muscle of G93A mice before disease onset The ALS mouse model is asymptomatic until after 3 months of age. To examine mitochondrial dynamics prior to ALS onset, experiments here were done on two-month old G93A and age-matched normal mice. A mitochondriatargeted photoactivatable green fluorescent MedChemExpress TMS protein was transfected into the FDB muscle of both G93A and control mice in 22576162 order to monitor mitochondrial dynamics in skeletal muscle. Seven days post-transfection, the transfected FDB muscles were collected and enzyme-digested to isolate individual 7884917 muscle fibers for confocal imaging studies. The isolated live muscle fibers were incubated with TMRE to visualize mitochondria. TMRE and mt-PAGFP were then excited at 543 nm and 488 nm respectively. Interleaved excitation and spectrally separated emission wavelengths permitted simultaneous recording of the TMRE and mt-PAGFP signals. Before photoactivation, mt-PAGFP showed no GFP fluorescence. Photoactivation of a small region of the muscle fiber by a 364 nm laser light evoked mt-PAGFP fluorescence in this region. Comparison of TMRE and mtPAGFP signals demonstrates that mt-PAGFP is indeed expressed inside mitochondria. Mitochondrial dynamics were evaluated by examining the time-dependent migration of mt-PAGFP out of the original photoactivation area. Representative control and G93A images are shown in Application of pharmacological reagents The mitochondrial fission inhibitor, Mdivi-1 was applied by intraperitoneal injection in the dose of 50 mg/kg/day for 7 days before FDB muscles were removed for analysis of mitochondrial dynamics. Mdivi-1 was dissolved in di-methyl sulfoxide at the concentration of 12.5%. Then 8 l Mdivi-1 solution was dissolved in 200 l saline for intraperitoneal injection. The same dilution of DMSO in normal saline was applied as vehicle control. In order to analyze the effect of mitochondrial membrane potential on mitochondrial dynamics, a proton ionophor was applied to muscle fibers in culture dishes to partially depolarize mitochondrial inner membrane potential. Chemicals were purchased from Sigma. Immunoblot assay Tibialis anterior muscles from normal and G93A mice were collected and homogenized in protein extraction buffer containing protease inhibitor cocktail using motorized homogenizer. Protein concentrations were determined by BCA protein assay. Protein samples were subject to 8% SDSpolyacrylamide gel electrophoresis. The protein was transferred into an Immobilon PVDF membrane. The primary antibodies used to probe the protein samples are anti-Mfn1 and anti-Mfn2 from Abcam; anti-Drp1 from Cell Signaling, and anti-tubulin from Santa Cruz Biotechnology. The C-SOD1 3 Mitochondrial Dynamics in ALS Skeletal Muscle doi: 10.1371/journal.pone.0082112.g001 The migration rates in th