Journal: Journal of Cachexia, Sarcopenia and Muscle
Article Title: Caveolin‐3 deficiency associated with the dystrophy P104L mutation impairs skeletal muscle mitochondrial form and function) Caveolin‐3 deficiency associated with the dystrophy P104L mutation impairs skeletal muscle mitochondrial form and function
Figure Lengend Snippet: Effects of Cav3 re‐expression on mitochondrial protein expression, membrane potential, superoxide and mitochondrial respiration. Myoblasts in which Cav3 had been deleted by CRISPR/Cas9 (Cav3KO) were infected with retroviral particles containing an empty vector (Cav3KO + EV) or a vector containing HA‐GFP tagged WT‐Cav3 (Cav3KO + Cav3) and stable transformants isolated by antibiotic selection prior to: (A) immunoblotting whole cell lysates (WCL) with antibodies against Cav3 or actin (used as a gel loading control) antibodies; (B) isolation of cytosolic (Cyto) and mitochondrial membranes and immunoblotting using antibodies to proteins indicated; (C) lysis and immunoblotting of WCL with antibodies to proteins shown and quantification of their abundance relative to GAPDH (used as a gel loading control) using Image J software; (D) determination of superoxide content using fluorescence intensity (FI) of MitoSOX and mitochondrial membrane potential monitored by spectral analysis of JC‐10 aggregate:monomer content from three separate experiments (for this experiment GFP would interfere with the JC‐10 probe and therefore wild type Cav3 was cloned into pcDNA3.1 using HindIII and XhoI Restriction sites and an empty vector or a vector containing Cav3 were transiently transfected in L6 muscle cells and selected for using G418 before JC‐10 probe incubation); and (E) analysis of mitochondrial respiration to assess basal oxygen consumption rate using Seahorse technology. Oligomycin (1 μM), FCCP (1 μM) and a mixture of Rotenone (1 μM)/Antimycin (2 μM) were added at the times indicated by dotted lines to determine basal, ATP‐linked and maximal respiration. The Seahorse trace shown in (E) is from a single experiment in which each point represents the mean ± SEM from triplicate analyses. All bar graph data in (E) represent the analysis of three individual experiments (values are mean ± SEM). Asterisks indicate significant change ( P
Article Snippet: Measurement of mitochondrial membrane potential, mitochondrial DNA, and citrate synthase activity For measurement of mitochondrial membrane potential, muscle cells were seeded and grown in dark‐walled 96‐well dishes until confluent for 4 days.
Techniques: Expressing, CRISPR, Infection, Plasmid Preparation, Isolation, Selection, Lysis, Software, Fluorescence, Clone Assay, Transfection, Incubation