Journal: Nature Communications

Article Title: Cytochrome c lysine acetylation regulates cellular respiration and cell death in ischemic skeletal muscle

doi: 10.1038/s41467-023-39820-8

Figure Lengend Snippet: A Annexin V/propidium iodide flow cytometry data for cells stably expressing WT, K39R, K39Q, K39E Cyt c , and EV after exposure to 400 µM H 2 O 2 for 16 h (n = 3), B 16 h of oxygen-glucose deprivation followed by 1 h of reoxygenation (n = 3), C or 1 mM thapsigargin for 24 h (n = 3). D Representative co-IP-Immunoblot experiment of Cyt c double knockout cells stably transfected with WT, K39R, K39Q, and K39E constructs immunoprobed for Cyt c and loading control Apaf-1 (n = 3). E Representative western blot of Cyt c double knockout cells stably transfected with EV, WT, K39R, K39Q, and K39E constructs immunoprobed for caspase-9 and loading control GAPDH after exposure to 1 µM staurosporine for 8 h (n = 3). F Representative western blot of Cyt c double knockout cells stably transfected with EV, WT, K39R, K39Q, and K39E constructs immunoprobed for caspase-3 and loading control GAPDH after exposure to 1 µM staurosporine for 8 h (n = 3). Data are represented as means ± standard deviation. A one-way ANOVA comparing the total cell death mean of each mutant with the total cell death mean of the control mutant (WT) with the Dunnett post-hoc test was used.

Article Snippet: After 48 h from the initial seeding, cells were trypsinized, washed twice with 1x PBS, counted, and a total of 1 x 10 6 cells were resuspended in 1 mL 1x annexin V binding buffer from the FITC annexin V apoptosis detection kit I (#556547; RRID: AB_2869082; BD Pharmingen).

Techniques: Flow Cytometry, Stable Transfection, Expressing, Co-Immunoprecipitation Assay, Western Blot, Double Knockout, Transfection, Construct, Standard Deviation, Mutagenesis