Journal: Redox Biology

Article Title: Mitochondrial permeability transition pore desensitization by a novel dispiranic derivative prevents cardiac reperfusion injury

doi: 10.1016/j.redox.2026.104097

Figure Lengend Snippet: Cytoprotective effects following H/R. (A) Calcein−cobalt assay in AC16 cells. The kinetics of PTP opening were evaluated at the moment of the reoxygenation in the presence of vehicle, 11d or 12c compounds. The raw values were expressed as a percentage of the vehicle. Each value is the mean of at least 10 cells from 3 biological and 3 technical replicates. (B) Quantification of AC16 cells positive to Propidium Iodide (PI + ) staining following H/R. Each value is the mean of 3 biological and 4 technical replicates. (C) Calcein-cobalt assay in differentiated HCM cells. Data has been collected at reperfusion time in the presence of vehicle or 11d compound. (D) Immunoblot detection of the main markers of apoptosis and necrosis such as Cleaved PARP, Cleaved Caspase 3 and Cleaved RIP. This is representative of 3 biological replicates. (E) Quantification of cell viability according to viable cells upon H/R staining with crystal violet. This is representative of at least 3 biological and 3 technical replicates. (F) Quantification of the MitoSox intensity of cells after H/R under the same experimental conditions. Each value is the mean of at least 15 cells from 3 technical and 3 biological replicates. (G) Mitochondrial calcium uptake in AC16 cells by using a mitochondrially targeted aequorin probe (mtAeq); [Ca 2+ ] is detected at the time of reoxygenation and after addition of 100 μM His and 100 μM Bk. Each value is the mean of at least of 3 biological and 3 technical replicates. Histograms of statistical and representative kinetics data are reported. One-way ANOVA was applied for statistical analysis for all graphs reported in the figure; (∗∗∗∗) p value < 0.0001; (∗∗∗) p value < 0.001; (∗∗) p value < 0.01; (∗) p value < 0.05.

Article Snippet: After electrophoretic separation, proteins were transferred onto nitrocellulose membranes that were incubated overnight with the following primary antibodies: cleaved PARP (Cell signaling, 9541, 1:1000), Caspase 3 (Cell signaling, 9662, 1:1000), RIP (Cell signaling, 3493, 1:1000), β-Actin (Merck, A1978, 1:5000), ATP5A (Abcam, ab14748, 1:5000).

Techniques: Cobalt Assay, Staining, Western Blot