Fig. 5.

Increased Cox6a2 levels, Complex IV activity, and mitochondrial ATP production following ivermectin treatment in HL-1 cardiomyocytes. (a) Fluorescence resonance energy transfer (FRET) ratio of mito-ATeam stable HL-1 cardiomyocytes treated with ivermectin for various time periods. Data are presented as means ± SD (n = 3 biologically independent samples). (b) Venn diagrams of the comparative analysis of differentially expressed genes identified from RNA-seq data on ivermectin (5 μM, 24 h) and importazole (10 μM, 24 h) treatment in HL-1 cardiomyocytes (n = 3 biologically independent samples for each condition, fold change [log₂] ≥ 1, p < 0.05 vs. vehicle treatment). Numbers in red indicate the total number of genes common to two conditions. Genes in red signify p < 0.01. (c) Heatmap of RNA-seq data of genes encoding subunits of mitochondrial respiratory chain complexes following compound treatment in HL-1 cardiomyocytes as in b (n = 3 biologically independent samples for each condition). Red, upregulated genes; green, downregulated genes; fold change (log₂) ≥ 1. (d) Western blot analysis of the indicated proteins in HL-1 cardiomyocytes treated with 5 μM ivermectin for 24 h. Data are presented as means ± SD (n = 3 biologically independent samples). ^⁎p < 0.05 vs. vehicle by two-tailed unpaired Aspin-Welch's t-test. (e) Complex IV activity of isolated mitochondria isolated from HL-1 cardiomyocytes treated with 5 μM ivermectin for 24 h. Data are presented as means ± SD (n = 4 or 6 biologically independent samples). ^⁎p < 0.05 vs. vehicle by two-tailed unpaired Aspin-Welch's t-test. (f) Mitochondrial ATP production in digitonin-permeabilized HL-1 cardiomyocytes treated with 5 μM ivermectin for 24 h. OA, oligomycin A (10 μM). Data are presented as means ± SD (n = 3 biologically independent samples). ^⁎⁎p < 0.01, ^⁎⁎⁎p < 0.001 vs. vehicle by two-tailed unpaired Student's t-test. (g) Mitochondrial ATP production rate in digitonin-permeabilized HL-1 cardiomyocytes transfected with control siRNA or Cox6a2 siRNA followed by treatment with 5 μM ivermectin for 24 h. Data are presented as means ± SD (n = 6 biologically independent samples). ^⁎⁎⁎p < 0.001 vs. vehicle by two-tailed unpaired Student's t-test.