Skip to main content
. 2024 Feb 9;70:103081. doi: 10.1016/j.redox.2024.103081

Fig. 7.

Fig. 7

Detection of Drp1-FBXL4 binding and FBXL4-Drp1 axis in ‘double-damage’-evoked mitochondrial fragmentation. A–B: Structure-based interface analysis between Drp1 and FBXL4. A: FBXL4-Drp1 complex structure with interaction hot-spot residues labeled using PRISM; B: Domain deletion of FBXL4 for Co-IP experiments, and predicted Drp1 interaction sites on FBXL4. Boxes indicate exons; white bars indicate deleted domains; C: IP analysis of FBXL4 tagged with Flag (FBXL4-Flag) and Drp1 tagged with His (Drp1-His) in AMCM cells from chow and HFpEF; D: IP analysis of FBXL4 domain mutations (FBXL4-Flag, FBXL4-ΔFbox, FBXL4-ΔLRR2) using Drp1-His in H9C2 cell line; E: H9C2 cells were divided into 4 groups (FBXL4-Flag, FBXL4-ΔFbox, FBXL4-ΔLRR2, Fbox) prior to transfection of Drp1 using plasmid DNA, Confocal image exhibiting H9C2 cells transfected with indicated plasmids, stained with MitoTracker (red), anti-FBXL4 (green), and anti-Drp1 (blue) antibodies; F–G: Colocalization and Pearson's correction coefficient; H–I: Assessment of mitochondrial structure using confocal microscopy in adult mouse cardiomyocytes (AMCM) transfected with Adv-FBXL4 for 48 h prior to DD challenge for 12 h; H: Representative mitochondrial immunofluorescence using Mito Tracker (red); and I: Quantification of mitochondrial length of Drp1CKI and Drp1hetCKO groups. J–M: Ubiquitylation level of Drp1 in heart tissues from chow and HFpEF with or without FBXL4 overexpression; N–P: Drp1 level in H9C2 cells transfected with FBXL4-siRNA or FBXL4-scramble, treated with or without MG132 for 4, 8, and 12 h; Q–S: Whole cell level of Drp1 treated with or without MG132 for 4, 8, and 12 h in AMCM cells from chow and HFpEF; Mean ± SEM, n = 3 mice per group (5 replicates per heart, panel E–I), or 3 replicates from 1 sample per group (panel J–M), or 2 replicates from 3 sample per group (panel N–S). Statistical significance was estimated by one-way ANOVA followed by the Tukey's multiple comparison test or repeated measures ANOVA in R and Bonferroni adjusted test. *p < 0.05, ***p < 0.001, ****p < 0.0001 between indicated groups.