Fig 2: Drp1 knockdown (NAG-Drp1si) in mice with NASH increases mitochondrial size and decreases mitochondrial oxidative function. (A-E) Mice fed regular chow (RC) or GAN diet to induce NASH for 24 weeks before being injected weekly with PBS (control vehicle) or NAG Drp1 siRNA (NAG-Drp1si) for 12 weeks. Data presented are the mean ± SD or ± SEM. RC/PBS n = 8, GAN/PBS n = 12, GAN/NAG-Drp1si n = 12, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 one-way ANOVA. (A) Complex 1 and 2 driven respiration, as well as total complex 4 activity were measured in liver homogenates. Each dot represents the average respiration of each mouse. (B) Mitochondria of liver cryosections stained with anti-Tomm20 antibodies from RC/PBS, GAN/PBS, and GAN/NAG-Drp1si mouse models (Top row). Mitochondria are color coordinated based on small (red), intermediate (white), and large (blue) area (bottom row). (C) Quantification of individual mitochondrion area. Each individual data point shown in the graph is the value of the area averaged for all individual mitochondria segmented in one field, as shown in panel A. A total of 20 fields were imaged for n = 6 mice/group and averaged to calculate SEM. (D) Expression analysis in total liver of genes controlling lipid metabolism and content in liver. (E) Total liver content of non-esterified fatty acids (NEFA), palmitate, oleate and triglycerides (TG) were measured by mass spectrometry.

Fig 3: Tilted light sheet imaging with long axial range point spread functions (PSFs). a 3D super-resolution (SR) reconstructions of mitochondria (TOM20) in a HeLa cell. The double-helix (DH) PSF was used for imaging of both single molecules and fiducial beads. b xy, xz, and yz views of the mitochondrion shown in the magenta rectangle in a, revealing the hollow cylinder structure of the mitochondrial outer membrane. c 3D SR reconstruction of the entire nuclear lamina (lamin B1) in a HeLa cell. Imaging of single molecules and fiducial beads was performed with the DH-PSF and a 6-µm tetrapod PSF, respectively. The xz view shows a 1.3-µm thick y-slice through the cell, where lamin meshwork enveloping an intranuclear channel is visible. The lower right inset shows the right cap of the reconstruction. All samples imaged were immunolabeled with Alexa Fluor 647. Scale bars are 5 µm in a and c, and 500 nm in b

Fig 4: HAS-G inhibits mitophagy compared with HAS. (A) ATP production was measured in HAS- or HAS-G-attached BMSCs pretreated with or without 10 µM NAC. (B) The hallmarks of mitophagy, including LC3 maturation, optineurin, p62, TIM23, TOM20 and HSP60 were measured by western blotting. (C) Confocal laser scanning microscopy colocalization images of TOM20 and EGFP-LC3B staining. *P<0.05 vs. HAS. HAS, hydroxyapatite scaffold; HAS-G, hydroxyapatite scaffold with a groove structure; BMSCs, bone marrow-derived stromal cells; NAC, N-acetylcysteine; LC3, microtubule-associated proteins 1A/1B light chain 3B; p62, nucleoporin p62; TIM23, translocase of the inner membrane 23; TOM20, translocase of the outer membrane 20; HSP60, heat shock protein 60; OPTN, optineurin; EGFP, enhanced green fluorescent protein.

Fig 5: FACS-based quantification of mito-FUNCAT (mito-FUNCAT-FACS). (A) Schematic representation of the mito-FUNCAT-FACS procedure. (B,C) Representative distribution (n = 4) of Cy3-conjugated HPG signals (B, mito-FUNCAT-FACS) and Alexa Fluor (AF) 647-labeled TOM20 signals (C) across C2C12 cells in FACS. Cells were analyzed in the presence or absence of translation inhibitors (ANS and CAP) and HPG. Data from 1 × 104 cells are shown. (D) Scatter plot for Cy3-conjugated HPG signals and AF647-labeled TOM20 signals across cells analyzed in B and C. ρ, Spearman's rank correlation coefficient. (E) The distribution of mito-FUNCAT-FACS signals (in B) normalized to the AF647-labeled TOM20 intensity (in C). (AU) Arbitrary unit.