ethidium bromide  (Bio-Rad)

Journal: bioRxiv

Article Title: It’s Not Rewarding for Mitochondria: Dopamine-Induced Mitochondrial Dysfunction Activates cGAS-STING to Drive IL-6 Secretion in Macrophages

doi: 10.64898/2026.04.23.719926

Figure Lengend Snippet: (A) Cells were treated with vehicle or dopamine and collected at 1 hr or 6 hrs post-treatment. Following mitochondrial and cytosolic fractionation, DNA was isolated from the cytoplasmic fraction, and qPCR was performed to measure cytochrome c mitochondrial DNA levels relative to 18S, indicating mtDNA release into the cytoplasm. (Biological replicates: N=7-12). Data are presented as mean±SEM. (B-D) hMDMs were pre-treated with Vehicle or EtBr (10 μM, t = −1 h) to deplete mitochondrial DNA, followed by treatment with vehicle or dopamine for 6 hrs. Cells were lysed and assessed for cGAS protein expression as well as phosphorylation levels of STING by western blotting. (Biological replicates: N=7). Data are presented as mean±SEM. (E) Supernatants from experiments in panel B-D were collected 24 hrs post-treatment, and IL-6 secretion was measured using AlphaLISA. (Biological replicates: N=10). Data are presented as box and whisker plot (min to max). (F,G) Using confocal live imaging, JC-1 stained cells were imaged every 1 minute, for 30 minutes. Following the first image, cells were treated with vehicle (PBS) (C-i), dopamine (C-ii), or a positive control (FCCP, 1.5 μM) (C-iii) (t:1 min). The scale bar represents 20 μm. The ratio of green fluorescence (488 nm) to red fluorescence (555 nm) was calculated as an indicator of mitochondrial membrane depolarization, (Biological replicates: N=10). Data are shown as mean of response in each condition at each specific timepoint. (H,I) Cells were treated with vehicle or dopamine in a time-course experiment (1 - 6 hrs) and then stained with MitoSOX. Using flow cytometry, the mean fluorescence intensity (MFI) of MitoSOX staining was measured in the live singlet population to assess mitochondrial superoxide production. (Biological replicates: N=10-15). Data are presented as mean±SEM and representive histogram shows MitoSox MFI overtime. (J-Q) Cells were treated with vehicle or dopamine for 6 hrs and stained with MitoTracker Orange CMTMRos. Following fixation, nuclei were stained with DAPI, and cells were imaged using confocal microscopy (30× objective) with 3D acquisition at a step size of 0.15 μm. Yellow arrows represent elongated mitochondria, while white arrows represent round-shape mitochondria (fission). The scale bar represents 20 μm. Images were processed using ImageJ, and representative images are shown as z-projections for each condition. Mitochondrial morphology analysis was performed using the Mitochondria Analyzer plug-in for FIJI, (Biological replicates: N=15). Data are presented as mean±SEM. Paired comparisons were analyzed using a paired t -test (non-parametric: Wilcoxon test) in panel H and K-Q or paired one-way ANOVA (non-parametric: Friedman test) in panel C-E, or unpaired one-way ANOVA (non-parametric: Kruskal Wallis test) in panel A. Significance levels are indicated as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).

Article Snippet: Dopamine hydrochloride (DA; #H8502, Sigma-Aldrich), lipopolysaccharide from Escherichia coli O55:B5 (LPS; #L2880, MilliporeSigma), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; #0453, Tocris), 2′3′-cGAMP (#HY-12512, MedChemExpress), Mdivi-1 (#HY-15886, MedChemExpress), G140 (#HY-133916, MedChemExpress), H-151 (#HY-112693, MedChemExpress), ethidium bromide (EtBr; #HY-D0021, MedChemExpress), BAY 11-7082 (#S2913, Selleck Chemicals) were purchased from the indicated vendors.

Techniques: Fractionation, Isolation, Expressing, Phospho-proteomics, Western Blot, Whisker Assay, Imaging, Staining, Positive Control, Fluorescence, Membrane, Flow Cytometry, Confocal Microscopy