Mitotracker red

rCPSCs were seeded on circle microscope cover glass in a 24-well plate at a density of 2.5 × 10⁴cells/well and then were cultured with or without chondrogenic induction medium (Chondrogenic basal medium, ITS supplement, TGF-β3, Sodium pyruvate, Ascorbate, Proline, and Dexamethasone). After 14 d of induction, the cells were incubated with Mito-Tracker Red (Beyotime, China) according to the manufacturer’s instructions. Fluorescence was captured using a Zeiss 510 Confocal microscope (Carl Zeiss, Germany).

Mitochondrial morphology was detected by Mito-tracker dyes. Mitochondria were labeled with the MitoTracker Red (C1049B, Beyotime, China) for 30 min in the dark. The mitochondrial morphology was photographed by a confocal microscope (LSM510; Zeiss, Germany).
The Δψm was analyzed using the fluorescent probe JC-1 assay kit (C2003S, Beyotime, China) according to the manufacturer’s instructions. JC-1 exhibits red fluorescence aggregates in the mitochondrial matrix in normal cells. When the Δψm is reduced, monomeric JC-1 displays green fluorescence. Therefore, the rate of green/red fluorescence was used to represent the Δψm in each cell sample. PDL pregenitors of different groups (Control, Force, and Force + GSK219) were cultured on the coverslips in 12-well plates and loaded with JC-1 (1:400 dilution) at 37 °C for 20 min. The images were observed and captured under a fluorescence microscope (Leica, Germany).

To determine the mitophagy status within ASCs in different groups, third-passage ASCs were seeded onto four-chamber 35 mm glass bottom dishes at 40-60% confluency. After 24 h culture in GM, ASCs were pretreated with 100 ng/ml irisin for 2 h and then treated with or without 40 μg/ml AGEs for another 48 h in the presence of irisin. ASCs were stained with MitoTracker Red (Beyotime, China) in the dark at 37°C for 30 min; then, ASCs of each group were fixed with 4% PFA for 15 min and permeabilized with 0.3% Triton X-100 for 10 min at room temperature, followed by blocking with 5% goat serum and 0.3% Triton X-100 for 60 min, and further incubated with anti-LC3 (1 : 100 dilution) overnight at 4°C. After washing with PBS, the cells were incubated for 1 h at room temperature with an Alexa Fluor 488-conjugated secondary antibody (1 : 500 dilution; Beyotime, Shanghai, China). Finally, the cells were washed with PBS and the nuclei were counterstained with 4′, 6′-diamidino-2-phenylindole (DAPI) for 5 min at room temperature. Images of the stained cells were acquired under CLSM.

Mitochondrial morphology under the confocal microscope was observed by Mito-tracker red staining (C1035, Beyotime) using a confocal microscope according to the previous research [35 (link)]. The length of mitochondria was determined by ImageJ software. At least 6 images per condition were analyzed.

The intracellular mitophagy initiation was evaluated by the fluorescent colocalization of mitochondria and autophagosomes. NP cells were transiently transfected with GFP-LC3 (Beyotime, China). MitoTracker Red (Beyotime, China) was used to label the mitochondria in cells, which carries a thiol-reactive chloromethyl group that covalently binds to the reduced thiols that present mitochondrial matrix protein. The numbers of total colocalizing GFP-LC3 puncta per cell (mitophagosomes) were counted using ImageJ.

Camptothecin (CPT) (>98%) and 2-(dimethylamino) ethyl methacrylate (DEA) were acquired from Giant Medical Technology Co., Ltd. (Chengdu, China), whereas N-(3-aminopropyl) methacrylamide hydrochloride was purchased from Bide Pharmaceutical Technology Co., Ltd. (Shanghai, China). Lyso-tracker Red was provided by Thermo Fisher Scientific (Shanghai, China). MitoTracker Red, Mitochondrial Membrane Potential Assay Kit, Reactive Oxygen Species Assay Kit, ATP Assay Kit, Caspase 3 and Caspase 9 Activity Assay Kit, and Tissue Mitochondria Isolation Kit were all obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China). All other reagents were of analytical grade or above.

Prussian blue staining: Paraffin-embedded lung tissue sections (3 μm) were dewaxed and rehydrated. Prussian blue staining was performed following the instructions provided with the Prussian blue staining kit (Cat# G1426; Solarbio, China). Iron deposition in the lung tissue was determined by the formation of a stable blue compound.
Cytoplasmic and mitochondrial iron deposition assays: The cells were first digested with trypsin. Mitochondria and cytoplasm were separated using the mitochondrial extraction kit described above. The iron content was determined by combining iron with Ferene S to form a colored compound. The absorbance at 593 nm was measured using a spectrophotometer, and the iron content was calculated using a standard curve from the iron content assay kit (Cat# ab83366; Abcam; England).
Mitochondrial iron deposition assays: MLE-12 cells were seeded in confocal dishes. Cells were labeled with Mito-Tracker Red (Cat# C1035; Beyotime, Chian). After drug treatment, the iron in mitochondria was labeled in green using Miito-FerroGreen (Cat# M489; DOJINDO; Japan) according to the manufacturer's instructions. Iron deposition in mitochondria was detected using confocal microscopy (ZEISS LSM 900 with Airyscan 2; Carl Zeiss; Germany).