Lsm750

The cells were plated on coverslips and treated with α-mangostin. After 24 h, the cells were stained with 50 nM MitoTracker Red at 37°C for 30 min. After washing twice with PBS, the cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min and washed three times with PBS. After permeabilization with 0.1% Triton X-100 and blocking with 1% BSA in PBS for 30 min, the cells were incubated with primary antibodies in 1% BSA overnight at 4°C. After washing with PBS, cells were incubated with FITC-conjugated secondary antibodies in 1% BSA-PBS for 1 hour and rinsed with PBS. The cells were imaged using a confocal microscopy (Zeiss LSM 750 laser-scanning confocal microscope; Carl Zeiss, Göettingen, Germany).

As previously described, the immunofluorescence was performed with minor modification (Q Y Zhang et al. 2019 (link)). Briefly, the tissue sections were blocked before an overnight incubation with primary antibodies (Table S4). Next, they were subjected to a 2-h incubation with the appropriate secondary antibody (Table S4). DAPI (Cat# C3606, Beyotime, Shanghai, China) was employed to stain the cell nuclei for 15 min. Lastly, the tissue sections were visualized by confocal microscopy (LSM 750, Zeiss, Gottingen, Germany).

Following the manufacturer’s instruction, the MMP change was determined using a JC-1 Assay Kit (Beyotime Biotechnology, Shanghai, China, Cat# C2003S). In brief, primary cortical neurons (5 × 10⁵ cells/well) cultured in 6-well plates were harvested. After washing, neurons were resuspended in a 500 μL growth medium and incubated with JC-1 working solution (2 μM final concentration) in the dark at 37°C for 20 min. For positive control, the mitochondrial uncoupler, CCCP (10 μM final concentration), was added simultaneously with JC-1. CCCP is a positive control group in JC-1, which is a powerful uncoupling agent for mitochondrial oxidative phosphorylation, which promotes the permeability of mitochondrial intima to H⁺, resulting in mitochondrial apoptosis. The loss of membrane potential on both sides of the intima induces apoptosis. Samples were then analyzed using a confocal microscope (LSM 750, Zeiss, Gottingen, Germany). Data were processed with the ImageJ software, and the results were represented as the relative ratio of green to red fluorescence intensity.

Frozen hearts were sectioned into 10 μm thick sections at the cryostat. After washing with PBS sections were fixed by incubating in 2% formaldehyde solution for 10 min. Primary antibodies against rabbit PCM-1 (1:100, Santa Cruz, sc-67204), mouse SMA-Cy3 (1:1000, SigmaAldrich, C6198), and biotinylated isolectin B4 (Vector labs, B-1205, 1:500) were diluted in blocking buffer (PBS, 4% donkey serum, 0.1% Triton X-100 in PBS, 2 mM EDTA) and sections were incubated overnight at RT. Sections were then washed 3x with PBS (15 min) and stained with matching secondary antibody anti-rabbit Alexa Fluor® 488 (1:500, Jackson ImmunoResearch, 711-546-152) and Streptavidin-Alexa Fluor 647® (1:1000, ThermoFisher, S21374) in PBS. Subsequently, Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 647 or 488 dyes (ThermoFisher, C10340 and C10637) was used to detect EdU. Slides were then washed and mounted using ProLong Gold Antifade Mountant with DAPI (ThermoFisher, P36931). Images were acquired using a Keyence BZ X800 fluorescence microscope (Keyence, Japan) and Zeiss LSM 750 confocal microscopy from at least three regions of the left ventricle per heart.

Cells were grown in 8-well glass chamber slides (Nunc). After fixation with paraformaldehyde (3.7% in PBS), cells were permeabilized with 0.05% Triton-X100 in PBS, blocked with FBS, and incubated (overnight, 4 °C) with mouse anti-B23 antibody (Sigma-Aldrich, B0556-100UL) diluted in PBS (1:200), washed thrice with PBS and incubated with secondary anti-mouse antibody conjugated with Alexa Fluor 488 for 1 h (RT). The slides were imaged under a confocal microscope (Zeiss LSM750).

Apoptosis was analyzed using TUNEL staining (Roche, 11684795910). Cells seeded in 8-well glass chambers were incubated with WP760 (24–48 h) and treated with TUNEL reaction mixture (slight modification). Briefly, 200 μL of TUNEL reaction mixture was added, cells were covered with coverslips and incubated (60 min/37 °C/dark) in a humidified atmosphere. Subsequently, cells were stained with DAPI (1 μg/mL) and analyzed using confocal microscopy (Zeiss LSM750). Apoptotic index was determined by counting at least 1000 neoplastic nuclei subdivided in 6–10 randomly chosen fields at 200× magnification. Apoptotic cells were identified by TUNEL staining status together with characteristic morphological features (cell shrinkage, membrane blebbing, and chromatin condensation).

Cryosectioning was at 5 μm using a Bright cryostat and specimens frozen in optimal cutting technology on cooled isopentane. Fluorescence-labeled sections were mounted in glycerol/PBS or in commercial antifade mountant and imaged within a few days on a Zeiss LSM750 (Imperial College Facility for Imaging by Light Microscopy). Standard UV/blue, 488, 560, and 750 channels were used, with additional differential interference contrast on the 488 channel. This instrument was also used for the fluorescence emission scans. Paraffin sectioning was at 5 μm, and histochemistry and immunohistochemistry is described in the Data Supplement.