Cyanine3 (cy3)

Freshly isolated PBMCs were incubated in the presence and absence (control) of rHcES-24 (5μg/ml) for 1 h at 37°C. Confirmation of binding was determined by an immunofluorescence assay (IFA) as described by Yuan et al. [54 (link)]. Briefly, washed cells (10⁵ / ml) were fixed with 4% paraformaldehyde on a poly-L-lysine-coated glass slide. The cells were then treated with blocking solution (4% BSA in PBS) for 30 min to minimize background staining. After sequential incubation with rat anti-rHcES-24 IgG (1:100) for 2 h and a secondary antibody (1:300) coupled to the fluorescent dye Cy3 (Beyotime, Jiangsu, China) for 1 h, nuclear staining with 2-(4-amidinophenyl)-6-indole carbamidinedihydrochloride (DAPI, 1.5 μM; Sigma, MO, USA) was performed for 6 min. Then, protein localization was determined by observing the staining patterns with a 100× oil objective lens on a laser scanning confocal microscope (L SM710, Zeiss, Jena, Germany). Digital images were captured using the Zeiss microscope software package ZEN 2012 (Zeiss, Jena, Germany).

The IF assay was carried out as described previously [27 (link)]. Cells were cultured on coverslips in 24-well plates for 24 h, fixed with 4% formaldehyde, blocked with 5% bovine serum albumin, and permeabilized with 0.5% Triton X-100. After that, the washed cells were incubated with polyclonal antibodies against TM4SF1 (Abcam, 1:50), SOX2 (Abcam, 1:80), Zo1 (Abcam, 1:100), and vimentin (Abcam, 1:50), followed by incubation with mouse/goat anti-rabbit antibodies labelled with Cy3 (Beyotime Institute of Biotechnology, 1:800). After incubation with DAPI (Biosharp Biotech, Hefei, China, 1:1000) for 5 min, the cells were observed under a fluorescence microscope within 4 h.

HepG2 cells were fixed in 4% paraformaldehyde at room temperature for 15 min, and these HepG2 cells were blocked with 5% BSA containing 0.3% TritonX-100 buffer at room temperature for 60 min. The primary antibodies used are listed in Table S2. Secondary antibodies containing either FITC or Cy3 (Beyotime, Shanghai, China) were used using immunofluorescence staining. An inverted microscope was used to analyze cell staining. For cell proliferation analysis, HepG2 cells were processed using the BeyoClickTM EdU Cell Proliferation Kit with Alexa Fluor555 (Beyotime, Shanghai, China) kit according to the instructions.

After treatment and fixation, cardiac fibroblasts were incubated with blocking solution at room temperature. Then, diluted primary anti-α-SMA (1:100), collagen I (1:200), and collagen III (1:200) (Boster Biological Technology, Dublin, CA, USA); ki67 (1:100), PCNA (1:200) (ABclonal Technology, Wuhan, China); and the DRP1, OPA1, or RIPK3 (1:50, Cell Signaling Technology, Danvers, MA, USA) antibody were added and incubated overnight at 4 °C. After washing, the cells were incubated by IgG conjugated with Alexa Fluor 488 or Cy3 (1:500, Beyotime, Shanghai, China) without light at room temperature for 2 h followed by DAPI staining for 15 s. The cells were observed and photographed with a laser confocal microscope. The protein expression, which is considered as the fluorescence intensity, was quantified using ImageJ software.

Apoptosis was evaluated in paraffin tissue sections of ovaries using the Bright Red Apoptosis Detect Kit for terminal deoxynucleotide transferase dUTP nick end labelling (TUNEL) (Vazyme, A113-02, Nanjing, China). Briefly, the ovary sections were heated at 60 °C for 2 h and after washing with xylene passed through a graded series of ethanol for rehydration and washed in PBS. Sections were incubated in Proteinase K for 15 min, then washed twice with PBS. The samples were incubated in the dark for 60 min at 37 °C in 100 μl TUNEL reaction mixture and counterstained with Hoechst33342.
Activated Caspase 3 was identified used Immunofluorescent staining and the ovaries were processed as described above. Briefly, after blocking for 1 hr with 10% BSA, the slides were incubated overnight at 4 °C with primary antibodies, anti: Caspase3 (Abcam, ab2302), diluted to optimal concentration. After thoroughly rinsing with TBS secondary antibodies (CY3, A0516, Beyotime) diluted 1:200 were applied at 37 °C for 1h in the dark. After washing three times with PBS, the samples were incubated with Hoechst33342 (Solarbio, China) and observed under a fluorescent microscope.

The group settings were the same as those used for the NO secretion assays. IFA was performed using STAT3 polyclonal antibody (1: 50; Affinity Biosciences) as primary antibody and goat anti-rabbit IgG conjugated with Cy3 (Beyotime; 1:500 dilution) as a secondary antibody. The procedure was the same as that for the PBMC binding assay.