Bz 710

Immunofluorescence-labeled cells or tissue samples were photographed by BZ-710 (KEYENCE, Osaka, Japan) and images were analyzed using ImageJ/FIJI (National Institutes of Health, United States). YAP1 nuclear localization was quantified using a colocalization analysis between YAP1-Alexa488 and Hoechst/DAPI. The colocalization analysis was performed by calculating Pearson’s correlation coefficient with the Coloc 2 plugin of ImageJ/FIJI. Data are presented as the mean ± S.D. Measurements were performed on at least three samples.

All the staining protocols were performed on 2 µm thick mouse tissue sections. Hematoxylin and eosin (HE) and Masson’s trichrome (MT) staining were performed according to standard protocols [10 (link)]. We performed immunohistochemistry (IHC) by first deparaffinizing the sections, following which we performed antigen retrieval at 121 °C in an autoclave for 10 min in a 10 mM sodium citrate buffer (pH 6.0). We then treated the sections with a 3% H₂O₂ solution (Envision Plus System; Dako, Santa Clara, CA, USA) to inhibit any endogenous peroxidases. Rabbit polyclonal LIF antibody (1:500, ab113262; Abcam) was used for IHC. The labeled antigens were visualized by chromogen 3,30-diaminobenzidine tetrahydrochloride; hematoxylin was used as a nuclear counterstain. Eventually, the slides were observed under a fluorescence microscope (BZ-710; Keyence).

We performed an in vitro macropinocytosis assay as previously described [20 (link)]. Briefly, 1 × 10⁵ cells were plated onto glass coverslips in 6-well plates for five days. We then incubated the cells for 30 min at 37 °C with 70-kDa fluoresceine isothiocyanate (FITC)-dextran (Sigma) directly added to the culture media at a final concentration of 1 mg/mL. Subsequently, we assessed the macropinocytic uptake of cells, and rinsed the cells five times on ice with ice-cold PBS. Thereafter, the cells were fixed with 3.7% formaldehyde and their nuclei were counterstained with DAPI. Eventually, the coverslips were mounted onto glass slides using an aqua-poly/mount (Polysciences, Inc., Warrington, PA, USA). Fluorescent images were captured using a fluorescence microscope (BZ-710; Keyence). Notably, each experimental condition was performed in triplicates.

We fixed AD-MSCs and Capan-1 cells with 100% methanol for 10 min at −20 °C. Subsequently, we washed them three times for 5 min each with an IF buffer solution (10 × stock: 38.0 g NaCl, 9.38 g Na₂HPO₄, 2.07 g NaH₂PO₄, 2.5 g NaN₃, 5.0 g bovine serum albumin (BSA), 10 mL Triton X-100, and 2.5 mL Tween-20 in 500 mL PBS). Thereafter, the cells were treated with a blocking solution (3% BSA in 1 × IF washing solution) for 30 min. They were then incubated for 1 h at 25 °C with a mouse SMA antibody (1:400, ab7817; Abcam, Cambridge, UK), rabbit anti IL-6 antibody (1:200, ab6672; Abcam), or rabbit anti-rat vimentin antibody (1:400, #280618; R&D Systems Inc., Minneapolis, MN, USA) in the 1 × IF buffer. Post this incubation, the cells were washed three times with the 1 × IF buffer. They were subsequently incubated for 1 h with Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen, Carlsbad, CA, USA), Alexa Fluor 568 goat anti-rat IgG antibody (Invitrogen), and Cy5 goat anti-rabbit IgG antibody (Invitrogen), all diluted to 1:400 in the 1 × IF buffer solution. The cell nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) and thereafter were washed thrice with the 1 × IF buffer. The slides were mounted and visualized under a fluorescence microscope (BZ-710, Keyence, Osaka, Japan).

Intracellular reduced GSH was analyzed using 4‐chloromethyl‐6,8‐difluoro‐7‐hydroxycoumarin (Cell Tracker Blue; Molecular Probes, Eugene, OR, USA). Ten oocytes from each group were incubated in the dark in 10 μM CellTracker Blue for 30 min at 37°C. The oocytes were rinsed twice with PBS (−) containing 0.1% (w/v) polyvinyl alcohol (PVA). Oocyte GSH levels were measured using an inverted fluorescence microscope with UV filter (Blue image, 370 nm, BZ‐710, Keyence).
Intracellular ROS levels were measured using 2′7′‐dichlorodihydrofluorescein diacetate (CM‐H₂DCFDA; Molecular Probes, Eugene, OR, USA). Ten oocytes from each group were incubated in the dark in 10 μM CM‐H₂DCFDA for 30 min at 37°C. The oocytes were rinsed twice with PBS (−) containing 0.1% (w/v) polyvinyl alcohol (PVA). To analyze ROS levels, 2,7‐dichlorofluorescein (DCF) fluorescence signals were detected using an inverted fluorescence microscope (green channel: 480 nm, BZ‐X710, Keyence).
In measuring ROS and GSH levels, the average fluorescence intensity of fresh oocytes was set to 1.0, and the reported values represent fold differences in fluorescence intensity.