Pi stain

Proliferation of T cells was determined by PKH26 staining in vitro. Individual groups of CAR-T and control Utd T cells (1 × 10⁶ cells/tube) were labeled with PKH26 (Sigma-Aldrich) at 37 °C for 5 min. After being washed, individual groups of cells were stimulated with the same number of CD105⁺ target cells that had been irradiated (100 Gy) for 120 h. The suspended T cells were collected and the percentages of proliferative T cells were determined by flow cytometry.
For cytotoxicity assays, individual groups of CAR-T and control Utd T cells were cultured in triplicate with each type of PKH26-labeled target cells at a ratio of 3 : 1, 1 : 1, or 1 : 3 for 16 h. After being washed, the adhered target cells were collected and stained with PI stain (Sigma), and the percentages of PKH26⁺PI⁺ dead cells in individual groups were determined by flow cytometry as % of specific lysis. In addition, anti-CD105 CAR-T cells were co-cultured with Bel7404 cells at a ratio of 3 : 1 for 16 h in the presence or absence of CD105 protein (eBioscience, Cat14-1051-85). The percentages of lysed target cells were determined by flow cytometry. Moreover, individual groups of CAR-T and control Utd T cells were cultured in triplicate with Bel7404 cells at a ratio of 3 : 1 for 12 h and stained with PE-anti-CD107a antibody (Biolegend,Cat304124). The percentages of CD107a⁺ T cells were determined by flow cytometry.

Bovine GCs were seeded on coverslips in a 12-well plate for 24 h. The cells attached with the bottom were fixed with absolute cold methanol for 20 min, and permeabilized with 200 μL 0.1% Triton X-100 (Sigma-Aldrich, St. Locus, MI, USA) in PBS for 5 min. After washing with PBS, cells were loaded with 5% goat serum for 1 h and incubated with anti-FSHR antibody (1:500, Sigma-Aldrich, St. Locus, MI, USA) at 4 °C overnight. Moreover, cells were exposed to FITC-conjugated secondary antibody (1:1000, Sigma-Aldrich, St. Locus, MI, USA) for 2 h in the dark. Cells were then washed two times with PBS and stained with PI stain (Sigma-Aldrich, St. Locus, MI, USA) for staining nuclear DNA. Cells were mounted and observed under a fluorescence microscope (Olympus, Tokyo, Japan).