Special 35 mm glass bottom plate

Cell suspensions were seeded on the central concavity of a special 35-mm glass-bottom plate (Nest, China) at 50% confluence. After three washes with ice-cold PBS, the cells were fixed with 4% paraformaldehyde (PFA, Dingguo, China) for 15 min and then permeabilized with 0.1% Triton X-100 (Dingguo) for 10 min. Subsequently, the cells were blocked with 5% bovine serum albumin (Thermo Fisher Scientific) for 1 h at room temperature and incubated with an appropriate concentration of primary antibodies against PD-L1 (1:500; Abcam, London, UK) overnight at 4 °C. Then, the cells were incubated with Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1:200, Abcam) or goat anti-mouse IgG (1:200, Abcam) secondary antibodies for 1 h. The stained cells were observed under a confocal (Leica TSC SP8, Mannheim, Germany). The antibodies used are listed in Table S2.

Cell suspensions were seeded on the central concavity of a special 35 mm glass bottom plate (Nest, China) at ~ 50% confluent density. The cells were cultured overnight with or without PLA. After three washes with ice-cold PBS, cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.1% Triton X-100 for 10 min. Subsequently, cells were blocked with 5% bovine serum albumin for 1 h at room temperature and incubated with an appropriate concentration of primary antibodies against Oct4, ABCG2, ALDH1, SOX2, or CD49f (Abcam, London, UK, 1:100 dilution) overnight at 4 °C. Then, the cells were incubated with Alexa Fluor^® 488 conjugated goat anti-rabbit IgG (Abcam, London, UK, 1:200 dilution) or goat anti-mouse IgG (Abcam, London, UK, 1:200 dilution) secondary antibodies for 1 h. The stained cells were observed under a fluorescence confocal microscope.