P0260

Placental tissues embedded in wax were sliced at a thickness of 5 μm. For the hematoxylin and eosin (H&E) assay, the slides were stained with hematoxylin for 5 min and eosin Y for 30 s (C0105S, Beyotime, Beijing, China). Images were captured using an Olympus DP71 microscope. For immunohistochemistry assay, antigen retrieval was carried out in citrate buffer pH 6.0 (P0083, Beyotime, Shanghai, China) using a pressure cooker for 3 min, followed by blocking endogenous peroxidases using 0.3% H₂O₂ for 15 min. After blocking 20 min by blocking buffer (P0260, Beyotime, Shanghai, China), the slides were incubated overnight at 4°C with an anti-CPT2 antibody (1:150, ab181114, Abcam, Cambridge, UK), anti-CPT1b antibody (1:150, 22170-1-AP, Proteintech, Wuhan, China). On the next day, the slides were washed and then incubated with a secondary antibody (PV9001, Zhongshan Gold Bridge Biotechnology Co, Beijing, China) for 20 min at room temperature. The slides were incubated with DAB (ZLI9018, Zhongshan Gold Bridge Biotechnology Co, Beijing, China) to detect side-specific antigen-antibody binding, followed by staining with hematoxylin. Then, the slides were dehydrated and sealed with neutral gum. Images were captured using an Olympus DP71 microscope. Five random views per tissue section were used to quantify the mean IOD (IOD/area) using Image-Pro Plus 6.0.

Dil dye (Beyotime, C1036) was added to 50 μL of exosome solution and then incubated at 37°C for 30 min. Dil-labeled exosome solution (1 μg/μL) was added to BM-derived MDSCs, followed by incubation at 37°C for 48 h with or without JPYZXZ. The culture medium was discarded, and fixed samples were blocked with blocking buffer (Beyotime, P0260) for 1 h, washed twice with PBS, and then incubated with anti-PD-L1 (Proteintech, 66248-1-Ig, 1:200) overnight. Next, the sections were incubated with fluorescent secondary antibodies and DAPI for 10 min. In addition, we analyzed tumor tissue and lung tissue sections from mice using the primary antibodies against CD11b (Abcam, ab13357, 1:100), anti-Gr-1 (BioLegend, 108448, 1:100), and anti-PD-L1 (Proteintech, 66248-1-Ig, 1:200). The sections were examined using fluorescence microscope and measured using ImageJ. Moreover, tumor tissues were fixed in 4% paraformaldehyde and embedded in paraffin, and then stained with H&E.

The sections were blocked with quickblock blocking buffer for immune staining (P0260, Beyotime, China) for 15 ​min at RT, followed by incubation with primary antibody against Pax7 (1:100, bs-22741R, Bioss, China), MyoD (1:100, ab203383, abcam, UK), MyoG (1:500, ab124800, abcam, UK), fast MyHC (1:1000, ab91506, abcam, UK), laminin (1:50, ab11575, abcam, UK) at 4 ​°C overnight and labeled with Alexa Fluor594-preabsorbed goat anti-rabbit IgG (ab150084, Abcam, 1:500, UK), Alexa Fluor 488 AffiniPure F(ab')₂ Fragment Goat Anti-Rabbit IgG (111-546-003, Jackson ImmunoResearch, 1:500, USA) respectively for 2 ​h at room temperature. Next, the nucleus was stained with DAPI.

OS cells were fixed using 4% paraformaldehyde (PFA) for 15 min and then permeabilized with 0.1% Triton X-100 for 10 min at room temperature. After blocked with blocking buffer (P0260, Beyotime, China) for 10 min at room temperature, the slices were incubated with antibodies against cleaved-caspase-3 (ab13847, Abcam, USA), cleaved-caspase-9 (ab202068, Abcam, USA) or LC3B (ab48394, Abcam, USA) overnight at 4 °C. The slices were imaged using confocal microscopy.

The GCs were fixed with 4% paraformaldehyde (PFA) for 30 min at 4 °C, and then incubated with 1% PBST (1% Triton X 100 dissolved in PBS) for 30 min at room temperature. The samples were then blocked with blocking solution (Beyotime, P0260) and then incubated with the primary antibodies (Table S1) in blocking solution overnight at 4 °C, followed by incubation with secondary antibodies at 37 °C for 1.5 h. The nuclei of cells were stained with Hoechst33342 (Beyotime, C1022). Fluorescent images were captured using a fluorescent microscope (Olympus, BX51, Japan). The relative fluorescence intensity per unit area was determined using Image J software (National Institutes of Health, USA).