Fig 2: M1/M2-associated protein level changes after JWH133, GP1a or AM630 treatment. a-d M1-associated protein levels, including IL-6, IL-12, CD86 and iNOS. e-h M2-associated protein levels, including IL-4, IL-10, CD206 and Arg-1. i and j The iNOS/Arg-1 and IL-12/IL-10 protein level. Data are mean ± SD. + P < 0.05, ++ P < 0.01, JWH133 versus vehicle; * P < 0.05, ** P < 0.01, GP1a versus vehicle; # P < 0.05, ## P < 0.01, AM630 versus vehicle

Fig 3: YAP/STAT3 regulated M2 macrophage polarization induced by BC cell supernatant. (A) YAP, STAT3, and the p-STAT3 expression level in cell lines were detected by WB. (B and C) M1 and M2 macrophages were identified using flow cytometry. (D) PD-L1 expression level in cell lines was detected by WB. (E) ELISA was adopted to test iNOS, IL-12, IL-10, TGF-ß, Arg-1, and CCL-22 expression in cell lines. (F) RT-qPCR was used to detect the expression of VEGF and VEGFR-2 in cell lines. (G) CoIP verified that YAP directly binds to STAT3 protein in cell lines. ***p < 0.001, **p < 0.01, *p < 0.05 vs the oe-NC group, ### p < 0.001, ## p < 0.01, # p < 0.05 vs the oe-YAP + si-NC group. All data were presented as mean ± SD (n = 3) from three independent experiments, each performed in triplicate. one-way ANOVA.

Fig 4: Inhibition of YAP could affect TME and tumor proliferation through the STAT3/VEGF/VEGFR-2 axis. (A) The detection of volume and weight of tumors of mice. (B) The tumor morphological changes of mice were observed by HE staining. (C) YAP expression of tumor tissues was tested by RT-qPCR. (D) CD3+CD4+, CD3+CD8+, and treg cells in tumor tissues were examined by flow cytometry. (E) The ratio of M1 and M2 macrophages in tumor tissues was tested by flow cytometry. (F) The iNOS, IL-12, IL-10, TGF-ß, Arg-1, and CCL-22 expressions were tested by ELISA. (G) IF staining was selected to examine the YAP, STAT3, and p-STAT3 expression levels in tumor tissues. The red signal represented positive staining for YAP, p-STAT3, and STAT3, while the blue signal indicated nuclear staining. (H) The levels of YAP, STAT3, p-STAT3, VEGF, VEGFR-2, and PD-L1 in tumor tissues were examined by WB. All data were presented as mean ± SD (n = 3) from three independent experiments, each performed in triplicate. ***p < 0.001, **p < 0.01, *p < 0.05 vs the model group. One-way ANOVA (A, left and B–H). Two-way ANOVA (A, right).

Fig 5: Inhibition of YAP reversed M2 macrophage polarization induced by the supernatant of BC cells. (A) RT-qPCR and WB were chosen to detect YAP expression in cell lines. (B) M1 and M2 macrophages were identified with flow cytometry with markers of CD16/32+F4/80+ and CD206+F4/80+, respectively. (C) The expression of PD-L1 protein in cell lines was detected through WB. (D) The iNOS, IL-12, IL-10, TGF-ß, Arg-1, and CCL-22 expressions in cell lines were tested with ELISA. (E) The expression of YAP in cell lines was examined with IF staining. The green signal indicated positive staining for YAP, while the blue signal indicated nuclear staining. All data were presented as mean ± SD (n = 3) from three independent experiments, each performed in triplicate. ***p < 0.001, **p < 0.01, *p < 0.05 vs the control group, ### p < 0.001, ## p < 0.01, # p < 0.05 vs the TAMs + si-NC group. one-way ANOVA (A–E).