Activation of STAT3 in neutrophils. (A) FACS analyses of phosphorylated STAT3 on primary BM neutrophils from WT and Myo1f^−/− B16F10 tumor models. (B) FACS analyses of phosphorylated STAT3 on cultured BM neutrophils from WT mice after 24 h of treatment with stattic (5 µM, N = 3). (C) FACS analyses of Ki-67 on cultured neutrophils from WT BM after 5 days of treatment with stattic (5 μM, N = 5). (D) FACS analyses of DCFH-DA–labeled BM neutrophils from WT and Myo1f^−/− tumor-free model at day 5 after treated with stattic (5 µM) for 24 h, (N = 5). (E) FACS analyses of PD-L1 on cultured BM neutrophils from WT and Myo1f^−/− tumor-free model at day 5 after treated with stattic (5 µM) for 24 h (N = 5). (F)Cybb and Cd274 mRNA level were detected by RT-qPCR on cultured BM neutrophils from WT and Myo1f^−/− tumor-free model at day 5 after treated with stattic (5 µM) for 24 h (N = 5). (G) FACS analyses of CD8⁺ (GZMB⁺) T cells from co-culture BM neutrophils from WT and Myo1f^−/− tumor-free model treated with stattic (5 μM) for 24 h (N = 5). (H) Schematic of cultured neutrophils with stattic (5 μM) treatment transfer from donor (CD45.1 WT and KO mice) to recipient (CD45.2 B16F10 tumor-bearing mice). (I) Recipient tumor growth curve over time (N = 5). (J) Schematic diagram of MYO1F regulates ROS and PD-L1 by inhibiting STAT3 activation. Data in A–I represent one experiment of three independent repeats. Data are presented as mean ± SD. P values were analyzed by one-way ANOVA test (D–G and I); two-tailed unpaired Student’s t test (A–C); *P < 0.05, **P < 0.01, and ***P < 0.001. Neu, neutrophil; TB, tumor bearing; TF, tumor free.