TGF-β1 induces downregulation of MYO1F level in neutrophils. (A) FACS analyses of CD11b+Ly6G+ neutrophils in primary BM from MC38 tumor-free and MC38 tumor-bearing models (N = 9). (B) FACS analyses of MYO1F on neutrophils and non-neutrophils in primary BM from tumor-free and MC38 tumor-bearing models (N = 9). (C) Representative fluorescence staining of neutrophils infiltration in human melanoma (blue, DAPI; grayish white, CD33; red, MYO1F), 30 (normal) and 50 (melanoma) representative images for each group were counted. Scale bar: 50 µm. (D) Statistical analyses of fluorescence intensity of MYO1F in neutrophils, Neu_Normal, N = 30; Neu_Melanoma, N = 50. (E) FACS analyses of MYO1F level by staining with fluorescent antibody-targeting MYO1F on BM neutrophils and tumor-infiltrating neutrophils at different time (N = 5). (F) Cultured neutrophils were treated with supernatant from cell lines; MYO1F protein levels were analyzed 72 h after treatment by immunoblotting. Myo1f mRNA levels were detected by RT-qPCR from cultured neutrophils treated with mixture supernatant from MC38 and Hepa1–6 cell lines (N = 5). TM: mixture supernatant from MC38 and Hepa1–6 cell lines. (G) Cultures neutrophils were treated with indicated commercial cytokines; MYO1F protein levels were analyzed 48 h after treatment by immunoblotting. TM: mixture supernatant from MC38 and Hepa1–6 cell lines was used as positive control. (H) Cultured neutrophils were treated with TGF-β1 (1, 2, and 5 ng/ml); Myo1f mRNA levels were detected by RT-qPCR (N = 3). (I) Neutrophils at day 2 treated with medium, TM, and TGF-β1 (5 ng/ml); Ki-67 was detected as proliferation marker by FACS 96 h after TGF-β1 treatment (N = 3). (J) Anti–TGF-β1 (0, 1, 2, and 5 µg/ml) used in TM-cultured neutrophils to neutralize supernumerary TGF-β1 in microenvironment; Ki-67 was detected as proliferation marker by FACS 96 h after anti–TGF-β1 treatment; isotype control antibody used as control; TM: mixture supernatant from MC38 and Hepa1–6 cell lines. (K) Violin plots of TGFB1 and MYO1F gene expression in tumor stages of 18 cancers: ACC, CESC, cholangio carcinoma (CHOL), COAD, glioblastoma multiforme (GBM), head and neck squamous carcinoma (HNSC), KICH, kidney renal papillary cell carcinoma (KIRP), acute myeloid leukemia (LAML), brain lower grade glioma (LGG), LIHC, LUAD, OV, sarcoma (SARC), testicular germ cell tumors (TGCT), THCA, THYM, and UCEC. (L) FACS analyses of CD11b+Ly6G+ neutrophils in BM CD45+ myeloid cells from sgRNA-transfected B16F10 models, tumor-free mice. (M) FACS analyses of MYO1F protein level on CD11b+Ly6G+ neutrophils. (N) ELISA detection of TGF-β1 level from peripheral blood serum (dilution in 400 µl RPMI1640 of 100 µl serum) and extract of total BM (dilution in 500 µl RPMI1640 of a tibia). Data in A, B, F–J, and L–N represent one experiment of three independent repeats; C–E represent one experiment of two independent repeats. Data are presented as mean ± SD. P values were analyzed by one-way ANOVA test (E and H–N); two-tailed unpaired Student’s t test (A, B, D, and F), *P < 0.05, **P < 0.01, and ***P < 0.001. ns, no significance; TM, tumor medium; TF, tumor free; TB, tumor bearing. Source data are available for this figure: SourceData F6.
Figure 6.

TGF-β1 induces downregulation of MYO1F level in neutrophils. (A) FACS analyses of CD11b+Ly6G+ neutrophils in primary BM from MC38 tumor-free and MC38 tumor-bearing models (N = 9). (B) FACS analyses of MYO1F on neutrophils and non-neutrophils in primary BM from tumor-free and MC38 tumor-bearing models (N = 9). (C) Representative fluorescence staining of neutrophils infiltration in human melanoma (blue, DAPI; grayish white, CD33; red, MYO1F), 30 (normal) and 50 (melanoma) representative images for each group were counted. Scale bar: 50 µm. (D) Statistical analyses of fluorescence intensity of MYO1F in neutrophils, Neu_Normal, N = 30; Neu_Melanoma, N = 50. (E) FACS analyses of MYO1F level by staining with fluorescent antibody-targeting MYO1F on BM neutrophils and tumor-infiltrating neutrophils at different time (N = 5). (F) Cultured neutrophils were treated with supernatant from cell lines; MYO1F protein levels were analyzed 72 h after treatment by immunoblotting. Myo1f mRNA levels were detected by RT-qPCR from cultured neutrophils treated with mixture supernatant from MC38 and Hepa1–6 cell lines (N = 5). TM: mixture supernatant from MC38 and Hepa1–6 cell lines. (G) Cultures neutrophils were treated with indicated commercial cytokines; MYO1F protein levels were analyzed 48 h after treatment by immunoblotting. TM: mixture supernatant from MC38 and Hepa1–6 cell lines was used as positive control. (H) Cultured neutrophils were treated with TGF-β1 (1, 2, and 5 ng/ml); Myo1f mRNA levels were detected by RT-qPCR (N = 3). (I) Neutrophils at day 2 treated with medium, TM, and TGF-β1 (5 ng/ml); Ki-67 was detected as proliferation marker by FACS 96 h after TGF-β1 treatment (N = 3). (J) Anti–TGF-β1 (0, 1, 2, and 5 µg/ml) used in TM-cultured neutrophils to neutralize supernumerary TGF-β1 in microenvironment; Ki-67 was detected as proliferation marker by FACS 96 h after anti–TGF-β1 treatment; isotype control antibody used as control; TM: mixture supernatant from MC38 and Hepa1–6 cell lines. (K) Violin plots of TGFB1 and MYO1F gene expression in tumor stages of 18 cancers: ACC, CESC, cholangio carcinoma (CHOL), COAD, glioblastoma multiforme (GBM), head and neck squamous carcinoma (HNSC), KICH, kidney renal papillary cell carcinoma (KIRP), acute myeloid leukemia (LAML), brain lower grade glioma (LGG), LIHC, LUAD, OV, sarcoma (SARC), testicular germ cell tumors (TGCT), THCA, THYM, and UCEC. (L) FACS analyses of CD11b+Ly6G+ neutrophils in BM CD45+ myeloid cells from sgRNA-transfected B16F10 models, tumor-free mice. (M) FACS analyses of MYO1F protein level on CD11b+Ly6G+ neutrophils. (N) ELISA detection of TGF-β1 level from peripheral blood serum (dilution in 400 µl RPMI1640 of 100 µl serum) and extract of total BM (dilution in 500 µl RPMI1640 of a tibia). Data in A, B, F–J, and L–N represent one experiment of three independent repeats; C–E represent one experiment of two independent repeats. Data are presented as mean ± SD. P values were analyzed by one-way ANOVA test (E and H–N); two-tailed unpaired Student’s t test (A, B, D, and F), *P < 0.05, **P < 0.01, and ***P < 0.001. ns, no significance; TM, tumor medium; TF, tumor free; TB, tumor bearing. Source data are available for this figure: SourceData F6.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal