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. 2020 Sep 24;11:4840. doi: 10.1038/s41467-020-18617-z

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Tumor growth and survival analysis of mice with 344SQR tumors treated with IgG ctrl (n = 5 animals) or anti-PD1 (10 mg/kg) (n = 5 animals) or 344SQR shBMP7 tumors treated with IgG (n = 5 animals) or anti-PD1 (10 mg/kg) (n = 5 animals) twice a week for 2 weeks. b Tumor growth and survival analysis of mice with 344SQR tumors (n = 5 animals) treated with IgG, anti-PD1 (10 mg/kg), follistatin (0.1 mg/kg), or follistatin (0.1 mg/kg) plus anti-PD1(10 mg/kg) for 2 weeks. For a, ctrl+ IgG vs. shBMP7 + αPD1, ****p < 0.0001, ctrl+ αPD1 vs. shBMP7 + αPD1, ****p < 0.0001 shBMP7+IgG vs. shBMP7 + αPD1, ****p < 0.0001, Two-way RM ANOVA. For b, IgG vs. foll+αPD1, **p = 0.0060, αPD1 vs. foll+αPD1, ***p = 0.0003, foll+IgG vs. foll+αPD1, ****p < 0.0001, Two-way RM ANOVA. Statistical significance was defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Mouse survival rates were analyzed by the Kaplan–Meier method and compared with log-rank tests. c–e Flow cytometry analysis of CD8⁺(*p = 0.0421), CD8⁺IFNG⁺(*p = 0.0475, *p = 0.0121), F4/80⁺CD206⁺, CD4⁺ (*p = 0.0199), CD4⁺IFNG⁺ T cells (*p = 0.0303) in tumor-infiltrating leukocytes (TILs) from 344SQR ctrl (n = 3 biologically independent samples) and 344SQR shBMP7 (n = 3 biologically independent samples) tumors treated with IgG or anti-PD1 (10 mg/kg) twice a week for 2 weeks. Data are presented as mean values ±SD. P values are from unpaired, two-sided t tests. f, g Representative images of immunohistochemical stains for CD206 (M2 macrophage marker) and CD4 (brown dots) in formalin-fixed paraffin-embedded tissue sections from BMP7-knockdown tumors treated with IgG or anti-PD1 compared with control. A representative staining image from each cohort (n = 3 biologically independent samples) is displayed. Data shown are representative of two reproducible independent experiments. Scale bar, 100 μm (×40 magnification). h, i Survival analysis of mice with 344SQR ctrl tumors or 344SQR shBMP7 tumors treated with IgG or anti-PDL1 (10 mg/kg) (n = 5 animals) or anti-CTLA4 (10 mg/kg) (n = 8 animals) twice a week for 2 weeks. Mouse survival rates were analyzed with the Kaplan–Meier method, and curves compared with log-rank tests. Statistical significance was defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.