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. 2021 Jul 19;6:273. doi: 10.1038/s41392-021-00597-1

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© The Author(s) 2021

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. 1 — a, b Plots of tumor growth rates and survival time of mice inoculated with either RHBDF1 knockout (R1KO) or mock-transfected 4T1 cells (MT). c Typical images of the overall extent of hypoxia in tumors detected by hypoxyprobe (left); the ratio of hypoxic area versus non-hypoxic area was determined by computer-assisted image analysis (right). d Typical images of immunofluorescence co-staining of 4T1 tumor specimens for fibroblast cell marker PDGFRβ and endothelial cell marker CD31; area of CD31 and PDGFRβ, and the dendritic number of vessels were determined by Image-Pro Plus, n = 5. e Typical images of 3D confocal microscopic analysis of MT and R1KO 4T1 tumor specimens demonstrating blood vessel structure and EndMT in progress; insets: details of the selected area (3× magnifications). f Crystal violet staining of the bottoms of the transwell chambers containing wildtype 4T1 cells co-cultured with HUVECs that were pre- co-cultured with either R1KO or MT 4T1 cells. g Co-fluorescent staining of collagen I (green) and aSMA for CAFs (red); the content of collagen is showed below, n = 5. h Masson staining of the tumor; blue area indicates tumor stroma, and the content is shown below, n = 6. i Fluorescent co-staining of CD8 and granzyme b of either R1KO or MT 4T1 tumors; the number of cytotoxic T cells (double-positive) was determined by image analysis, n = 6. j Quantitative analysis of the transcription level of the indicated genes by qPCR, n = 6. k AP-1 transcription activation assay by a luciferase-based reporter system. l Western blotting analysis of c-Jun phosphorylation in whole cell or nucleus of MT or R1KO 4T1 cells. m Western blotting analysis of JNK1/2/3 and c-Jun phosphorylation after PTX treatment (48 h). n Co-immunoprecipitation analysis in MCF-7 cells of co-transfected JNK1, 2 or 3 and various RHBDF1 fragments (amino acid residues 1–411 and 406–855). o Schematic representation of RHBDF1-supported activation of transcription factor AP-1 and the impact on tumor stroma modulation. Data are means ± SD. Statistics, Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001