Fig 2: TM4SF1 is involved in B7-H3-associated resistance to DOX-induced senescence.A Western blot analysis of TM4SF1, and p21 in control cells or shB7-H3 CRC cells after treatment with TM4SF1 overexpression vectors and DOX. ß-Actin served as a loading control. B, C SA-ß-Gal activity of control cells or shB7-H3 CRC cells after treatment with TM4SF1 overexpression vectors and DOX was examined. Scale bar, 100 µm. One representative image from three reproducible experiments is shown. The percentages of SA-ß-gal-positive cells are shown in the bar graph. D, E SAHF activity of control cells or shB7-H3 CRC cells after treatment with TM4SF1 overexpression vectors and DOX was examined. Scale bar, 50 µm. One representative image from three replicate experiments is shown. The percentages of SAHF-positive cells are shown in the bar graph. F The cell viability of control cells or shB7-H3 CRC cells with TM4SF1 overexpression vectors and DOX treatment after 24, 36, 48, and 96 h was examined by CCK-8 assays. G The colony formation of control cells or shB7-H3 CRC cells after treatment with TM4SF1 overexpression vectors and DOX was examined. One representative image from three reproducible experiments is shown. The number of colonies is shown in the bar graph. H Cell cycle analysis by PI staining in control cells or shB7-H3 CRC cells after treatment with TM4SF1 overexpression vectors and DOX was examined through flow cytometry. The data represent the means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: B7-H3 promotes TM4SF1 expression and inhibits cellular senescence dependent on the AKT pathway in CRC.A Western blot analysis of AKT, p-AKT, TM4SF1 and p21 in control cells or B7-H3 CRC cells cotreated with perifosine and DOX. ß-Actin served as a loading control. B, C SA-ß-Gal activity of control cells or B7-H3 CRC cells cotreated with perifosine and DOX was examined in CRC cells. Scale bar, 100 µm. One representative image from three reproducible experiments is shown. The percentages of SA-ß-gal-positive cells are shown in the bar graph. D, E SAHF activity of control cells or B7-H3 CRC cells cotreated with perifosine and DOX was examined. Scale bar, 50 µm. One representative image from three replicate experiments. F The cell viability of control cells or B7-H3 CRC cells cotreated with perifosine and DOX after 24, 36, 48, and 96 h was examined by CCK-8 assays. G Colony formation assay in control cells or B7-H3 CRC cells cotreated with perifosine and DOX. One representative image from three reproducible experiments is shown. The number of colonies is shown in the bar graph. H Cell cycle analysis by PI staining in control cells or B7-H3 CRC cells with perifosine and DOX cotreatment was examined through flow cytometry. The data represent the mean ± SEM. **P < 0.01; ***P < 0.001.

Fig 4: B7-H3 promoted angiogenesis via NF-?B/VEGFA pathway in Matrigel plugs in vivo.a, b CD31 (a) and VEGFA (b) protein expression based on their IHC staining index results in subcutaneous tumors formed by sh-NC-HCT116 and shB7-H3-HCT116 cells. N = 5. c, d CD31 (c) and VEGFA (d) protein expression based on their IHC staining index results in subcutaneous tumors formed by EV-HCT116 and B7-H3-HCT116 cells. N = 5. e, f CD31 (e) and VEGFA (f) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors treated with BAY11–7082 (6 mg/kg). g, h CD31 (g) and VEGFA (h) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors treated with bevacizumab (1 mg/kg). i, j CD31 (i) and VEGFA (j) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors co-treated with 3E8 (5 mg/kg) and BAY11–7082 (6 mg/kg) or bevacizumab (1 mg/kg). N = 5. The data represent the means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 5: Interfering B7-H3 expression in CAFs-blocked cytokine secretion and decreased migration and invasion ability. (A) immunofluorescence images of vimentin, CD31, B7-H3, FAP-a, and a-SMA in CAFs and NFs. DAPI fluorescence is shown as blue. Vimentin, CD31, B7-H3, FAP-a, and a-SMA immunofluorescence is shown as red. (B) The Fluorescence intensity statistical results of vimentin, CD31, B7-H3, FAP-a, and a-SMA. Mean ± SEM, *P < 0.05 and #P < 0.01 by Student's t-test. (C) B7-H3 protein expression in fibroblasts after treatment with B7-H3 siRNA. (D) The relative mRNA expression levels of B7-H3 and cytokines are indicated after treatment with B7-H3 siRNA. Mean ± SEM, *P < 0.05 and #P < 0.01 by Student's t-test. (E) The IL-6 and VEGF expression levels as detected by ELISA. Mean ± SEM, *P < 0.05 and #P < 0.01 by Student's t-test. (F) The transwell tests were conducted to assess the migration and invasion ability of fibroblasts.