Journal: Cell Death & Disease

Article Title: CXCL1 derived from tumor-associated macrophages promotes breast cancer metastasis via activating NF-κB/SOX4 signaling

doi: 10.1038/s41419-018-0876-3

Figure Lengend Snippet: a TAMs were isolated from breast tumors by differential adhesion technique and validated by F4/80 + /CD206 + staining. b Cytokine array revealed that CXCL1 had the highest expression in the supernatants of TAMs. c Primary mammary tumors and lung metastatic lesions were collected from MMTV-PyVT +/- mice, respectively, and validated by HE staining. d CXCL1 expression was significantly enhanced in the lung metastatic lesions compared with its primary tumors, accompanied by increased expression levels of vimentin, N-cadherin and β-catenin, whereas the epithelial marker E-cadherin was reduced, indicating that CXCL1 expression was closely correlated with EMT process. Meanwhile, ARG1 expression was also increased in the lung metastasis lesions, implying that the enhanced CXCL1 expression might be correlated with increased M2 macrophage phenotype. (All values from three independent experiments are quantified as mean ± SD, * P < 0.05, ** P < 0.01)

Article Snippet: The slides were then subjected to incubation with primary antibodies including CXCL1 (no. A5802, ABclonal), Vimentin (no. A2666, ABclonal), N-cadherin (no. A0443, ABclonal), ARG-1 (no. A1847, ABclonal), SOX4 (no. ab80261, ABclonal) and p-P65 (no. AP0475, ABclonal).

Techniques: Isolation, Staining, Expressing, Marker

Journal: Cell Death & Disease

Article Title: CXCL1 derived from tumor-associated macrophages promotes breast cancer metastasis via activating NF-κB/SOX4 signaling

doi: 10.1038/s41419-018-0876-3

Figure Lengend Snippet: a CXCL1 had little influence on the proliferation of breast cancer cells including 4T1, MDA-MB-231 and MCF-7 in both dose- and time-dependent manner. b Would-healing and Transwell assay revealed that CXCL1 could promote the migration and invasion ability of 4T1 cells. c Western blotting results showed that CXCL1 administration promotes the EMT process of 4T1 cells, presenting as dose-dependent increase of vimentin, N-cadherin, β-catenin and gradual downregulation of E-cadherin. Gelatin zymography also indicated that CXCL1 treatment results in increased secretion of MMP-9 and MMP-2 from 4T1 cells. d The mouse macrophage cell line Raw264.7 was induced to TAMs by administrating IL-4 and IL-13, resulting in the increased expression of CD206 and Arg-1, and reduction of iNOS; Meanwhile, ELISA assay demonstrated that CXCL1 expression in M2-Raw264.7 supernatants was significantly higher than that in either M0-Raw164.7 or 4T1 cancer cells. Notably, the CM of TAMs did not increase CXCL1 expression in 4T1 cancer cells. e TAMs-CM treatment led to increased migration and invasion ability of 4T1 cells, whereas was blocked by administration of CXCL1-neutralizing antibody. f, g CXCR2 silencing in 4T1 cancer cells did not block CXCL1-induced EMT and invasion ability. (All values from three independent experiments are quantified as mean ± SD, ** P < 0.01)

Article Snippet: The slides were then subjected to incubation with primary antibodies including CXCL1 (no. A5802, ABclonal), Vimentin (no. A2666, ABclonal), N-cadherin (no. A0443, ABclonal), ARG-1 (no. A1847, ABclonal), SOX4 (no. ab80261, ABclonal) and p-P65 (no. AP0475, ABclonal).

Techniques: Transwell Assay, Migration, Western Blot, Zymography, Expressing, Enzyme-linked Immunosorbent Assay, Blocking Assay

Journal: Cell Death & Disease

Article Title: CXCL1 derived from tumor-associated macrophages promotes breast cancer metastasis via activating NF-κB/SOX4 signaling

doi: 10.1038/s41419-018-0876-3

Figure Lengend Snippet: a Would-healing and Transwell assay revealed that CXCL1 could promote the migration and invasion ability of both MDA-MB-231 and MCF-7 cells. b Western blotting results showed that CXCL1 administration dose dependently promotes the EMT process of both breast cancer cells, presenting as increased expression of vimentin, N-cadherin, β-catenin and gradual downregulation of E-cadherin. c The enhanced expression levels of CD206, Arg1 and CXCL1 validated the successful induction of TAMs by administrating IL-4. Meanwhile, shCXCL1 was applied to knockdown its expression in M2-THP1 macrophages. d CXCL1 silencing in M2-THP1 macrophages suppressed the enhanced migration and invasion abilities of MDA-MB-231 and MCF-7 cells induced by TAMs-CM. (All values from three independent experiments are quantified as mean ± SD, ** P < 0.01)

Article Snippet: The slides were then subjected to incubation with primary antibodies including CXCL1 (no. A5802, ABclonal), Vimentin (no. A2666, ABclonal), N-cadherin (no. A0443, ABclonal), ARG-1 (no. A1847, ABclonal), SOX4 (no. ab80261, ABclonal) and p-P65 (no. AP0475, ABclonal).

Techniques: Transwell Assay, Migration, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: CXCL1 derived from tumor-associated macrophages promotes breast cancer metastasis via activating NF-κB/SOX4 signaling

doi: 10.1038/s41419-018-0876-3

Figure Lengend Snippet: a qPCR screening assay revealed SOX4 as the highest responsive gene following CXCL1 administration in both MDA-MB-231 and MCF-7 cells. b Immunofluorescence assay revealed that CXCL1 administration resulted in increased SOX4 expression and its nucleus transportation ( × 400). c Western blotting assay indicated that CXCL1 overexpression resulted in SOX4 upregulation, accompanied by enhanced EMT process. By contrast, CXCL1 silencing in both breast cancer cells led to the downregulation of SOX4 and inhibited EMT process. d SOX4 silencing inhibited the EMT process in both breast cancer cells induced by CXCL1 administration, presenting as decreased expression of vimentin, β-catenin and upregulation of E-cadherin. (All values from three independent experiments are quantified as mean ± SD, ** P < 0.01 vs. control)

Article Snippet: The slides were then subjected to incubation with primary antibodies including CXCL1 (no. A5802, ABclonal), Vimentin (no. A2666, ABclonal), N-cadherin (no. A0443, ABclonal), ARG-1 (no. A1847, ABclonal), SOX4 (no. ab80261, ABclonal) and p-P65 (no. AP0475, ABclonal).

Techniques: Screening Assay, Immunofluorescence, Expressing, Western Blot, Over Expression

Journal: Cell Death & Disease

Article Title: CXCL1 derived from tumor-associated macrophages promotes breast cancer metastasis via activating NF-κB/SOX4 signaling

doi: 10.1038/s41419-018-0876-3

Figure Lengend Snippet: a , b In situ breast cancer xenograft showed that TAMs co-injection promotes breast cancer growth, whereas CXCL1 silencing in TAMs significantly suppressed tumor growth (values represents as mean ± SD, n = 4, * P < 0.05, ** P < 0.01). c Immunohistochemistry assay showed that TAMs co-injection significantly elevates the expression of vimentin, SOX4 and p-P65 in tumors, whereas E-cadherin expression was inhibited. By contrast, CXCL1 silencing in TAMs led to opposite effects (scale bars indicate 50 μm). d In vivo luciferase imaging model showed that TAMs significantly promotes the metastatic colonies formation, whereas CXCL1 silencing in TAMs significantly blocks breast cancer lung colony growth. e Immunofluorescence assay revealed that TAMs co-injection significantly elevates vimentin and SOX4 expression in the metastatic lesions, whereas CXCL1 silencing in TAMs results in an inhibition of vimentin and SOX4 expression ( × 400)

Article Snippet: The slides were then subjected to incubation with primary antibodies including CXCL1 (no. A5802, ABclonal), Vimentin (no. A2666, ABclonal), N-cadherin (no. A0443, ABclonal), ARG-1 (no. A1847, ABclonal), SOX4 (no. ab80261, ABclonal) and p-P65 (no. AP0475, ABclonal).

Techniques: In Situ, Injection, Immunohistochemistry, Expressing, In Vivo, Luciferase, Imaging, Immunofluorescence, Inhibition