il 8  (R&D Systems)

Journal: International Journal of Biological Sciences

Article Title: Identification of a novel cancer stem cell subpopulation that promotes progression of human fatal renal cell carcinoma by single-cell RNA-seq analysis

doi: 10.7150/ijbs.46645

Figure Lengend Snippet: Immunohistochemistry and survival analysis of CDRCC. A , t-NSE plot shows the expression distribution of BIRC5, PTTG1, CENPF and CDKN3 in 15208 cells and the corresponding immunohistochemical images using the 10× and 40× objective. B , IHC images show the expression of Cancer cluster-associated genes ATF3, PDZK1, VTN, and CXCL8 in CDRCC tissues. C , Kaplan-Meier survival curves for CDRCC patients show the relationship between gene expression level in ( A ) and ( B ) and survival prognosis (n=17 ,p<0.01).

Article Snippet: The following antibodies were used to represent the expression of the selected genes: anti-PARP1 (rabbit, 1:500, Abcam, ab32138), anti-PIGF (rabbit, 1:300, Proteintech, 10642-1-AP), anti-HDAC2 (rabbit, 1:500, Abcam, 32117), anti-FGFR3 (rabbit, 1:200, Abcam, ab137084), anti-BIRC5 (rabbit, 1:500, Abcam, ab76424), anti-PTTG1 (rabbit, 1:1000, Abcam, ab79546), anti-CENPF (rabbit, 1:500, Abcam, ab223847), anti-CDKN3 (rabbit, 1:500, Abcam, ab206314), anti-ATF3 (rabbit, 1:1000, Novusbio, nbp1-85816), anti-PDZK1 (mouse, 1:200, R&Dsystems, af4997), anti-VTN (rabbit, 1:300, Abcam, ab45139), anti-CXCL8 (mouse, 1:500, R&Dsystems, af-208-na)(Figure , ).

Techniques: Immunohistochemistry, Expressing, Immunohistochemical staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A positive feedback loop between gastric cancer cells and tumor-associated macrophage induces malignancy progression

doi: 10.1186/s13046-022-02366-6

Figure Lengend Snippet: Hypoxia promoted macrophage-derived CXCL8 secretion. A , B Differential genetic analysis of the transcription data of macrophages cultured under hypoxia and normoxia showed that hypoxia could promote CXCL8 expression. (A. volcano; B. heatmap). C MACS obtained human CD14 + monocytes and induced them into macrophages through M-CSF. D , E Hypoxia promoted human-derived macrophage CXCL8 expression at both transcription and protein levels. F . Hypoxia promoted the secretion of macrophage-derived cytokine CXCL8 secretion. G An evident co-localization of macrophages and CXCL8 in human GC tissue specimens. H Schematic of gating strategy of flow cytometry analysis. GC tissue were dissociated to obtain a single-cell suspension and stained with antibodies. Cells were first gated to exclude debris and dead cells (Sup Fig. 1D), then GC cells and macrophages were selected. Cells were further gated by cluster of CXCL8 expression. I GC cells (green) and macrophages (red) were co-cultured under hypoxia to mimics the microenvironment. J CXCL8 was mainly derived from macrophages and IL-10 was expressed by GC cells in the co-culture system

Article Snippet: Then, they were incubated with species-appropriate rabbit/mouse secondary antibodies coupled with AlexaFluor dyes (488, 594, 1:200, Invitrogen, A32814, A32723, A32740), DAPI (1:1000, Dojindo, KT013) at room temperature for 1 h. CXCL8 (5 μg/mL, R&D Systems, AF-208-NA), CD68 , CD163 , CD86 (1:200, Abcam, ab213363, ab182422, ab270719).

Techniques: Derivative Assay, Cell Culture, Expressing, Flow Cytometry, Suspension, Staining, Co-Culture Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A positive feedback loop between gastric cancer cells and tumor-associated macrophage induces malignancy progression

doi: 10.1186/s13046-022-02366-6

Figure Lengend Snippet: CXCL8 overexpression in GC promotes poor survival. A , B CXCL8 was up-regulated in GC tissue both in TCGA-GC and GEO-GC cohort. C In matched GC and adjacent tissues, CXCL8 was highly expressed in cancer tissues. D Representative image of CXCL8 expression in GC tissue. (Left: high-expression; Right: weak-expression). E , F CXCL8 promoted poor DFS and OS in the GC cohort. (E. DFS; F. OS)

Article Snippet: Then, they were incubated with species-appropriate rabbit/mouse secondary antibodies coupled with AlexaFluor dyes (488, 594, 1:200, Invitrogen, A32814, A32723, A32740), DAPI (1:1000, Dojindo, KT013) at room temperature for 1 h. CXCL8 (5 μg/mL, R&D Systems, AF-208-NA), CD68 , CD163 , CD86 (1:200, Abcam, ab213363, ab182422, ab270719).

Techniques: Over Expression, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A positive feedback loop between gastric cancer cells and tumor-associated macrophage induces malignancy progression

doi: 10.1186/s13046-022-02366-6

Figure Lengend Snippet: CXCL8 - CXCR1/2 deteriorates the malignant phenotype of GC. A Migration patterns of GC cells on Matrigel compared to cells cultured with CXCL8 and/or CXCL8-CXCR1/2 inhibitor repertaxin. B Migration trail of GC cells cultured with CXCL8 or CXCL8 and CXCL8-CXCR1/2 inhibitor. C Migration average distances of GC cells in each group. D Transwell exhibited the differences in the number of permeable cells in each group. E , F CCK-8, and colony formation assay confirmed CXCL8 could promote GC proliferation which could be inhibited by CXCL8-CXCR1/2 inhibitor. G CXCL8 increased the subcutaneous tumor size and weight. H IHC representative image of subcutaneous tumors showing that CXCL8 could promote Ki67 and inhibit the expression of Capcase3 ; the inhibitors could counteract this effect. I CXCL8 induced ascites formation and intra- metastasis of GC

Article Snippet: Then, they were incubated with species-appropriate rabbit/mouse secondary antibodies coupled with AlexaFluor dyes (488, 594, 1:200, Invitrogen, A32814, A32723, A32740), DAPI (1:1000, Dojindo, KT013) at room temperature for 1 h. CXCL8 (5 μg/mL, R&D Systems, AF-208-NA), CD68 , CD163 , CD86 (1:200, Abcam, ab213363, ab182422, ab270719).

Techniques: Migration, Cell Culture, CCK-8 Assay, Colony Assay, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A positive feedback loop between gastric cancer cells and tumor-associated macrophage induces malignancy progression

doi: 10.1186/s13046-022-02366-6

Figure Lengend Snippet: CXCL8-CXCR1/2 activated JAK/STAT1 signal pathway and promoted GC progression. A CXCL8 activated the JAK/STAT1 signal pathway. The STAT1 specific inhibitor (Fludarabine) could antagonize the effects of CXCL8 . B - D Migration patterns, migration trail, and average traveled distances of GC cells treated with CXCL8 or CXCL8 and Fludarabine together. E Fludarabine limited the GC invasion. F , G CCK-8, and colony formation assay revealed that the Fludarabine could resist CXCL8’s promotion of GC proliferation

Article Snippet: Then, they were incubated with species-appropriate rabbit/mouse secondary antibodies coupled with AlexaFluor dyes (488, 594, 1:200, Invitrogen, A32814, A32723, A32740), DAPI (1:1000, Dojindo, KT013) at room temperature for 1 h. CXCL8 (5 μg/mL, R&D Systems, AF-208-NA), CD68 , CD163 , CD86 (1:200, Abcam, ab213363, ab182422, ab270719).

Techniques: Migration, CCK-8 Assay, Colony Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A positive feedback loop between gastric cancer cells and tumor-associated macrophage induces malignancy progression

doi: 10.1186/s13046-022-02366-6

Figure Lengend Snippet: The mechanism graph of the positive feedback loop between GC cells and TAMs. Hypoxia-induced macrophage-derived CXCL8 secretion activated CXCL8- CXCR1/2-JAK/STAT1 signaling pathway and deteriorated the GC malignant phenotype. The transcription factor STAT1 could directly promote IL-10 expression. The latter activated the macrophage IL-10/ NF-κB signaling pathway and induced M2 polarization. The transcription factor NFKB1 directly upregulated CXCL8 expression

Article Snippet: Then, they were incubated with species-appropriate rabbit/mouse secondary antibodies coupled with AlexaFluor dyes (488, 594, 1:200, Invitrogen, A32814, A32723, A32740), DAPI (1:1000, Dojindo, KT013) at room temperature for 1 h. CXCL8 (5 μg/mL, R&D Systems, AF-208-NA), CD68 , CD163 , CD86 (1:200, Abcam, ab213363, ab182422, ab270719).

Techniques: Derivative Assay, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A positive feedback loop between gastric cancer cells and tumor-associated macrophage induces malignancy progression

doi: 10.1186/s13046-022-02366-6

Figure Lengend Snippet: Cytokines IL-10 positive feedback induces macrophage M2 polarization and upregulates macrophage-derived CXCL8 expression. A IL-10 promoted macrophages CD163 expression and induced M2 polarization. B IL-10 activated NF-κB signaling pathway. C The NF-κB pathway inhibitor (Sarsasapogenin) could limit IL-10 induced M2 polarization. D E. IL-10 upregulated CXCL8 expression, and it could be prevented by NF-κB pathway inhibitor. F . IL-10 could promote CXCL8 secretion, and Sarsasapogenin could antagonize the effect of IL-10 . G Deletion and selective mutation analysis recognized the NFKB1 -responsive regions in the CXCL8 promoter. Luciferase reporter plasmids containing serially truncated or mutated CXCL8 promoter constructs were co-transfected with OE-NFKB1 into macrophage cells, and relative luciferase activities were detected. The − 1829 ~ − 1819 and − 91 ~ − 81 sequences were all the binding sites

Article Snippet: Then, they were incubated with species-appropriate rabbit/mouse secondary antibodies coupled with AlexaFluor dyes (488, 594, 1:200, Invitrogen, A32814, A32723, A32740), DAPI (1:1000, Dojindo, KT013) at room temperature for 1 h. CXCL8 (5 μg/mL, R&D Systems, AF-208-NA), CD68 , CD163 , CD86 (1:200, Abcam, ab213363, ab182422, ab270719).

Techniques: Derivative Assay, Expressing, Mutagenesis, Luciferase, Construct, Transfection, Binding Assay