Journal: International Journal of Cancer

Article Title: Cancer‐associated fibroblasts promote PD‐L1 expression in mice cancer cells via secreting CXCL5

doi: 10.1002/ijc.32278

Figure Lengend Snippet: CAFs‐derived CXCL5 promotes PD‐L1 expression in cancer cells. ( a ) The expression of PD‐L1 in B16, CT26, A375 and HCT116 was determined by flow cytometry after treated with different concentrations of recombined CXCL5. ( b ) The mRNA expression of PD‐L1 in B16, CT26, A375 and HCT116 was detected by RT‐PCR. * p < 0.05. ( c ) Western blot showed the protein levels of CXCR2 in B16 and CT26 after transfected with siCXCR2. ( d ) Flow cytometry showed the expression of PD‐L1 in B16 and CT26 cocultured with CAFs after transfected with siCXCR2. ( e ) The protein levels of CXCR2 in A375 and HCT116 after transfected with siCXCR2 were analyzed by western blotting. ( f ) The expression of PD‐L1 in A375 and HCT116 treated with CXCL5 after transfected with siCXCR2 was determined by flow cytometry. ( g ) CXCL5 and α‐SMA expression in 3T3 cells were detected by western after transfected with CXCL5‐overexpression plasmid. ( h ) PD‐L1 protein expression in B16 and CT26 cocultured with/without 3T3‐CXCL5 cells was analyzed by western blot and the normalized protein expression was analyzed by Image J. * p < 0.05.

Article Snippet: The following antibodies were used for IHC, flow cytometry and western blotting: rabbit monoclonal anti‐PD‐L1 (1:200, ab205921, Abcam, Cambridge, MA), mouse monoclonal anti‐α‐smooth muscle actin (α‐SMA; 1:200, BM0002, BOSTER Biological Technology, Pleasanton, CA), rabbit polyclonal anti‐CXCR2 (1:200, BA0732‐2, BOSTER).

Techniques: Derivative Assay, Expressing, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Over Expression, Plasmid Preparation

Journal: International Journal of Cancer

Article Title: Cancer‐associated fibroblasts promote PD‐L1 expression in mice cancer cells via secreting CXCL5

doi: 10.1002/ijc.32278

Figure Lengend Snippet: The correlation of CXCR2, PD‐L1 and p‐AKT in tumor tissues in vivo . ( a ) The expression level of CXCR2 and PD‐L1 in human melanoma and CRC tissues were detected by IHC and evaluated. ( b ) The correlation of CXCR2 and PD‐L1 in xenograft tumor tissues was analyzed by IHC and evaluated. ( c ) The correlation of p‐AKT and PD‐L1 in xenograft tumor tissues was detected by IHC and quantized. ( d ) Representative immunostaining images of PD‐L1, CXCR2 and p‐AKT in B16 and CT26 xenograft tumor tissues. Scale bar, 200 μm.

Article Snippet: The following antibodies were used for IHC, flow cytometry and western blotting: rabbit monoclonal anti‐PD‐L1 (1:200, ab205921, Abcam, Cambridge, MA), mouse monoclonal anti‐α‐smooth muscle actin (α‐SMA; 1:200, BM0002, BOSTER Biological Technology, Pleasanton, CA), rabbit polyclonal anti‐CXCR2 (1:200, BA0732‐2, BOSTER).

Techniques: In Vivo, Expressing, Immunostaining

Journal: Journal of Orthopaedic Translation

Article Title: Unveiling the role of CXCL8/CXCR2 in intervertebral disc degeneration: A path to promising therapeutic strategies

doi: 10.1016/j.jot.2024.08.022

Figure Lengend Snippet: The expressions of CXCL8 and CXCR2 were up-regulated in nucleus pulposus(NP) tissues of IVDD. (a–c) qRT-PCR analysis of CXCL8, CXCR2 and CXCR1 mRNA expression levels in NP tissues of Mild group and Severe Group (N = 24 for each group). (d) ELISA analysis of CXCL8 concentrations of Mild group and Severe Group (N = 24 for each group). (e–f) Western blot detection (e) and grey value analysis (f) of CXCL8 and CXCR2 level in Mild group and Severe Group (N = 3 biological samples for each group). (g) Representative images of hematoxylin-eosin staining (H&E) and immunohistochemical staining (IHC) (N = 3 slides for each group; scale bar: 4x left; 40x right). (h–k) Pearson's correlation of CXCL8 concentrations with VAS scores, ODI index, disk height(mm) and BMI(kg/m 2 ) of enrolled individuals (N = 48 for each group). (l–o) Pearson's correlation of CXCL8 concentrations with LMM and PM degeneration, CSA and Goutallier grade (N = 48 for each group). Data are represented as mean ± s.e.m and were analysed using two-tailed unpaired t-test(a,b,c,d,f), two-tailed Pearson's correlation analysis (h–o). Significant differences between the groups are indicated as ∗∗p < 0.01, ns: no significance. Notes: VAS: Visual Analogue Scale, ODI: Oswestry Disability Index, BMI: body mass index, LMM: lumbar multifidus muscle, PM: psoas major muscle,CSA: cross-sectional area.

Article Snippet: The primary antibodies used for incubation were as follows: Human anti-CXCL8 antibody (1:500, R&D Systems), Human and mouse anti-CXCR2 antibody (1:1000, R&D Systems), Rabbit anti-GAPDH antibody (1:5000, PEPROTECH), Rabbit anti-MMP13 antibody (1:1000, Abcam), Rabbit anti-COL2A1 antibody (1:1000, Abcam), Rabbit anti-P65 antibody (1:2000, CST), Rabbit anti-p-P65 antibody (1:2000, CST), Rabbit anti-IKBa antibody (1:2000, CST), Rabbit anti-p-IKBa antibody (1:2000, CST).

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Immunohistochemical staining, Two Tailed Test

Journal: Journal of Orthopaedic Translation

Article Title: Unveiling the role of CXCL8/CXCR2 in intervertebral disc degeneration: A path to promising therapeutic strategies

doi: 10.1016/j.jot.2024.08.022

Figure Lengend Snippet: CXCL8 neutralizing antibody alleviated intervertebral disc degeneration (a) Schematic diagram illustrating the design of the CXCL8 Ab experiments. (b–c) Representative X-ray images (b, red arrow represents the modeled segment) and %DHI (c) in control group, IVDD group and CXCL8 Ab group (N = 6 for each group). (d) Representative images of Safranin O-fast green staining (SO-FG) and IHC. Regions within black boxes (left) were amplified at right. Scale bar (left) = 400 μm. Scale bar(right) = 50 μm. (e) CXCL8 content in different groups of NP (N = 6 for each group). (f–i) The histological score of the sections, the number of CXCR2/MMP13 positive cells and COL2A1 positive area in each group (N = 6 for each group). (j–m) Western blot detection (j) and grey value analysis (k–m) of CXCR2/MMP13/COL2A1 level in control group, IVDD group and CXCL8 Ab group (N = 3 biological samples for each group). Data are represented mean ± s.e.m and were analysed using one-way ANOVA followed by Tukey's multiple-comparison test(c, e, g-i, k-m), histological score was analysed by Kruskal–Wallis with Dunn's post-test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001, ∗∗∗∗p < 0.00001 were considered significant differences. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies used for incubation were as follows: Human anti-CXCL8 antibody (1:500, R&D Systems), Human and mouse anti-CXCR2 antibody (1:1000, R&D Systems), Rabbit anti-GAPDH antibody (1:5000, PEPROTECH), Rabbit anti-MMP13 antibody (1:1000, Abcam), Rabbit anti-COL2A1 antibody (1:1000, Abcam), Rabbit anti-P65 antibody (1:2000, CST), Rabbit anti-p-P65 antibody (1:2000, CST), Rabbit anti-IKBa antibody (1:2000, CST), Rabbit anti-p-IKBa antibody (1:2000, CST).

Techniques: Control, Staining, Amplification, Western Blot, Comparison

Journal: Journal of Orthopaedic Translation

Article Title: Unveiling the role of CXCL8/CXCR2 in intervertebral disc degeneration: A path to promising therapeutic strategies

doi: 10.1016/j.jot.2024.08.022

Figure Lengend Snippet: CXCL8/CXCR2 signaling is involved in mediating extracellular matrix degradation of the IVDD model (a) Schematic picture depicting the time points of SB225002 (selective CXCR2 antagonist) administration and SO-FG/IHC/WB tests. (b–f) Representative images of SO-FG and IHC (f). Regions within black boxes on the left were magnified on the right. Scale bar (left) = 400 μm. Scale bar (right) = 50 μm. Histological scores(b) for the sections, the number of CXCR2 (c)/MMP13 (d) positive cells and COL2A1 positive area (e) in control group, IVDD group and SB225002 group (N = 5 for each group). (g–j) Western blot detection (g) and grey value analysis (h–j) of CXCR2/MMP13/COL2A1 level (N = 3 biological samples for each group). (k) Schematic diagram illustrating the design of the loss-gain function experiments. (l–n) Representative X-ray images (l), %DHI (m) and histological score (n) of the sections in IVDD group, IVDD + r-CXCL8 group and IVDD + r-CXCL8+SB225002 group (N = 6 for each group). Data are represented mean ± s.e.m and were analysed using one-way ANOVA followed by Tukey's multiple-comparison test (c-e, h-j, m), histological score was analysed by Kruskal–Wallis with Dunn's post-test (b and n). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001, ∗∗∗∗p < 0.00001 were considered significant difference.

Article Snippet: The primary antibodies used for incubation were as follows: Human anti-CXCL8 antibody (1:500, R&D Systems), Human and mouse anti-CXCR2 antibody (1:1000, R&D Systems), Rabbit anti-GAPDH antibody (1:5000, PEPROTECH), Rabbit anti-MMP13 antibody (1:1000, Abcam), Rabbit anti-COL2A1 antibody (1:1000, Abcam), Rabbit anti-P65 antibody (1:2000, CST), Rabbit anti-p-P65 antibody (1:2000, CST), Rabbit anti-IKBa antibody (1:2000, CST), Rabbit anti-p-IKBa antibody (1:2000, CST).

Techniques: Control, Western Blot, Comparison

Journal: Journal of Orthopaedic Translation

Article Title: Unveiling the role of CXCL8/CXCR2 in intervertebral disc degeneration: A path to promising therapeutic strategies

doi: 10.1016/j.jot.2024.08.022

Figure Lengend Snippet: Macrophage is among one of the cellular mchanisms for CXCL8 production in IVDD (a) Schematic protocol illustrating time points for RAW264.7 and Thioglycollate-elicited macrophages (TPMs) cell harvest. (b–d) Expression of CXCL8 mRNA in RAW264.7 cells after LPS (1 μg/ml) incubation by qRT-PCR (b). CXCL8 concentration in RAW264.7 cells and TPMs after LPS incubation by ELISA (N = 5 for each group). (e–i) Representative images of SO-FG and IHC (e). Regions within black boxes on the left were magnified on the right. Scale bar (left) = 400 μm. Scale bar (right) = 50 μm. Histological scores (f) for the sections, the number of CXCR2 (g)/MMP13 (h) positive cells and COL2A1 positive area (i) in control group, IVDD group and IVDD + Clodronate group (N = 5 IVD slices for each group). (j–l) Immunofluorescence (IF) assays for CXCR2 (green), F4/80 (red), and Nucleus (blue) in IVD. Scale bar = 400 μm (above), Scale bar = 100 μm (below). Co-localization analysis by evaluating relative fluorescence intensity (j and k). Data are represented mean ± s.e.m and were analysed using two-tailed unpaired t-test(b,c,d), one-way ANOVA followed by Tukey's multiple-comparison test(g-k), histological score was analysed by Kruskal–Wallis with Dunn's post-test (f). ∗∗p < 0.01, ∗∗∗p < 0.0001, ∗∗∗∗p < 0.00001 were considered significant difference. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies used for incubation were as follows: Human anti-CXCL8 antibody (1:500, R&D Systems), Human and mouse anti-CXCR2 antibody (1:1000, R&D Systems), Rabbit anti-GAPDH antibody (1:5000, PEPROTECH), Rabbit anti-MMP13 antibody (1:1000, Abcam), Rabbit anti-COL2A1 antibody (1:1000, Abcam), Rabbit anti-P65 antibody (1:2000, CST), Rabbit anti-p-P65 antibody (1:2000, CST), Rabbit anti-IKBa antibody (1:2000, CST), Rabbit anti-p-IKBa antibody (1:2000, CST).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Immunofluorescence, Fluorescence, Two Tailed Test, Comparison

Journal: Journal of Orthopaedic Translation

Article Title: Unveiling the role of CXCL8/CXCR2 in intervertebral disc degeneration: A path to promising therapeutic strategies

doi: 10.1016/j.jot.2024.08.022

Figure Lengend Snippet: CXCL8/CXCR2 signaling participates in oxidative stress modulation and promotes ROS production (a-b) Gene Set Enrichment Analysis (GSEA) showed significant enriched in reactive oxygen species (ROS) production and apoptosis. (c–f) Summarized data showed H2O2 content (c), MDA content (d), SOD activity (e) and GSH-Px activity (f) in IVDs (N = 7 biological samples for each group). (g–j) Representative images of TUNEL assay (g and i) and quantification of apoptotic cells (h and j) by fluorescent TUNEL assay (N = 7 IVDs for each group). (k–n) Summarized data showed H2O2 content (k), MDA content (l), SOD activity (m) and GSH-Px activity (n) in IVDs after rescue experiment (N = 7 biological samples for each group). Data are represented mean ± s.e.m and were analysed using one-way ANOVA followed by Tukey's multiple-comparison test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001, ∗∗∗∗p < 0.00001were considered significant difference, ns: non-significance.

Article Snippet: The primary antibodies used for incubation were as follows: Human anti-CXCL8 antibody (1:500, R&D Systems), Human and mouse anti-CXCR2 antibody (1:1000, R&D Systems), Rabbit anti-GAPDH antibody (1:5000, PEPROTECH), Rabbit anti-MMP13 antibody (1:1000, Abcam), Rabbit anti-COL2A1 antibody (1:1000, Abcam), Rabbit anti-P65 antibody (1:2000, CST), Rabbit anti-p-P65 antibody (1:2000, CST), Rabbit anti-IKBa antibody (1:2000, CST), Rabbit anti-p-IKBa antibody (1:2000, CST).

Techniques: Activity Assay, TUNEL Assay, Comparison

Journal: Journal of Orthopaedic Translation

Article Title: Unveiling the role of CXCL8/CXCR2 in intervertebral disc degeneration: A path to promising therapeutic strategies

doi: 10.1016/j.jot.2024.08.022

Figure Lengend Snippet: CXCL8 promotes NPCs apoptosis and the overproduction of ROS in vitro (a-d) Western blot analysis and quantification for CXCR2/MMP13/COL2A1 (N = 3 biological samples for each group). (e–f) Quantification of NP cell apoptosis by flow cytometry (N = 3 for each group). (g and i-j) IF assays showed the co-localization images of CXCR2 (red) and cleaved caspase-3 (green). Scale bar = 50 μm. Co-localization analysis (i and j) by evaluating relative fluorescence intensity. (h) Intracellular ROS levels detected by flow cytometry. (k) Quantification of ROS level (N = 3 for each group). Data are represented mean ± s.e.m and were analysed using one-way ANOVA followed by Tukey's multiple-comparison test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001, ∗∗∗∗p < 0.00001 were considered significant difference, ns: non-significance. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies used for incubation were as follows: Human anti-CXCL8 antibody (1:500, R&D Systems), Human and mouse anti-CXCR2 antibody (1:1000, R&D Systems), Rabbit anti-GAPDH antibody (1:5000, PEPROTECH), Rabbit anti-MMP13 antibody (1:1000, Abcam), Rabbit anti-COL2A1 antibody (1:1000, Abcam), Rabbit anti-P65 antibody (1:2000, CST), Rabbit anti-p-P65 antibody (1:2000, CST), Rabbit anti-IKBa antibody (1:2000, CST), Rabbit anti-p-IKBa antibody (1:2000, CST).

Techniques: In Vitro, Western Blot, Flow Cytometry, Fluorescence, Comparison

Journal: Journal of Orthopaedic Translation

Article Title: Unveiling the role of CXCL8/CXCR2 in intervertebral disc degeneration: A path to promising therapeutic strategies

doi: 10.1016/j.jot.2024.08.022

Figure Lengend Snippet: CXCL8/CXCR2 regulate NPCs apoptosis and ROS via NF-κB signalling pathway (a-c) Western blot detection (a) and grey value analysis (b–c) of p-p65 and p-IKBa level (N = 3 biological samples for each group). (d–f) IF assays showed the co-localisation of CXCR2 (red) and p65 (green), Scale bar = 50 μm. Co-localization analysis (e and f) by evaluating relative fluorescence intensity (N = 3 IVDs for each group). (g–j) Western blot analysis and quantification for p-p65/MMP13/COL2A1 (N = 3 biological samples for each group). (k) Quantification of NP cell apoptosis after PDTC administration by flow cytometry (N = 3 for each group). (l) Intracellular ROS levels detected by flow cytometry. (m) Quantification of ROS level (N = 3 for each group). Data are represented mean ± s.e.m and were analysed using one-way ANOVA followed by Tukey's multiple-comparison test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001, ∗∗∗∗p < 0.00001 were considered significant difference, ns: non-significance. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies used for incubation were as follows: Human anti-CXCL8 antibody (1:500, R&D Systems), Human and mouse anti-CXCR2 antibody (1:1000, R&D Systems), Rabbit anti-GAPDH antibody (1:5000, PEPROTECH), Rabbit anti-MMP13 antibody (1:1000, Abcam), Rabbit anti-COL2A1 antibody (1:1000, Abcam), Rabbit anti-P65 antibody (1:2000, CST), Rabbit anti-p-P65 antibody (1:2000, CST), Rabbit anti-IKBa antibody (1:2000, CST), Rabbit anti-p-IKBa antibody (1:2000, CST).

Techniques: Western Blot, Fluorescence, Flow Cytometry, Comparison

Journal:

Article Title: Downregulation of CXCR1 and CXCR2 Expression on Human Neutrophils by Helicobacter pylori : a New Pathomechanism in H. pylori Infection?

doi: 10.1128/IAI.72.12.6773-6779.2004

Figure Lengend Snippet: H. pylori downregulates the CXCR1 and CXCR2 receptors on purified human neutrophils. Incubation of neutrophils with a cag+ H. pylori strain as well as with the corresponding cag deletion mutant resulted in complete downregulation of both chemokine receptors after only 0.5 h, as determined by FACS analysis. Neutrophils incubated with medium alone (labeled “medium control”) did not show any receptor downregulation. The x axis indicates fluorescence intensity measured on log10 scale, and the y axis indicates event counts per channel on a linear scale.

Article Snippet: The blotted membranes were blocked with phosphate-buffered saline containing 0.1% (vol/vol) Tween 20 and 3% (wt/vol) low-fat milk powder, followed by a 1-h incubation with the primary antibodies polyclonal rabbit anti-human CXCR1 and CXCR2 (Santa Cruz, Heidelberg, Germany) at a dilution of 1:200.

Techniques: Purification, Incubation, Mutagenesis, Labeling, Fluorescence

Journal:

Article Title: Downregulation of CXCR1 and CXCR2 Expression on Human Neutrophils by Helicobacter pylori : a New Pathomechanism in H. pylori Infection?

doi: 10.1128/IAI.72.12.6773-6779.2004

Figure Lengend Snippet: Purified LPS from H. pylori does not downregulate the CXCR1 and CXCR2 receptors on human neutrophils. Incubation of neutrophils with LPS from H. pylori did not downregulate the CXCR1 and CXCR2 receptors compared with the medium control, as determined by FACS analysis.

Article Snippet: The blotted membranes were blocked with phosphate-buffered saline containing 0.1% (vol/vol) Tween 20 and 3% (wt/vol) low-fat milk powder, followed by a 1-h incubation with the primary antibodies polyclonal rabbit anti-human CXCR1 and CXCR2 (Santa Cruz, Heidelberg, Germany) at a dilution of 1:200.

Techniques: Purification, Incubation

Journal:

Article Title: Downregulation of CXCR1 and CXCR2 Expression on Human Neutrophils by Helicobacter pylori : a New Pathomechanism in H. pylori Infection?

doi: 10.1128/IAI.72.12.6773-6779.2004

Figure Lengend Snippet: CXCR1 and CXCR2 receptor downregulation on neutrophils cannot be attributed to increased IL-8 and TNF-α concentrations. Human neutrophils did not express IL-8 (a) and TNF-α (b) 0.5 h after incubation with H. pylori, as determined by ELISA. Three hours after incubation with H. pylori, IL-8 was strongly increased and TNF-α was moderately increased in the neutrophil cell culture. The IL-8 level was higher after incubation with the cag+ than with the cag deletion H. pylori strain. IL-8 measurements were performed in triplicate, and TNF-α measurements were performed in duplicate. Mean values and standard deviations are shown.

Article Snippet: The blotted membranes were blocked with phosphate-buffered saline containing 0.1% (vol/vol) Tween 20 and 3% (wt/vol) low-fat milk powder, followed by a 1-h incubation with the primary antibodies polyclonal rabbit anti-human CXCR1 and CXCR2 (Santa Cruz, Heidelberg, Germany) at a dilution of 1:200.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal:

Article Title: Downregulation of CXCR1 and CXCR2 Expression on Human Neutrophils by Helicobacter pylori : a New Pathomechanism in H. pylori Infection?

doi: 10.1128/IAI.72.12.6773-6779.2004

Figure Lengend Snippet: H. pylori downregulates the CXCR1 and CXCR2 receptors on neutrophils, as shown by confocal microscopy. Purified human neutrophils showed strong CXCR1 and CXCR2 expression at the cell membrane and in the cytoplasm after 0.5 h of incubation in RPMI medium. Incubation with a cag+ and a cag deletion H. pylori strain for 0.5 h resulted in a strong reduction of the fluorescence signals, with complete loss of the signals at the cell membrane and only some CXCR1 and CXCR2 signal in the cytoplasm. Fluorescence microscopy is shown in the lower part of the figure, and transmission light microscopy of the same cells is shown in the upper part of the figure.

Article Snippet: The blotted membranes were blocked with phosphate-buffered saline containing 0.1% (vol/vol) Tween 20 and 3% (wt/vol) low-fat milk powder, followed by a 1-h incubation with the primary antibodies polyclonal rabbit anti-human CXCR1 and CXCR2 (Santa Cruz, Heidelberg, Germany) at a dilution of 1:200.

Techniques: Confocal Microscopy, Purification, Expressing, Incubation, Fluorescence, Microscopy, Transmission Assay, Light Microscopy

Journal:

Article Title: Downregulation of CXCR1 and CXCR2 Expression on Human Neutrophils by Helicobacter pylori : a New Pathomechanism in H. pylori Infection?

doi: 10.1128/IAI.72.12.6773-6779.2004

Figure Lengend Snippet: cag+ H. pylori induced more severe downregulation of CXCR1 and CXCR2 mRNAs in purified human neutrophils than cag deletion H. pylori. Neutrophils incubated in RPMI for 0.5 and 3 h strongly expressed CXCR1 and CXCR2 mRNAs (lanes 1 and 4). H. pylori downregulated CXCR1 and CXCR2 mRNAs in neutrophils 3 h after incubation (lanes 5 and 6). CXCR1 and CXCR2 mRNAs were not downregulated 0.5 h after coincubation with H. pylori (lanes 2 and 3). The H. pylori cag+ strain (lane 5) downregulated CXCR1 and CXCR2 mRNAs significantly more strongly than the cag deletion mutant (lane 6).

Article Snippet: The blotted membranes were blocked with phosphate-buffered saline containing 0.1% (vol/vol) Tween 20 and 3% (wt/vol) low-fat milk powder, followed by a 1-h incubation with the primary antibodies polyclonal rabbit anti-human CXCR1 and CXCR2 (Santa Cruz, Heidelberg, Germany) at a dilution of 1:200.

Techniques: Purification, Incubation, Mutagenesis

Journal:

Article Title: Downregulation of CXCR1 and CXCR2 Expression on Human Neutrophils by Helicobacter pylori : a New Pathomechanism in H. pylori Infection?

doi: 10.1128/IAI.72.12.6773-6779.2004

Figure Lengend Snippet: Downmodulation of the CXCR1 receptor on neutrophils may also occur during H. pylori infection in the human stomach. Neutrophils in the lamina propria expressed the CXCR1 receptor, whereas neutrophils infiltrating gastric epithelium or localized in the foveolar lumen (arrows), where they are in closer contact with H. pylori, lacked CXCR1 expression.

Article Snippet: The blotted membranes were blocked with phosphate-buffered saline containing 0.1% (vol/vol) Tween 20 and 3% (wt/vol) low-fat milk powder, followed by a 1-h incubation with the primary antibodies polyclonal rabbit anti-human CXCR1 and CXCR2 (Santa Cruz, Heidelberg, Germany) at a dilution of 1:200.

Techniques: Infection, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Concomitant NAFLD Facilitates Liver Metastases and PD-1-Refractory by Recruiting MDSCs via CXCL5/CXCR2 in Colorectal Cancer

doi: 10.1016/j.jcmgh.2024.04.008

Figure Lengend Snippet: NAFLD promotes CXCL5 and recruits CXCR2 + MDSCs accumulation in liver metastases. ( A ) Relative CXCL5 level in peripheral blood of each tumor-bearing C57BL/6 mice models, n = 4. Student t test. ( B ) Quantification of CXCL5 concentration in peripheral blood of mice in each group, n = 5. One-way ANOVA. ( C ) Quantification of CXCL5 concentration in liver homogenate of mice in each group, n = 4. One-way ANOVA. ( D ) Quantification of CXCL5 concentration in tumor tissues of each tumor-bearing C57BL/6 mice models, n = 3. Student t test. ( E ) UMAP visualization of liver metastatic tissues, paratumoral tissues, and liver tissues profiled in all groups ( F ) colored by sample or subpopulation ( red rectangles represent granulocytes), n = 6. ( G ) Graph of cellular identification results, UMAP cluster map revealing 8 specific clusters representing the major tissue types. ( H ) UMAP cluster map showing CXCR2 expression of all samples’ cell types. Violet dots give the fields of the main clusters of interest. ( I ) Representative histograms of CXCR2 expression in MDSCs (CD45 + CD11b + Gr-1 + ) and percentages (%) of CXCR2 expression in each group were analyzed by FC, n = 6, one-way ANOVA. All data are presented as mean ± SD. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001, and ns, not significant.

Article Snippet: Rabbit anti-human CXCR2 , N/A , Proteintech; 20634-1-AP.

Techniques: Concentration Assay, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Concomitant NAFLD Facilitates Liver Metastases and PD-1-Refractory by Recruiting MDSCs via CXCL5/CXCR2 in Colorectal Cancer

doi: 10.1016/j.jcmgh.2024.04.008

Figure Lengend Snippet: NAFLD promotes CXCL5 and recruits CXCR2 + MDSCs accumulation in liver metastases. ( A ) Mouse XL Cytokine Array detects multiple cytokines, chemokines, growth factors, and other soluble proteins in serum. Data shown are from a 5-minute exposure to Bio-Rad, top: CRLM group, bottom : NAFLD with CRLM group. ( B ) Relative cytokine levels in serum of each tumor-bearing C57BL/6 mice models. ( C ) Localization of CXCL5 and CXCR2 reactivity in tissue samples of ob/ob and WT group, acquired by confocal imaging. Scale bars represent 100 μm, upper panel ; 20 μm, lower panel .

Article Snippet: Rabbit anti-human CXCR2 , N/A , Proteintech; 20634-1-AP.

Techniques: Imaging

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Concomitant NAFLD Facilitates Liver Metastases and PD-1-Refractory by Recruiting MDSCs via CXCL5/CXCR2 in Colorectal Cancer

doi: 10.1016/j.jcmgh.2024.04.008

Figure Lengend Snippet: CXCR2 + MDSCs are recruited to liver via CXCL5 secreted by Kupffer cells. ( A ) Cell clusters of CXCL5 source. Representative images of mIF for CXCL5 and candidate clusters (CD146/SMA/CD31/F4/80) expression in the liver. Scale bars represent 50 μm. ( B ) Representative images of immunohistochemical staining for CXCL5 and CXCR2 expression in the liver. Scale bars represent 50 μm. Mean intensity of CXCL5 and CXCR2 expression in the livers was analyzed by TG tissue imaging cytometry system, n = 4, one-way ANOVA. ( C ) Correlation between the mean intensity of CXCL5 and CXCR2 in each group. Pearson test. ( D ) Localization of CXCL5 and CXCR2 reactivity in tissue samples of each group, acquired by confocal imaging. Scale bars represent 100 μm, upper panel ; 20 μm, lower panel . ( E ) Macroscopic views of representative livers are shown of CXCL5 KO+NAFLD+CRLM and CXCL5 KO+CRLM groups. Yellow arrow indicates tumors. ( F ) Liver metastasis foci number of CXCL5 KO+NAFLD+CRLM and CXCL5 KO+CRLM groups, n = 4–5, Student t test. ( G ) Representative data show that CXCL5 KO+NAFLD+CRLM and CXCL5 KO+CRLM groups of CD45 + /CD11b + /Gr-1 + MDSCs expression on day 12 in the liver, which percentages (%) of MDSCs (CD45 + C11b + Gr-1 + ) in CD45 + cells were analyzed by FC, n = 4–5, Student t test. ( H ) Percentages (%) of CXCR2 expression in MDSCs (CD45 + CD11b + Gr-1 + ) in each group were analyzed by flow cytometry, n = 4–5, Student t test. All data are presented as mean ± SD. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001, and ns, not significant.

Article Snippet: Rabbit anti-human CXCR2 , N/A , Proteintech; 20634-1-AP.

Techniques: Expressing, Immunohistochemical staining, Staining, Imaging, Cytometry, Flow Cytometry

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Concomitant NAFLD Facilitates Liver Metastases and PD-1-Refractory by Recruiting MDSCs via CXCL5/CXCR2 in Colorectal Cancer

doi: 10.1016/j.jcmgh.2024.04.008

Figure Lengend Snippet: CXCR2 + MDSCs are recruited to liver via CXCL5 secreted by Kupffer cells. ( A ) Cell clusters of CXCL5 source were analyzed by Akoya system. One-way ANOVA. ( B ) Liver weights of CXCL5 KO+NAFLD+CRLM and CXCL5 KO+CRLM groups, n = 4–5, Student test. ( C ) Liver/body weight ratio of CXCL5 KO+NAFLD+CRLM and CXCL5 KO+CRLM groups, n = 4–5, Student t test. ( D ) Liver weights of CXCL5 KO+NAFLD+CRLM and WT+NAFLD+CRLM groups, n = 4–5, Student test. ( E ) Liver/body weight ratio of CXCL5 KO+NAFLD+CRLM and WT+NAFLD+CRLM groups, n = 4–5, Student t test. ( F ) Liver metastasis foci number of CXCL5 KO+NAFLD+CRLM and WT+NAFLD+CRLM groups, n = 4–5, Student t test. ( G ) Representative data show that CXCL5 KO+NAFLD+CRLM and WT+NAFLD+CRLM groups of CD45 + /CD11b + /Gr-1 + MDSCs expression on day 12 in the liver, which percentages (%) of MDSCs (CD45 + C11b + Gr-1 + ) in CD45 + cells were analyzed by FC, n = 4–5, Student t test. ( H ) Percentages (%) of CXCR2 expression in MDSCs (CD45 + CD11b + Gr-1 + ) in each group were analyzed by FC, n = 4–5, Student t test. ( I ) Localization of CXCL5 and CXCR2 reactivity in tissue samples of CXCL5 KO+NAFLD+CRLM and CXCL5 KO+CRLM groups, acquired by confocal imaging. Scale bars represent 100 μm, upper panel ; 20 μm, lower panel . All data are presented as mean ± SD. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001, and ns, not significant.

Article Snippet: Rabbit anti-human CXCR2 , N/A , Proteintech; 20634-1-AP.

Techniques: Expressing, Imaging

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Concomitant NAFLD Facilitates Liver Metastases and PD-1-Refractory by Recruiting MDSCs via CXCL5/CXCR2 in Colorectal Cancer

doi: 10.1016/j.jcmgh.2024.04.008

Figure Lengend Snippet: Reparixin inhibits CXCR2 + MDSC infiltration and CRLM growth. ( A ) Treatment experimental scheme. After Luci-MC38 cell inoculation, PBS or Reparixin was injected at days 3, 5, 7, 9, and 11, Mice were euthanized on day 12 for analyses. ( B ) In vivo bioluminescence imaging of the 4 groups (CRLM, CRLM+Reparixin, NAFLD+CRLM, and NAFLD+CRLM+Reparixin) at indicated time points of day 6. ( C ) Macroscopic views of representative livers are shown of 4 groups (CRLM, CRLM+Reparixin, NAFLD+CRLM, and NAFLD+CRLM+Reparixin). Yellow arrow indicates tumors. ( D ) Liver weights of each group. Samples were collected on day 12 after Luciferase-MC38 cell inoculation and treatment; n = 5–6. One-way ANOVA. ( E ) Liver/body weight ratio of each group; n = 5–6. One-way ANOVA. ( F ) Representative data show that each group of CD45 + /CD11b + /Gr-1 + MDSCs expression on day 12 in the liver, which percentages (%) of MDSCs (CD45 + C11b + Gr-1 + ) in CD45 + cells were analyzed by FC. One-way ANOVA. ( G ) Representative histograms of CXCR2 expression in MDSCs (CD45 + CD11b + Gr-1 + ) and percentages (%) of CXCR2 expression in each group were analyzed by FC. One-way ANOVA. ( H ) Co-localization of CXCL5 and CXCR2 reactivity in tissue samples of liver metastases, NAFLD with liver metastasis, and their separately treated liver tissues, acquired by confocal imaging. Scale bars represent 100 μm, upper panel ; 20 μm, lower panel , n = 6. ( I ) Therapeutic effects of Reparixin-21d on NAFLD. Macroscopic views of representative liver tissues, Oil Red O staining, and immunohistochemistry staining of liver sections for each group. Scale bars represent 100 μm. Mean density of immunohistochemical staining in the Reparixin-21d on NAFLD, n = 4, Student t test. All data are presented as mean ± SD. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001, and ns, not significant.

Article Snippet: Rabbit anti-human CXCR2 , N/A , Proteintech; 20634-1-AP.

Techniques: Injection, In Vivo, Imaging, Luciferase, Expressing, Staining, Immunohistochemistry, Immunohistochemical staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Concomitant NAFLD Facilitates Liver Metastases and PD-1-Refractory by Recruiting MDSCs via CXCL5/CXCR2 in Colorectal Cancer

doi: 10.1016/j.jcmgh.2024.04.008

Figure Lengend Snippet: Reparixin inhibits CXCR2 + MDSC infiltration and CRLM growth. ( A ) Differential therapeutic effects of Reparixin-12d or 15d on NAFLD. Macroscopic views of representative liver tissues and Oil Red O staining of liver sections for each group. Scale bars represent 50 μm. Oil Red O staining positive area rate of NAFLD and the 12d ( B ) or 15d ( C ) treatment group, n = 4, Student t test. All data are presented as mean ± SD. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001, and ns, not significant.

Article Snippet: Rabbit anti-human CXCR2 , N/A , Proteintech; 20634-1-AP.

Techniques: Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Concomitant NAFLD Facilitates Liver Metastases and PD-1-Refractory by Recruiting MDSCs via CXCL5/CXCR2 in Colorectal Cancer

doi: 10.1016/j.jcmgh.2024.04.008

Figure Lengend Snippet: Reparixin overcomes anti-PD-1 treatment refractory and achieves long-term survival over half mice with CRLM. ( A ) Scheme of combination treatment with or without antibody to PD-1. ∗Treatment intraperitoneally, after Luci-MC38 cell inoculation, isotype-IgG, anti-PD-1 antibodies, Reparixin were injected every other day starting on day 3, with the combination treatment group also received at these time points. ( B ) Body weight trends of mice in the combination treatment group. n = 9. ( C ) Representative liver tissues stained with 5 markers (CD8, programmed death-ligand 1 [PD-L1], CXCL5, CXCR2, and CD11b). The image acquisition of all markers occurs simultaneously. Individual markers (or select combinations of markers) can then be displayed. Five-color overlay ( top left ) and 2/3-color insets (border) of a representative sampling of the simultaneously acquired markers. Scale bars represent 20 μm. Top : PD-1 treatment group, bottom : Reparixin treatment group. ( D ) Quantification of CD8 + PD-L1 + lymphocytes by AKOYA, n = 3/group, 5 views, one-way ANOVA. ( E ) Quantification of CXCR2 + CD11b + cluster by AKOYA, n = 3/group, 3 views, one-way ANOVA. All data are presented as mean ± SD. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001, and ns, not significant.

Article Snippet: Rabbit anti-human CXCR2 , N/A , Proteintech; 20634-1-AP.

Techniques: Injection, Staining, Sampling

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Concomitant NAFLD Facilitates Liver Metastases and PD-1-Refractory by Recruiting MDSCs via CXCL5/CXCR2 in Colorectal Cancer

doi: 10.1016/j.jcmgh.2024.04.008

Figure Lengend Snippet: Reparixin overcomes anti-PD-1 treatment refractory and achieves long-term survival over half mice with CRLM. ( A ) In vivo bioluminescence imaging of each treatment group at indicated time points, n = 6–9. ( B ) In vivo bioluminescence imaging of the combination treatment group at day 12 and day 58, n = 6. ( C ) Body weight trends of NAFLD with CRLM mice treated with different protocols, n = 6–9. ( D ) Survival curves of NAFLD+CRLM mice, n = 6–9. Log-rank (Mantel-Cox) test compared with the group of NAFLD+CRLM+isotype-IgG. ( E ) Survival curves of CRLM mice, n = 7–8. Log-rank (Mantel-Cox) test compared with the group of CRLM+isotype-IgG. ( F and G ) Liver weights of each group, n = 6–9, one-way ANOVA. ( H ) Macroscopic views of representative livers are shown of 4 groups (NAFLD+CRLM+isotype-IgG, NAFLD+CRLM+PD-1, NAFLD+CRLM+Reparixin, and NAFLD+CRLM+PD-1+Reparixin), n = 6–9. ( I ) Representative images of H&E staining of liver sections are shown of each group. Black box indicates lymphocytes. Scale bars represent 1 mm, upper panel ; 100 μm, lower panel. ( J ) Representative liver tissues stained with 5 markers (CD8, PD-L1, CXCL5, CXCR2, and CD11b). The image acquisition of all markers occurs simultaneously. Individual markers (or select combinations of markers) can then be displayed. Six-color overlay ( top left ) and 2/3-color insets (border) of a representative sampling of the simultaneously acquired markers. Left: control group, right: combination treatment group. Scale bars represent 20 μm. Quantification of CD8 + lymphocytes in liver metastases of mice in each treatment group, one-way ANOVA. All data are presented as mean ± SD. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001, and ns, not significant.

Article Snippet: Rabbit anti-human CXCR2 , N/A , Proteintech; 20634-1-AP.

Techniques: In Vivo, Imaging, Staining, Sampling, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Concomitant NAFLD Facilitates Liver Metastases and PD-1-Refractory by Recruiting MDSCs via CXCL5/CXCR2 in Colorectal Cancer

doi: 10.1016/j.jcmgh.2024.04.008

Figure Lengend Snippet: NAFLD increases risk of liver metastases in CRC patients. ( A–C ) Representative immunohistochemistry-stained liver sections of CXCL5 ( A ), CXCR2 ( B ), and CD11b + ( C ) expression in each group. Scale bars represent 100 μm: upper panel ; 20 μm: lower panel . ( D ) Representative H&E-stained sections of liver tissues and adipocyte infiltration were assessed between liver metastasis with fatty liver (n = 6) or without fatty liver (n = 5). Scale bars represent 100 μm: upper panel ; 20 μm: lower panel . Mean ± SD, Student t test. ( E ) Comparison of BMI in synCRLM + and synCRLM - cohorts. Mean ± standard error of the mean, Student t test. ( F ) Multivariate logistic regression analysis of the significant predictors for synCRLM. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001, and ns, not significant.

Article Snippet: Rabbit anti-human CXCR2 , N/A , Proteintech; 20634-1-AP.

Techniques: Immunohistochemistry, Staining, Expressing, Comparison

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Concomitant NAFLD Facilitates Liver Metastases and PD-1-Refractory by Recruiting MDSCs via CXCL5/CXCR2 in Colorectal Cancer

doi: 10.1016/j.jcmgh.2024.04.008

Figure Lengend Snippet: NAFLD increases the risk of liver metastases in CRC patients. ( A–C ) Representative immunohistochemistry-stained liver sections of CXCL5 ( A ), CXCR2 ( B ), and CD11b + ( C ) expression between liver metastasis with fatty liver (n = 9) or without fatty liver (n = 6). Scale bars represent 100 μm: upper panel ; 20 μm: lower panel . ( D ) Prevalence of synCRLM in normal weight and obesity cohort. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001, and ns, not significant.

Article Snippet: Rabbit anti-human CXCR2 , N/A , Proteintech; 20634-1-AP.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Concomitant NAFLD Facilitates Liver Metastases and PD-1-Refractory by Recruiting MDSCs via CXCL5/CXCR2 in Colorectal Cancer

doi: 10.1016/j.jcmgh.2024.04.008

Figure Lengend Snippet: Immunohistochemical Staining

Article Snippet: Rabbit anti-human CXCR2 , N/A , Proteintech; 20634-1-AP.

Techniques: Immunohistochemical staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Concomitant NAFLD Facilitates Liver Metastases and PD-1-Refractory by Recruiting MDSCs via CXCL5/CXCR2 in Colorectal Cancer

doi: 10.1016/j.jcmgh.2024.04.008

Figure Lengend Snippet: Fluorescence-Activated Cell Sorting

Article Snippet: Rabbit anti-human CXCR2 , N/A , Proteintech; 20634-1-AP.

Techniques: Fluorescence

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Concomitant NAFLD Facilitates Liver Metastases and PD-1-Refractory by Recruiting MDSCs via CXCL5/CXCR2 in Colorectal Cancer

doi: 10.1016/j.jcmgh.2024.04.008

Figure Lengend Snippet: mIF Staining

Article Snippet: Rabbit anti-human CXCR2 , N/A , Proteintech; 20634-1-AP.

Techniques: Concentration Assay