Journal: Advanced Science

Article Title: Transcriptome Landscape of Cancer‐Associated Fibroblasts in Human PDAC

doi: 10.1002/advs.202415196

Figure Lengend Snippet: Cell–cell interactions between CAF subtypes and myeloid cells. A) Boxplot showing the percentage of MKI67 + cells in each cell type within tumor tissues. Each dot corresponds to each sample. B) Boxplot showing the frequency of each CAF subtype in tumors. Each dot corresponds to each sample. C) Heatmap showing the Spearman correlation coefficient between the abundance of CAF subtypes and the proliferation percent of other cells in tumors. Correlation test p values are indicated, *** p < 0.001, ** p < 0.01, and * p < 0.05. D) Cell–cell interaction network showing interactions between CAF subtypes and other cells in tumors. The dot color indicates the cell type, the dot size indicates the interaction number of a given cell type, and the line thickness indicates the interaction number of a given cell type pair. E) Bar plots showing the interaction number of a given cell type. F) Dot plot showing GO terms of ligands and receptors within specific myeloid cells‐CAFs interactions and specific lymphocytes‐CAFs interactions. G) Dot plot showing the expression level of ligand‐receptor gene pair related with chemotaxis and cytokine within myeloid cell‐CAF interactions. The dot color and size indicate the expression levels and statistical significance, respectively. Red words indicate the ligands (row) expressed in the corresponding cell type (column), and blue words indicate the receptors (row) expressed in the corresponding cell type (column). H) Bar plot showing the fold change of neutrophil migration obtained following the addition of the CAFs/NFs or the CM derived from CAFs/NFs in lower chamber after 1 h. For each group, n = 7 biological replicates. Data are shown as mean value ± SD. One‐way ANOVA p values are calculated. * p < 0.05 and *** p < 0.001. I) Neutrophils were treated with or without CXCL4 inhibitor Plerixafor (25 µM). Neutrophil migration assay was performed by adding control medium or culture medium of CAFs into lower champers, or seeding CAFs into lower champers with or without Plerixafor (25 µM). Bar plot showing the fold change of neutrophil migration after 1 h. For each group, n = 7 biological replicates. Data are shown as mean value ± SD. One‐way ANOVA p values are calculated. ** p < 0.01, **** p < 0.0001, and ns, not significant. J) Cartoon depicting CXCL12‐CXCR4 as a major interaction axis between CAFs and myeloid cells.

Article Snippet: Primary antibodies: CXCL12 (Cat# 17402‐1‐AP, Proteintech), PDPN (Cat# ab236529, Abcam), CK18 (Cat# 10830‐1‐AP, Proteintech), ISG15 (Cat# 15981‐1‐AP, Proteintech), α‐SMA (Cat# ab5694, Abcam), COLL1A1 (Cat# 72026, CST), HLA‐DR (Cat# ab20181, Abcam), CD74 (Cat# ab108393, Abcam), FTL (Cat# 10727‐1‐AP, Proteintech), CDCP1 (Cat# ab252947, Abcam), HIF‐1α (Cat# 20960‐1‐AP, Proteintech), LUM (Cat# ab168343, Abcam), CD3 (Cat# ab5690, Abcam), CD19 (Cat# ab31947, Abcam), CD16 (Cat# 16559‐1‐AP, Proteintech), TPSAB1 (Cat# ab151757, Abcam).

Techniques: Expressing, Chemotaxis Assay, Migration, Derivative Assay, Control

Journal: Oncology Letters

Article Title: Mechanisms of CXCR7 induction in malignant melanoma development

doi: 10.3892/ol.2017.6720

Figure Lengend Snippet: CXCL12 cytokine induces M14 cell invasive activity, and CXCR7 knocked down inhibits CXCR12-induced M14 cell invasive activity. M14 cells (10,000 cells/2 ml) were seeded in each Transwell chamber with 0, 10 or 100 ng/ml CXCL12 for 24 h. The cells that passed through the Transwell chamber and bound to the matrigel were recorded and counted. (A) Images of M14 cells bound to the matrigel membrane (magnification, ×400 (B) CXCL12 increased M14 cell invasive activity. The M14 cell count in the Transwell matrigel membrane was 41.67±5.51 (0 ng/ml CXCL12), 56.67±2.52 (10 ng/ml CXCL12) and 82.23±6.11 (100 ng/ml CXCL12). P-values were obtained for the comparisons between the indicated treatments and 0 ng/ml CXCR12 (**P<0.01; ***P<0.001). (C) CXCR7 shRNA significantly reduced M14 cell counts in in the matrigel membrane. The M14 cell count in the Transwell matrigel membrane was 43.63±2.15 (control), 42.00±6.02 (vector) and 20.62±1.50 (CXCR7 shRNA). **P<0.01 was obtained for the comparison between vector and CXCR7 shRNA. There were not significantly different cell counts between the control and vector groups. These results indicated that CXCR7 downregulation inhibited M14 cell migration and invasion. (D) Images of M14 cells in the matrigel membrane (magnification, ×400). Untreated M14 cells (control), empty vector shRNA-transfected M14 cells (vector) and CXCR7 shRNA-transfected M14 cells (10,000 cells/2 ml/well) were plated in Transwell chamber wells with 10 ng/ml CXCR12 for 24 h. The cells that passed through the membrane and bound to the matrigel were recorded and counted. A lower cell number was observed in CXCR7 shRNA-transfected M14 cells compared with that in control and vector-transfected M14 cells. CXCR7, C-X-C chemokine receptor type 7; shRNA, small hairpin RNA; CXCL12, C-X-C motif chemokine ligand 12.

Article Snippet: Recombinant Human CXCR12 (cat. no., 350-NS/CF, R&D Systems Inc., Minneapolis, MN, USA) (10 ng/ml) was added to all wells.

Techniques: Activity Assay, Membrane, Cell Counting, shRNA, Control, Plasmid Preparation, Comparison, Migration, Transfection

Journal: Molecular Medicine Reports

Article Title: CP-25 exerts anti-angiogenic effects on a rat model of adjuvant-induced arthritis by promoting GRK2-induced downregulation of CXCR4-ERK1/2 signaling in endothelial cells

doi: 10.3892/mmr.2019.10765

Figure Lengend Snippet: Expression of CXCL12 level in the synovium and the wall of blood vessel from rats. Representative immunohistochemical analyses of CXCL12 expression in the (A) synovium and (B) blood vessel walls, illustrating alterations in the joints of each group of rats (magnification, ×400). ODVs of CXCL12 in the (C) synovium and (D) vessel were markedly decreased in rats with AA following administration of CP-25 and MTX (>10 microscopic fields were observed in each section). (E) Levels of CXCL12 in the synovium assessed by ELISA. (F) Representative western blotting demonstrating CXCR4 expression in rat synovium. Western blotting data are expressed as the means ± standard deviation of three independent experiments. **P<0.01 vs. AA group (n=8–10 per group). (G and H) Correlation between pathological alterations in the pannus score and the expression of CXCL12 and CXCR4 in synovium. AA, adjuvant-induced arthritis; CP-25, paeoniflorin-6′-O-benzene sulfonate; CXCL12, C-X-C motif chemokine ligand 12; CXCR4, C-X-C chemokine receptor type 4; MTX, methotrexate; ODV, optical density value.

Article Snippet: Cell Counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. An ELISA kit for CXCL12 (cat. no. CSB-E08729r) was purchased from Cusabio, Inc. Antibodies against α-smooth muscle actin (α-SMA) (cat. no. ab32575), CXCR4 (cat. no. ab124824), GRK2 (cat. no. ab228705), ERK1/2 (cat. no. ab79853), phosphorylated (p)-ERK1/2 (cat. no. ab214362), CXCL12 (cat. no. ab9797), and β-actin (cat. no. ab115777) were purchased from Abcam.

Techniques: Expressing, Immunohistochemical staining, Enzyme-linked Immunosorbent Assay, Western Blot, Standard Deviation, Adjuvant

Journal: Molecular Medicine Reports

Article Title: CP-25 exerts anti-angiogenic effects on a rat model of adjuvant-induced arthritis by promoting GRK2-induced downregulation of CXCR4-ERK1/2 signaling in endothelial cells

doi: 10.3892/mmr.2019.10765

Figure Lengend Snippet: Effects of CP-25 on CXCL12-induced HUVEC proliferation, migration and tube formation. (A-C) Quantitative analysis of HUVEC proliferation, migration and tube formation induced by various concentrations of CXCL12 for 24 h. (D-F) Quantitative analysis of HUVEC proliferation, migration and tube formation following treatment with CXCL12 alone or in combination with CP-25 for 24 h. (G and H) Representative images of the Transwell and tube formation assays of HUVECs (magnification, ×200). Data are expressed as the means ± standard deviation of three independent experiments. # P<0.05, ## P<0.01 vs. control; *P<0.05, **P<0.01 vs. CXCL12. CP-25, paeoniflorin-6′-O-benzene sulfonate; CXCL12, C-X-C motif chemokine ligand 12; CXCR4, C-X-C chemokine receptor type 4.

Article Snippet: Cell Counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. An ELISA kit for CXCL12 (cat. no. CSB-E08729r) was purchased from Cusabio, Inc. Antibodies against α-smooth muscle actin (α-SMA) (cat. no. ab32575), CXCR4 (cat. no. ab124824), GRK2 (cat. no. ab228705), ERK1/2 (cat. no. ab79853), phosphorylated (p)-ERK1/2 (cat. no. ab214362), CXCL12 (cat. no. ab9797), and β-actin (cat. no. ab115777) were purchased from Abcam.

Techniques: Migration, Standard Deviation, Control

Journal: Molecular Medicine Reports

Article Title: CP-25 exerts anti-angiogenic effects on a rat model of adjuvant-induced arthritis by promoting GRK2-induced downregulation of CXCR4-ERK1/2 signaling in endothelial cells

doi: 10.3892/mmr.2019.10765

Figure Lengend Snippet: Effects of CP-25 on GRK2 and CXCR4 expression in HUVECs treated with CXCL12. Representative images of western blotting of (A) total, (D) cytoplasmic and (G) membrane expression of GRK2 and CXCR4. (B, C, E, F, H and I) Western blotting semi-quantification of GRK2 and CXCR4 expression. Data are expressed as the means ± standard deviation of three independent experiments. # P<0.05, ## P<0.01 vs. control; *P<0.05, **P<0.01 vs. CXCL12. CP-25, paeoniflorin-6′-O-benzene sulfonate; CXCL12, C-X-C motif chemokine ligand 12; CXCR4, C-X-C chemokine receptor type 4; GRK2; G protein-coupled receptor kinase 2.

Article Snippet: Cell Counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. An ELISA kit for CXCL12 (cat. no. CSB-E08729r) was purchased from Cusabio, Inc. Antibodies against α-smooth muscle actin (α-SMA) (cat. no. ab32575), CXCR4 (cat. no. ab124824), GRK2 (cat. no. ab228705), ERK1/2 (cat. no. ab79853), phosphorylated (p)-ERK1/2 (cat. no. ab214362), CXCL12 (cat. no. ab9797), and β-actin (cat. no. ab115777) were purchased from Abcam.

Techniques: Expressing, Western Blot, Membrane, Standard Deviation, Control

Journal: Molecular Medicine Reports

Article Title: CP-25 exerts anti-angiogenic effects on a rat model of adjuvant-induced arthritis by promoting GRK2-induced downregulation of CXCR4-ERK1/2 signaling in endothelial cells

doi: 10.3892/mmr.2019.10765

Figure Lengend Snippet: Effects of CP-25 on ERK1/2 expression in HUVECs treated with CXCL12. (A) Representative images of western blotting of the expression of ERK1/2 and p-ERK1/2. (B and C) Western blotting semi-quantification of ERK1/2 and p-ERK1/2. (D) Ratio of p-ERK/total-ERK. (E) Representative images of western blotting for the co-expression of GRK2 and CXCR4, and GRK2 and p-ERK1/2. (F and G) Western blotting semi-quantification of the binding between GRK2 and CXCR4, and GRK2 and p-ERK1/2. Data are expressed as the means ± standard deviation of three independent experiments. ## P<0.01 vs. control; *P<0.05, **P<0.01 vs. CXCL12. CP-25, paeoniflorin-6‰-O-benzene sulfonate; CXCL12, C-X-C motif chemokine ligand 12; CXCR4, C-X-C chemokine receptor type 4; GRK2; G protein-coupled receptor kinase 2; p, phosphorylated.

Article Snippet: Cell Counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. An ELISA kit for CXCL12 (cat. no. CSB-E08729r) was purchased from Cusabio, Inc. Antibodies against α-smooth muscle actin (α-SMA) (cat. no. ab32575), CXCR4 (cat. no. ab124824), GRK2 (cat. no. ab228705), ERK1/2 (cat. no. ab79853), phosphorylated (p)-ERK1/2 (cat. no. ab214362), CXCL12 (cat. no. ab9797), and β-actin (cat. no. ab115777) were purchased from Abcam.

Techniques: Expressing, Western Blot, Binding Assay, Standard Deviation, Control

Journal: Molecular Medicine Reports

Article Title: CP-25 exerts anti-angiogenic effects on a rat model of adjuvant-induced arthritis by promoting GRK2-induced downregulation of CXCR4-ERK1/2 signaling in endothelial cells

doi: 10.3892/mmr.2019.10765

Figure Lengend Snippet: Regulation of CXCR4 activity and signaling. (A) Upon ligand binding, CXCR4 could activate numerous signaling cascades, which may result in increased GRK2 membrane localization, weakening the inhibitory effect of GRK2 on ERK1/2 in the cytoplasm and enhancing ERK1/2 phosphorylation. (B) CP-25 could inhibit ERK1/2 phosphorylation by reducing the membrane localization of GRK2 and enhancing the inhibitory effect of GRK2 on ERK1/2 in the cytoplasm. CP-25, paeoniflorin-6′-O-benzene sulfonate; CXCL12, C-X-C motif chemokine ligand 12; CXCR4, C-X-C chemokine receptor type 4; GRK2; G protein-coupled receptor kinase 2; p, phosphorylated.

Article Snippet: Cell Counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. An ELISA kit for CXCL12 (cat. no. CSB-E08729r) was purchased from Cusabio, Inc. Antibodies against α-smooth muscle actin (α-SMA) (cat. no. ab32575), CXCR4 (cat. no. ab124824), GRK2 (cat. no. ab228705), ERK1/2 (cat. no. ab79853), phosphorylated (p)-ERK1/2 (cat. no. ab214362), CXCL12 (cat. no. ab9797), and β-actin (cat. no. ab115777) were purchased from Abcam.

Techniques: Activity Assay, Ligand Binding Assay, Membrane, Phospho-proteomics