rs504393  (Absource Diagnostics GmbH)

Journal: Journal of Advanced Research

Article Title: The fatty acid receptor CD36 promotes macrophage infiltration via p110γ signaling to stimulate metastasis

doi: 10.1016/j.jare.2024.10.006

Figure Lengend Snippet: CD36 mediates the recruitment of macrophages through CCL2/CCR2 axis. A, The mRNA levels of chemokines in normal liver or LLC metastatic liver measured by RT-PCR (n = 4). c p < 0.05, b p < 0.01, a p < 0.001 vs normal liver. B, C, The mRNA levels of chemokines (B) and chemokine receptors (C) in tumor cells, BMDMs, CD11b + myeloid cells and blood monocytes (n = 4–6). c p < 0.05, b p < 0.01, a p < 0.001 vs the LLC group. D, E, The mRNA levels of Ccl2 or Mcsf (D) and their receptor Ccr2 or Csf1r (E) were measured in WT and Cd36 MKO mice with liver metastasis by RT-PCR (n = 5–6). F, G, The Ccr2 or Csf1r gene expression in WT and Cd36 -/- BMDMs (F), or in NC and CD36 OE THP-1 cells (G) cultured with TCM (n = 4–6). H, I, The MFI of CCR2 was measured in macrophages (H) and monocytes (I) isolated from metastatic liver tumors of WT and Cd36 MKO mice (n = 5). J, Migration assay was performed in WT or Cd36 -/- BMDMs treated with or without CCL2 or MCSF (n = 5). K-O, WT (n = 5) and Cd36 MKO (n = 5) mice were injected with LLC cells, following intraperitoneal injection of CCR2 inhibitor, RS504393, once a day from the 4th day to 14th day. HE-stained liver tissues along with the quantification of tumor area from WT and Cd36 MKO mice after treated with RS504393 (K, M, n = 3). F4/80-stained metastatic liver tissues from WT and Cd36 MKO mice after treated with RS504393 (L, M, n = 5). TSNE analysis of CD45 + cells isolated from the liver based on FCM (N). The percentage of indicated cells was measured (O, n = 5). Values for n represent biologically independent samples. Values for n represent biologically independent samples. Data are presented as mean ± SEM. *p < 0.05, ** p < 0.01, and *** p < 0.001 vs the control group; # p < 0.01, ## p < 0.01, and ### p < 0.001 vs the WT group. Unpaired two-tailed t -test (A, D-J), Unpaired two-tailed t -test with Mean-Whitney test (O) and ANOVA (B, C, M).

Article Snippet: For the treatment group, mice were individually treated with RS504393 (5 mg/kg, i.p., #HY-15418, MedChemExpress, Princeton, NJ, USA) or AS252424 (10 mg/kg, i.p., #HY-13532, MedchemExpress) once a day for 10 days, while the control group was given vehicle instead.

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Cell Culture, Isolation, Migration, Injection, Staining, Control, Two Tailed Test

Journal: Advanced Science

Article Title: CCN1 Enhances Tumor Immunosuppression through Collagen‐Mediated Chemokine Secretion in Pancreatic Cancer

doi: 10.1002/advs.202500589

Figure Lengend Snippet: Collagens modulate chemokine expression in PDAC cells under IFNγ stimulation. A) Tumor growth of sgCtrl and sgCcn1 KPC cells inoculated subcutaneously in C57B/L6 mice. Tumor‐bearing mice were treated with CCR2 inhibitor RS504393 and CXCR2 inhibitor SB225002. n = 6 mice per group. B) Representative images of tumors (left) and quantification of tumor weight (right) in mice inoculated with sgCtrl or sgCcn1 KPC cells. Tumor‐bearing mice were treated with the CCR2 inhibitor RS504393 and the CXCR2 inhibitor SB225002. C) Flow cytometric analysis of MDSC proportions in subcutaneous sgCtrl and sgCcn1 KPC tumors. D) mRNA levels of collagens, including Col4a1, Cola4a2, Col5a1, Col6a1, Col6a2, Col6a3, and Col8a1, in KPC cells after their total knockdown. E) mRNA levels of chemokines, including Ccl2, Ccl20, Cxcl1, Cxcl3, Cxcl5, Csf1, Csf2, and Csf 3 , in KPC cells after their total knockdown. F–H) mRNA levels of chemokines, including Ccl2, Ccl7, Ccl20, Cxcl1, Cxcl3, Cxcl5, Csf1, Csf2, and Csf3, in KPC cells after Col5a1 knockdown (F), Col6a3 knockdown (G), and Col8a1 knockdown (H). I) mRNA levels of collagens, including Col4a1, Cola4a2, Col5a1, Col6a1, Col6a2, Col6a3, and Col8a1, in sgCtrl KPC cells after treatment with 100 ng mL −1 IFNγ. J) mRNA levels of Col5a1, Col6a3, and Col8a1 measured in siCol5a1, siCol6a3, and siCol8a1 KPC cells after treatment with 100 ng mL −1 IFNγ. K–M) mRNA levels of chemokines including the Ccls (K), Cxcls (L), and Csfs (M), in siCol5a1, siCol6a3, and siCol8a1 KPC cells after treatment with 100 ng mL −1 IFNγ. N) mRNA level of Il6 in siCol5a1, siCol6a3, and siCol8a1 KPC cells after treatment with 100 ng mL −1 IFNγ.

Article Snippet: CCR2 inhibitor RS504393 (S9738, Selleck), CXCR2 inhibitor SB225002 (S7651, Selleck), and gemcitabine (S1714, Selleck) were purchased from Selleckchem.

Techniques: Expressing, Knockdown

Journal: Advanced Science

Article Title: CCN1 Enhances Tumor Immunosuppression through Collagen‐Mediated Chemokine Secretion in Pancreatic Cancer

doi: 10.1002/advs.202500589

Figure Lengend Snippet: Ccn1 regulates the TME in PDAC. A) Schematic representation of macrophage isolation from mouse bone marrow. B) Macrophage invasion evaluated by the chemotactic effect of sgCtrl and sgCcn1 KPC cells in a Transwell invasion assay. C) Macrophage polarization assessed by flow cytometry in macrophages cocultured with sgCtrl and sgCcn1 KPC cells. D) Macrophage polarization assessed by flow cytometry in macrophages cocultured with sgCtrl and sgCcn1 KPC cells after treatment with CCR2 inhibitor RS504393 and CXCR2 inhibitor SB225002. E) Survival of sgCtrl and sgCcn1 KPC cells cocultured with macrophages, assessed by CCK‐8 assay. F) Cell death of sgCtrl and sgCcn1 KPC cells cocultured with macrophages, assessed by flow cytometry. G) Protein expression levels of TNFα in macrophages cocultured with sgCtrl and sgCcn1 KPC cells. H) mRNA expression levels of TNFα in macrophages cocultured with sgCtrl and sgCcn1 KPC cells. I) Protein expression level of c‐Caspase 3 in sgCtrl and sgCcn1 KPC cells after treatment with 100 ng mL −1 TNFα. J) Protein expression level of c‐Caspase 3 in sgCtrl and sgCcn1 KPC cells after treatment with 100 ng mL −1 TNFα, mouse CCN1 protein, and cilengitide (αVβ3 and αVβ5 integrin inhibitor). K) Nucleus localization of CCN1 in KPC cells with CCN1 (red) and DAPI (blue) fluorescence. L) Protein expression level of p‐STAT3 in sgCtrl and sgCcn1 KPC cells after treatment with 100 ng mL −1 INFγ. M) mRNA levels of Ccl7, Cxcl3, and Csf3 in sgCtrl and sgCcn1 KPC cells after treatment with 100 ng mL −1 INFγ. N) mRNA level of Il6 measured in sgCtrl and sgCcn1 KPC cells after treatment with 100 ng mL −1 INFγ.

Article Snippet: CCR2 inhibitor RS504393 (S9738, Selleck), CXCR2 inhibitor SB225002 (S7651, Selleck), and gemcitabine (S1714, Selleck) were purchased from Selleckchem.

Techniques: Isolation, Transwell Invasion Assay, Flow Cytometry, CCK-8 Assay, Expressing, Fluorescence

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Roles played by IL-8 in altering dynamics of trabecular meshwork cells after human cytomegalovirus infection

doi: 10.3389/fcimb.2025.1550509

Figure Lengend Snippet: Network analysis of the roles of CXCR2 and CCR2 in human trabecular meshwork cells (HTMCs) after HCMV infection. HCMV-infected HTMCs blocked by CXCR2 or CCR2 were analyzed using RNA-seq analysis. The significantly induced or repressed genes were assessed for canonical analysis using Ingenuity pathway analysis. Associations with detected canonical pathways were assessed by the z-scores. (A) The top repressed canonical pathways by CXCR2 blockade by SB225002 were the translocation of SLC2A4 (GLUT4) to the plasma membrane, remodeling of adherens junctions, Rho GTPases activated IQGAPs, aggrephagy, carboxyterminal post-translational modification of tubulin, viral replication pathway, Rho GTPases activates Formins, kinesins, and Gap junction trafficking and regulations (blue bars). (B) The repressed canonical pathways by CCR2 blockade by RS504393 were the cell cycle checkpoints and synthesis of DNA.

Article Snippet: For blocking CCR2, 5 μM RS504393 (CCR2 antagonist, Tocris, Bristol, UK) was used.

Techniques: Infection, RNA Sequencing, Translocation Assay, Clinical Proteomics, Membrane, Modification

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Roles played by IL-8 in altering dynamics of trabecular meshwork cells after human cytomegalovirus infection

doi: 10.3389/fcimb.2025.1550509

Figure Lengend Snippet: HCMV-induced cell motility of human trabecular meshwork cells (HTMCs). HTMCs infected with TB40/E were stained for actin using SiR-actin. (A) HCMV infected HTMCs were assessed for cell motility. HCMV infection significantly stimulated speed of cell movement. Exposure to SB225002 (500 nM) abolished HCMV-induced cell motility. The blockade of CCR2 by RS504393 (5 μM) partially reduced cell motility by HCMV infection. The data and images represent the speed of cell movement at 6 hours post-infection. For analysis, 4 three-dimensional frames of 1410 μm × 1060 μm × 90 μm were randomly selected from the wells by a masked observer, and cells whose movement could be traced within the same frame over 24 hours were extracted for digitization. Next, a randomly selected eight representative cells per group were analyzed by a masked observer for their speed of cell movement at each hourly interval using a multivariate linear regression analysis. P-values were adjusted using the Scheffe test. Data represent repeated experiments. (B) HCMV-infected HTMC were accompanied by an increased formation of lamellipodia (arrowhead) and filopodia (arrow). Exposure to SB225002 (500 nM) suppressed formation of lamellipodia and filopodia.

Article Snippet: For blocking CCR2, 5 μM RS504393 (CCR2 antagonist, Tocris, Bristol, UK) was used.

Techniques: Infection, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Roles played by IL-8 in altering dynamics of trabecular meshwork cells after human cytomegalovirus infection

doi: 10.3389/fcimb.2025.1550509

Figure Lengend Snippet: HCMV induced contraction of trabecular meshwork cells. HCMV-infected HTMCs were embedded in collagen gel, and assessed for cell contraction at 6 h. Cell contraction was calculated by the percentage of collagen gel area subtracted from total area of the well. HCMV (TB40/E) infection significantly induced cell contraction. Cell contraction was suppressed by blocking CXCR2 by SB225002 (500 nM). In contrast, cell contraction was not affected by RS504393 exposure. N=6/group. ANOVA and Tukey test. Data represent repeated experiments.

Article Snippet: For blocking CCR2, 5 μM RS504393 (CCR2 antagonist, Tocris, Bristol, UK) was used.

Techniques: Infection, Blocking Assay

Journal: The Journal of Clinical Investigation

Article Title: Inhibition of aortic CX3CR1 + macrophages mitigates thoracic aortic aneurysm progression in Marfan syndrome in mice

doi: 10.1172/JCI178198

Figure Lengend Snippet: ( A – D ) Six-week-old Fbn1 C1041G/+ mice were intraperitoneally injected with RS504393 (4 mg/kg/d) until age 26 weeks. Age-matched WT mice were used as a normal control. ( A ) Flow cytometry analysis of Ly6C + monocytes and CX3CR1 + macrophages in aortic root and ascending aortas. n = 4. * P < 0.05 by 1-way ANOVA followed by Tukey’s test for post hoc comparison. ( B ) Representative transthoracic echocardiographic images of the aortic root and ascending aortas. Scale bars: 2 mm. White arrows depict the sinus of Valsalva measurements. Yellow arrows depict ascending aorta measurement. ( C ) Quantification of transthoracic echocardiography. n = 4 for WT and n = 8 for Fbn1 C1041G/+ mice. * P < 0.05 by repeated-measures ANOVA with the Greenhouse-Geisser adjustment followed by Bonferroni’s post hoc comparisons. ( D ) EVG staining of the aortic roots. n = 5. * P < 0.05 by Kruskal-Wallis test followed by Dunn’s test for elastin degradation grade and 1-way ANOVA followed by Tukey’s test for post hoc comparison for aortic thickness. ( E – H ) Twenty-four-week-old Fbn1 C1041G/+ mice were intraperitoneally injected with RS504393 (4 mg/kg/d) until age 36 weeks. Age-matched WT mice were used as a normal control. ( E ) Flow cytometry analysis of Ly6C + monocytes and CX3CR1 + macrophages in aortic root and ascending aortas. n = 5. * P < 0.05 by 1-way ANOVA followed by Tukey’s test for post hoc comparison. ( F ) Representative transthoracic echocardiographic images of the aortic root and ascending aortas. Scale bars: 2 mm. White arrows depict the sinus of Valsalva measurements. Yellow arrows depict ascending aorta measurement. ( G ) Quantification of transthoracic echocardiography. n = 5. * P < 0.05 by repeated-measures ANOVA with the Greenhouse-Geisser adjustment followed by Bonferroni’s post hoc comparisons. ( H ) EVG staining of aortic roots. n = 5. * P < 0.05 by Kruskal-Wallis test followed by Dunn’s test for elastin degradation grade and 1-way ANOVA followed by Tukey’s test for post hoc comparison for aortic thickness.

Article Snippet: Tamoxifen (HY-13757A), adalimumab (HY-P9908), teprotumumab (HY-P99165), and the CCR2 inhibitor RS504393 (HY-15418) were purchased from MedChem Express.

Techniques: Injection, Control, Flow Cytometry, Comparison, Staining

Journal: The Journal of Clinical Investigation

Article Title: Inhibition of aortic CX3CR1 + macrophages mitigates thoracic aortic aneurysm progression in Marfan syndrome in mice

doi: 10.1172/JCI178198

Figure Lengend Snippet: CX3CR1 + macrophages mainly located in the aortic intima and originated from circulating Ly6C + monocytes. Intimal CX3CR1 + macrophages upregulated inflammatory genes, including IL6 , MCP1 , and CXCL2 , in VSMCs by producing and secreting TNF-α and IGF1, thereby aggravating TAA development in MFS. Either elimination of intimal CX3CR1 + macrophages or administration of a CCR2 inhibitor to suppress monocyte recruitment efficiently alleviated TAA progression in MFS.

Article Snippet: Tamoxifen (HY-13757A), adalimumab (HY-P9908), teprotumumab (HY-P99165), and the CCR2 inhibitor RS504393 (HY-15418) were purchased from MedChem Express.

Techniques: