Journal:

Article Title: Protection against inflammation- and autoantibody-caused fetal loss by the chemokine decoy receptor D6

doi: 10.1073/pnas.0607514104

Figure Lengend Snippet: Chemokines in the LPS model of fetal loss. (A–E) Serum chemokine concentrations after LPS treatment in WT and D6−/− male mice. WT (open symbols) and D6−/− (filled symbols) mice were injected i.p. with 1.35 mg/kg LPS. At the indicated time points, circulating chemokine concentrations were measured by ELISA. Data are from seven mice for each time point. (F–J) Serum chemokine concentrations. WT (open columns) and D6−/− (filled columns) mice at day 10 of pregnancy were injected i.p. with 0.4 mg/kg LPS. Circulating chemokine concentrations were measured at 8 h postinjection by ELISA. Data are from nine WT and eight D6−/− mice. (K–O) Chemokine levels in placenta. WT (open columns) and D6−/− (filled columns) mice at day 10 of pregnancy were injected i.p. with 0.4 mg/kg LPS. Chemokine concentrations (expressed as nanograms of chemokine per milligram of total proteins of the lysates) were measured at 8 h postinjection by ELISA. Data are from nine WT and eight D6−/− mice. Results are reported as mean ± SEM. (A, F, and K) CCL22. (B, G, and L) CCL2. (C, H, and M) CCL11. (D, I, and N) CCL3. (E, J, and O) CXCL2.

Article Snippet: To block inflammatory chemokines, animals were treated with a mixture of goat antibodies to the mouse CC chemokines CCL3L1 (catalog no. AB450NA), CCL4 (catalog no. AB451NA), CCL2 (catalog no. AB479NA) and rat mAb anti-mouse CCL5 (catalog no. MAB478) purchased lyophilized from R&D Systems, resuspended in PBS, and mixed.

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Cell research

Article Title: Localized, highly efficient secretion of signaling proteins by migrasomes.

doi: 10.1038/s41422-024-00992-7

Figure Lengend Snippet: Fig. 6 Migrasomes are enriched with cytokines. a, b L929-GFP-VAMP2 cells were stained with M-CSF (a) or CCL2 (b) antibody and then visualized. Scale bar, 20 μm. The right panels show enlarged migrasomes. Scale bar, 2 μm. c, d Immunostaining of endogenous M-CSF (c) or CCL2 (d) in WT, Myo5a OE and Myo5a KO L929 cells. Scale bar, 20 µm. Right panels, enlarged ROI. Scale bar, 2 µm. Statistical analysis of the number of M-CSF (c) and CCL2 (d) puncta in migrasomes per cell is shown as the means ± SEM. n > 100 cells from three independent experiments were analyzed using the two-tailed unpaired t-test (right panel). **P < 0.01, ***P < 0.001. e Migrasomes were purified from the indicated L929 cells. Cell lysates and migrasomes were normalized with total protein and subjected to western blot analysis using the indicated antibodies. PIGK was used as a migrasome marker in L929 cells. Representative densitometry analysis of western blot gray values is shown. Three independent experiments were conducted.

Article Snippet: Anti-CCL2 (#AF-479-NA) antibody was from R&D SYSTEMS.

Techniques: Staining, Immunostaining, Two Tailed Test, Western Blot, Marker

Journal: Cell research

Article Title: Localized, highly efficient secretion of signaling proteins by migrasomes.

doi: 10.1038/s41422-024-00992-7

Figure Lengend Snippet: Fig. 7 Migrasomes are the major sites of secretion in migrating cells. a Secretion analysis of M-CSF and CCL2 in the indicated L929 cells. The concentrated media were collected and normalized with the numbers of cells, and were then subjected to western blot analysis using the indicated antibodies. Representative densitometry analysis of western blot gray values is shown. The ratios of secreted cytokines vs those in the cell body were quantified and shown as the means ± SEM from three independent experiments. Two-tailed unpaired t-test was used for statistical analyses (lower panels). **P < 0.01, ***P < 0.001. b, c L929 cells, either untreated or treated with 10 μM BAPTA-AM for 10 h, were stained with M-CSF (b) or CCL2 (c) antibody. Z-stack images were acquired by confocal microscopy, and z-projection was shown as the max intensity. Scale bar, 20 μm. The lower panels show statistical analysis of relative fluorescence intensity of control-cell body (C-C), control- migrasome (C-M), BAPTA-AM-cell body (B-C) and BAPTA-AM-migrasome (B-M). Data are means ± SEM. n > 100 cells from three independent experiments. Two-tailed unpaired t-test was used for statistical analyses. ***P < 0.001. d Diagram showing how migrasomes are involved in secretion in migrating cells.

Article Snippet: Anti-CCL2 (#AF-479-NA) antibody was from R&D SYSTEMS.

Techniques: Western Blot, Two Tailed Test, Staining, Confocal Microscopy, Control

Journal: Cancer immunology research

Article Title: B7-H3 Suppresses Antitumor Immunity via the CCL2-CCR2-M2 Macrophage Axis and Contributes to Ovarian Cancer Progression.

doi: 10.1158/2326-6066.CIR-21-0407

Figure Lengend Snippet: Figure 4. B7-H3 suppression downregulates CCL2 production, in part, via the STAT3 pathway in ovarian cancer cells. A, Differential gene expression based on RNA sequencing (RNA-seq) between controls (n ¼ 3) and B7-H3 KO (n ¼ 3) groups in HM-1 and ID8 cells and HM-1 intradermal tumors. CCL2 protein in the culture supernatants (B) and tumors derived from HM-1 B7-H3 KO (top), ID8 B7-H3 KO (bottom), and their respective controls (n ¼ 5; C). D, CCL2 protein in the culture supernatants of the OVCAR3 shB7-H3 (top), OVCA420 shB7-H3 (bottom), and their respective controls (n ¼ 5). Representative Western blot analysis of nuclear phosphorylated STAT3 (p-STAT3) in HM-1 and ID8 (E) and OVCAR3 and OVCA420 (F) cells. Bottom bar graphs show the signal intensity of p-STAT3 relative to the TBP control in HM-1 and ID8 cells (both n ¼ 10) and OVCAR3 and OVCA420 cells (both n ¼ 7). qPCR for expression of Ccl2 in HM-1 and ID8 cells (G) and CCL2 in OVCAR3 and OVCA420 cells (H) treated with 1.25 and 5 mmol/L of the STAT3 inhibitors C188-9 (G) and Stattic (H), respectively. DMSO was used as the control (n ¼ 3). Data are presented as the mean SEM; , P < 0.05; , P < 0.01; , P < 0.001; N.S., not significant, unpaired t test in B, C, D (OVCA420), E, and F and one-way ANOVA with Tukey multiple comparisons test in D (OVCAR3), G, and H.

Article Snippet: Mouse bone marrowmonocytes (isolated and cultured as described in the “Macrophage and monocyte selection” section) were plated in the upper compartment of 0.4 mm cell culture inserts (catalog no. 353095, Corning) at a density of at 1 105, and 500 mL of TCM of HM-1-B7-H3 KO cells, ID8-B7-H3 KO cells or their control cells (generated as described in the “ELISAs” section) supplemented with 20 mg/mL anti-mouse CCL2 (catalog no. AB-479-NA, RRID: AB_354366; R&DSystems) or goat IgG control (catalog no. AB-108-C, RRID:AB_354267; R&D Systems) was added to the lower compartment.

Techniques: Gene Expression, RNA Sequencing, Derivative Assay, Western Blot, Control, Expressing

Journal: Cancer immunology research

Article Title: B7-H3 Suppresses Antitumor Immunity via the CCL2-CCR2-M2 Macrophage Axis and Contributes to Ovarian Cancer Progression.

doi: 10.1158/2326-6066.CIR-21-0407

Figure Lengend Snippet: Figure 5. The CCL2–CCR2–M2 macrophage axis contributes to B7-H3–mediated tumor progression. A, Chemotaxis of mouse monocytes (left) and generated M2 macrophages (right) in response to HM-1 control or B7-H3 KO TCM. Monocytes or M2 macrophages were pretreated with 2 mmol/L RS504393 (CCR2 antagonist) or DMSO before plating. MEM-Alpha was used as the negative control (n ¼ 3). B, Differentiation of mouse monocytes into M2 macrophages in response to HM-1 control or B7-H3 KO TCM supplemented with 20 mg/mL anti-CCL2 or control IgG. MEM-Alpha was used as the negative control (n ¼ 3). Ab, antibody. C, Tumor growth in mice intradermally injected with HM-1 B7-H3 KO cells or control cells and treated with RS504393 (2 mg/kg body weight) or DMSO daily following tumor inoculation. D, Flow cytometric analysis of the treated tumors in C (n ¼ 6). Data are presented as the mean SEM (, P < 0.05; , P < 0.01; , P < 0.001; N.S., not significant, one-way ANOVA with Tukey multiple comparisons test in A and B, and unpaired t test in C and D).

Article Snippet: Mouse bone marrowmonocytes (isolated and cultured as described in the “Macrophage and monocyte selection” section) were plated in the upper compartment of 0.4 mm cell culture inserts (catalog no. 353095, Corning) at a density of at 1 105, and 500 mL of TCM of HM-1-B7-H3 KO cells, ID8-B7-H3 KO cells or their control cells (generated as described in the “ELISAs” section) supplemented with 20 mg/mL anti-mouse CCL2 (catalog no. AB-479-NA, RRID: AB_354366; R&DSystems) or goat IgG control (catalog no. AB-108-C, RRID:AB_354267; R&D Systems) was added to the lower compartment.

Techniques: Chemotaxis Assay, Generated, Control, Negative Control, Injection

Journal: Cancer immunology research

Article Title: B7-H3 Suppresses Antitumor Immunity via the CCL2-CCR2-M2 Macrophage Axis and Contributes to Ovarian Cancer Progression.

doi: 10.1158/2326-6066.CIR-21-0407

Figure Lengend Snippet: Figure 6. B7-H3 expression associates with an M2 macrophage–rich, IFNgþCD8 T cell–poor TME and a poor prognosis in patients with HGSOC. A, Correlation between CCL2 expression and B7-H3 protein in primary HGSOC tumors (n ¼ 28) analyzed by Spearman correlation. B, Representative images of HGSOC samples showing B7-H3 expression and CD206þ, CD8þ, and IFNgþCD8þ cells. Cells were stained with hematoxylin and eosin (H&E) or immunostained with the corresponding antibodies. Scale bar, 100 mm. Positive cells were stained brown in IHC, and red (CD8), green (IFNg), and blue (DAPI) in immunofluorescence slides. The boxed area represents the zoomed image shown in the top right corner. C, Correlation between the infiltration of CD206þ cells and B7-H3 expression in the corresponding primary tumors of HGSOC (n ¼ 62) analyzed using Jonckheere–Terpstra test. Data are presented as the medianwith 95% confidenceintervals. D, Correlation between tumor-infiltrating CD8þ and IFNgþCD8þ T cells (n ¼ 62) analyzed using Spearman correlation. Blue and pink dots represent B7-H3–low and B7-H3–high expression cases, respectively. E, Comparisons of the number of tumor-infiltrating CD8þ T cells (left) and IFNgþCD8þ T cells (right) in B7-H3–low and B7-H3–high groups by immunostaining (n ¼ 62). Data are presented as the medianwith 95% confidence intervals; , P < 0.05; N.S., not significant, Mann–Whitney U test. F, PFS (left) and OS(right) of patients with HGSOC (n ¼ 62). Patients were classified into a B7-H3–low group (n ¼ 31) and a B7-H3–high group (n ¼ 31). , P < 0.05; N.S., not significant, log-rank test.

Article Snippet: Mouse bone marrowmonocytes (isolated and cultured as described in the “Macrophage and monocyte selection” section) were plated in the upper compartment of 0.4 mm cell culture inserts (catalog no. 353095, Corning) at a density of at 1 105, and 500 mL of TCM of HM-1-B7-H3 KO cells, ID8-B7-H3 KO cells or their control cells (generated as described in the “ELISAs” section) supplemented with 20 mg/mL anti-mouse CCL2 (catalog no. AB-479-NA, RRID: AB_354366; R&DSystems) or goat IgG control (catalog no. AB-108-C, RRID:AB_354267; R&D Systems) was added to the lower compartment.

Techniques: Expressing, Staining, Immunostaining, MANN-WHITNEY