Journal: eLife

Article Title: Subventricular zone/white matter microglia reconstitute the empty adult microglial niche in a dynamic wave

doi: 10.7554/eLife.66738

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Ccl12 (Goat polyclonal) , R and D Systems , Cat# AF428, RRID: AB_2070875 , 100 ug/0.5 ml sterile HBSS.

Techniques: Sterility, Plasmid Preparation, Software

Journal: JCI Insight

Article Title: Epithelial-macrophage interactions determine pulmonary fibrosis susceptibility in Hermansky-Pudlak syndrome

doi: 10.1172/jci.insight.88947

Figure Lengend Snippet: (A) Macrophage TGF-β1 production. Mice were challenged with IT bleomycin, and bronchoalveolar lavage (BAL) was performed 24 hours later. Macrophages were separated by adherence to cell culture plates and then cultured for 24 hours in vitro. Total TGF-β1 was quantitated in the cell media by ELISA. Data are presented as box-and-whisker Tukey plots; n = 7 WT and HPS2/CCR2–/–, n = 6 WT/CCR2–/–, n = 5 HPS2, and n = 4/group for HPS1, HPS1/CCR2–/–, and HPS2/TG+. Comparisons were conducted by 2-way ANOVA with Bonferroni’s post-hoc test, *P < 0.05 vs. WT and CCR2–/–, **P < 0.05 vs. HPS2/TG+. (B) MCP-1 augments TGF-β production by WT macrophages. Macrophages were isolated by BAL from unchallenged WT mice and cultured with recombinant MCP-1 (10 ng/ml) or vehicle for 60 minutes. Total TGF-β1 was quantitated in the cell media by ELISA. Data are presented as box-and-whisker Tukey plots; n = 6/group. Comparisons between groups were conducted by Mann-Whitney U analysis, *P < 0.05.

Article Snippet: Cytokine levels were measured in cell culture media supernatant or BAL fluid by ELISA (R&D Systems : MCP-1, #MJE00; MCP-5, #MCC120; MIP-1α, # MMA00; GM-CSF, #MGM00; RANTES, #MMR00; TGFβ1, MB100B; and Abcam: MCP-3, #ab205571; and M-CSF, #ab155457).

Techniques: Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Whisker Assay, Isolation, Recombinant, MANN-WHITNEY

Journal: JCI Insight

Article Title: Epithelial-macrophage interactions determine pulmonary fibrosis susceptibility in Hermansky-Pudlak syndrome

doi: 10.1172/jci.insight.88947

Figure Lengend Snippet: (A–C) Mice deficient in myeloid TGF-β1 (LysM.Cre/TGFb1f/f; denoted WT/TGF-βΔMye) were studied in comparison to LysM.Cre– littermate controls (LysM.Cre–/TGFb1f/f; denoted WT). (A) Macrophages were isolated by bronchoalveolar lavage (BAL), and total TGF-β1 was measured in the cell culture media by ELISA after 24 hours in culture (mean ± SEM); n = 3/group; *P < 0.05 by Mann-Whitney U analysis. (B) BAL cell counts from unchallenged mice; n = 5 WT and n = 7 WT/ TGF-βΔMye, P = NS by Mann-Whitney U analysis. (C) Total TGF-β1 in lung homogenates from unchallenged mice. Data are presented as box-and-whisker Tukey plots; n = 4/group, P = NS by Mann-Whitney U analysis. (D–H) TGF-βΔMye mice were bred onto the HPS1 background to generate HPS1/TGF-βΔMye mice or HPS1/Cre– littermate controls (denoted HPS1). (D) Total TGF-β1 was determined by ELISA in BAL fluid from unchallenged mice; n = 15 WT/TGF-βΔMye, n = 8 HPS1/TGF-βΔMye, n = 7 WT controls, and n = 11 HPS1 controls. Comparisons between groups were conducted by Kruskal-Wallis test with Dunn’s multiple comparisons post-test, *P < 0.01 vs. WT, **P < 0.05 vs. HPS1/TGF-βΔMye. (E) Representative H&E histologic images (original magnification, ×10) of lung sections at 7 days after IT bleomycin. (F) Lung collagen content of the left lung in unchallenged mice and at 7 days after bleomycin (mean ± SEM). For unchallenged groups, n = 6. For bleomycin-challenged groups, n = 3 WT, n = 6 WT/TGF-βΔMye, n = 21 HPS1, and n = 15 HPS1/TGF-βΔMye mice. Comparisons between bleomycin-challenged groups were conducted by Kruskal-Wallis test with Dunn’s multiple comparisons post-test, *P < 0.01 vs. other bleomycin-challenged groups. (G) Fibrosis scoring on trichrome-stained lung tissue (mean ± SEM); n = 3/group for WT and n = 6/group for HPS1, *P < 0.05. (H) TUNEL+ alveolar epithelial cells in lung sections from mice 24 hours after bleomycin challenge (mean ± SEM); n = 10 WT/LysM.Cre– (WT) and n = 12 for other groups. Comparisons between groups were conducted by Kruskal-Wallis test with Dunn’s multiple comparisons post-test, *P < 0.01 vs. WT, **P < 0.05 vs. HPS1/ TGF-βΔMye.

Article Snippet: Cytokine levels were measured in cell culture media supernatant or BAL fluid by ELISA (R&D Systems : MCP-1, #MJE00; MCP-5, #MCC120; MIP-1α, # MMA00; GM-CSF, #MGM00; RANTES, #MMR00; TGFβ1, MB100B; and Abcam: MCP-3, #ab205571; and M-CSF, #ab155457).

Techniques: Comparison, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Whisker Assay, Staining, TUNEL Assay

Journal: JCI Insight

Article Title: Epithelial-macrophage interactions determine pulmonary fibrosis susceptibility in Hermansky-Pudlak syndrome

doi: 10.1172/jci.insight.88947

Figure Lengend Snippet: HPS1 mice were bred to homozygosity with SPC.Cre+/TGFBR2f/f mice (denoted HPS1/TGFBR2ΔAEC) and studied in comparison to HPS1/SPC.Cre– littermate controls (denoted HPS1). (A–D) Representative histologic images of lung sections from mice at 7 days after bleomycin. (A and C) H&E images (original magnification, ×10). (B and D) Trichrome images (original magnification, ×20). (E) Lung collagen content of left lung in unchallenged mice or at 7 days after bleomycin (mean ± SEM); n = 5 WT unchallenged, n = 4 WT bleomycin, n = 5 WT/TGFBR2 ΔAEC; n = 13 HPS1/TGFBR2ΔAEC; and n = 18 HPS1. Comparisons between groups were conducted by Kruskal-Wallis test with Dunn’s multiple comparisons post-test, *P < 0.01 vs. unchallenged groups and WT bleomycin groups, **P < 0.05 vs. HPS1/ TGFBR2ΔAEC. (F) Fibrosis scoring on trichrome-stained lung tissue. Data are presented as box-and-whisker Tukey plots; n = 6 WT/TGFBR2ΔAEC, n = 5 HPS1/ TGFBR2ΔAEC, n = 4 WT/Cre– littermate controls, and n = 6 HPS1 controls, *P < 0.05 vs. all other groups. (G) TUNEL+ alveolar epithelial cells (AECs) in lung sections from mice 24 hours after bleomycin challenge; n = 10 HPS1/TGFBR2ΔAEC and n = 6 HPS1. Comparison between groups was assessed using Mann-Whitney U analysis, *P < 0.01. (H) Total TGF-β1 in BAL from unchallenged mice quantitated by ELISA; n = 14 HPS1/TGFBR2ΔAEC and n = 13 Cre– controls; Mann-Whitney U analysis, *P < 0.001. (I) MCP-1 production from type II AECs isolated from unchallenged mice and cultured for 24 hours; n = 9 WT/TGFBR2 ΔAEC and HPS1/TGFBR2ΔAEC and n = 6/group for WT and SPC.Cre– littermate controls. Comparisons between groups were conducted by Kruskal-Wallis test with Dunn’s multiple comparisons post-test, *P < 0.05 vs. WT, **P < 0.01 vs. HPS1/TGFBR2ΔAEC. (J) MCP-1 production from WT type II AECs after exposure to TGF-β. WT AECs were cultured for 24 hours in the presence of 20 ng/ml TGF-β or vehicle control, and MCP-1 levels were measured in the cell culture media by ELISA (mean ± SEM); n = 4 WT + vehicle and n = 8 WT + TGF-β, *P < 0.001.

Article Snippet: Cytokine levels were measured in cell culture media supernatant or BAL fluid by ELISA (R&D Systems : MCP-1, #MJE00; MCP-5, #MCC120; MIP-1α, # MMA00; GM-CSF, #MGM00; RANTES, #MMR00; TGFβ1, MB100B; and Abcam: MCP-3, #ab205571; and M-CSF, #ab155457).

Techniques: Comparison, Staining, Whisker Assay, TUNEL Assay, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Isolation, Cell Culture

Journal: Experimental & Molecular Medicine

Article Title: CCL12 induces trabecular bone loss by stimulating RANKL production in BMSCs during acute lung injury

doi: 10.1038/s12276-023-00970-w

Figure Lengend Snippet: a Chemokine levels in the peripheral blood of ALI mice on Days 0, 2, 8, and 14 after LPS administration were determined by an antibody array ( n = 6). b Schematic diagram of mice treated with 5 mg/kg LPS (i.t.) and 4 mg/kg neutralizing antibodies against CCL12 via the tail vein ( n = 8). Saline was used as a negative control for LPS. Isotype IgG was used as a negative control for the neutralizing antibody. The day when LPS was administered was defined as “Day 0”. A neutralizing antibody against CCL12 was injected for the first time before LPS administration on Day 0. Then, the antibody was injected every 4 days. Femurs were collected on Day 14. c Representative 3D reconstruction images of the distal femur trabecular bone of ALI mice after the administration of the neutralizing antibody against CCL12 were determined by micro-CT. d BV/TV, Tb.N, and Tb.Sp of the distal femur in ALI mice after the administration of the neutralizing antibody against CCL12 ( n = 8). e H&E staining and histomorphometric analysis of the Ob.N/B.Pm and Ob.S/BS in the femurs of ALI mice after the administration of the neutralizing antibody against CCL12 ( n = 8). Scale bar: 40 μm. f TRAP staining and histomorphometric analysis of the Oc.N/B.Pm and Oc.S/BS in the femurs of ALI mice after the administration of the neutralizing antibody against CCL12 ( n = 8). Scale bar: 40 μm. The data are representative of three independent experiments. The data are shown as the means ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001, ns no significance.

Article Snippet: BMSCs were treated in vitro with 50 and 100 ng/ml recombinant mouse CCL12 (R&D Systems, Minneapolis, MN).

Techniques: Ab Array, Saline, Negative Control, Injection, Micro-CT, Staining

Journal: Experimental & Molecular Medicine

Article Title: CCL12 induces trabecular bone loss by stimulating RANKL production in BMSCs during acute lung injury

doi: 10.1038/s12276-023-00970-w

Figure Lengend Snippet: a CCL12 levels in the peripheral blood of wild-type (WT) and CCL12 −/− mice were determined by ELISA ( n = 8). b , c CCL12 protein expression in the bone marrow of WT and CCL12 −/− mice was determined by immunohistochemistry ( b ) and ELISA ( c ) ( n = 8). B: bone. Scale bar: 20 μm. d Representative 3D reconstruction images of the distal femur trabecular bones of WT and CCL12 −/− mice were determined by micro-CT. e BV/TV, Tb.N, and Tb.Sp of the distal femurs of WT and CCL12 −/− mice ( n = 8). f H&E staining and histomorphometric analysis of the Ob.N/B.Pm and Ob.S/BS in the femur ( n = 8). Scale bar: 40 μm. g TRAP staining and histomorphometric analysis of the Oc.N/B.Pm and Oc.S/BS in the femurs of WT and CCL12 −/− mice (n = 8). Scale bar: 40 μm. The data are representative of three independent experiments. The data are shown as the means ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001, ns no significance.

Article Snippet: BMSCs were treated in vitro with 50 and 100 ng/ml recombinant mouse CCL12 (R&D Systems, Minneapolis, MN).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemistry, Micro-CT, Staining

Journal: Experimental & Molecular Medicine

Article Title: CCL12 induces trabecular bone loss by stimulating RANKL production in BMSCs during acute lung injury

doi: 10.1038/s12276-023-00970-w

Figure Lengend Snippet: a RANKL levels in peripheral blood and bone marrow of ALI mice were determined by ELISA ( n = 8). b RANKL protein expression in the femur osteocytes, osteoblasts, and bone marrow of ALI mice was determined by immunohistochemistry ( n = 8). B: bone. Scale bar: 20 μm. c OPG levels in the peripheral blood and bone marrow of ALI mice were determined by ELISA ( n = 8). d OPG protein expression in the femur osteocytes, osteoblasts, and bone marrow of ALI mice was determined by immunohistochemistry ( n = 8). B: bone. Scale bar: 20 μm. e RANKL levels in the peripheral blood and bone marrow of ALI mice with global deletion of CCL12 were determined by ELISA ( n = 8). f RANKL protein expression in the femur osteocytes, osteoblasts, and bone marrow of ALI mice with global deletion of CCL12 was determined by immunohistochemistry ( n = 8). B: bone. Scale bar: 20 μm. The data are representative of three independent experiments. The data are shown as the means ± s.d. ** p < 0.01, *** p < 0.001, ns no significance.

Article Snippet: BMSCs were treated in vitro with 50 and 100 ng/ml recombinant mouse CCL12 (R&D Systems, Minneapolis, MN).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemistry

Journal: Experimental & Molecular Medicine

Article Title: CCL12 induces trabecular bone loss by stimulating RANKL production in BMSCs during acute lung injury

doi: 10.1038/s12276-023-00970-w

Figure Lengend Snippet: a RANKL mRNA and protein expression in RANKL + CCR2 + BMSCs from ALI mice ( n = 8). b RANKL mRNA and protein expression in primary BMSCs and ST2 cells cultured with 0, 50, and 100 ng/ml recombinant CCL12 for 48 h in vitro ( n = 8). c RANKL mRNA and protein expression in RANKL + CCR2 + BMSCs from ALI mice with global deletion of CCL12 ( n = 8). d RANKL protein expression in the femur osteocytes, osteoblasts, and bone marrow of ALI mice with conditional deletion of CCR2 in BMSCs was determined by immunohistochemistry ( n = 8). B: bone. Scale bar: 20 μm. e RANKL mRNA and protein expression in BMSCs with CCR2 deletion from ALI mice was determined by flow cytometry ( n = 8). f TRAP staining and histomorphometric analysis of the Oc.N/B.Pm and Oc.S/BS in the femurs of ALI mice with conditional deletion of CCR2 in BMSCs ( n = 8). Scale bar: 40 μm. g Representative 3D reconstruction images and the BV/TV, Tb.N, and Tb.Sp of the distal femur trabecular bones of ALI mice with conditional deletion of CCR2 in BMSCs were determined by micro-CT ( n = 8). α-Tubulin was used as an internal control for qPCR. The data are representative of three independent experiments. The data are shown as the means ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001, ns no significance.

Article Snippet: BMSCs were treated in vitro with 50 and 100 ng/ml recombinant mouse CCL12 (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Cell Culture, Recombinant, In Vitro, Immunohistochemistry, Flow Cytometry, Staining, Micro-CT, Control

Journal: Experimental & Molecular Medicine

Article Title: CCL12 induces trabecular bone loss by stimulating RANKL production in BMSCs during acute lung injury

doi: 10.1038/s12276-023-00970-w

Figure Lengend Snippet: a Diagram of the mouse RANKL promoter. Analysis of transcription factor binding sites in the RANKL promoter revealed putative STAT3 and STAT4 binding sites around the transcription start site. b STAT3 binding to the ChIP-1 and ChIP-2 regions within the RANKL promoter in BMSCs in response to 0, 50, and 100 ng/ml recombinant CCL12 treatment for 24 h, as determined by ChIP. c STAT4 binding to the ChIP-1 and ChIP-2 regions within the RANKL promoter in BMSCs in response to 0, 50, and 100 ng/ml recombinant CCL12 treatment for 24 h, as determined by ChIP. d Lentivirus-mediated stable overexpression (OE) of STAT3 with an HA tag and STAT4 with a Flag tag in BMSC cultures was determined by western blotting. e STAT3 binding to the ChIP-1 region within the RANKL promoter in BMSCs with STAT3 overexpression was determined by ChIP. f STAT4 binding to the ChIP-2 region within the RANKL promoter in BMSCs with STAT4 overexpression was determined by ChIP. g Lentivirus-mediated stable knockdown of STAT3 and STAT4 in BMSC cultures was determined by western blotting. h STAT3 binding to the ChIP-1 region within the RANKL promoter in BMSCs treated with recombinant CCL12 for 24 h and/or STAT3 knockdown was determined by ChIP. i STAT4 binding to the ChIP-2 region within the RANKL promoter in BMSCs treated with recombinant CCL12 for 24 h and/or STAT4 knockdown was determined by ChIP. Isotype IgG was used as a negative control in the ChIP assay. α-Tubulin was used as a loading control for western blotting. The data are representative of three independent experiments. The data are shown as the means ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BMSCs were treated in vitro with 50 and 100 ng/ml recombinant mouse CCL12 (R&D Systems, Minneapolis, MN).

Techniques: Binding Assay, Recombinant, Over Expression, FLAG-tag, Western Blot, Knockdown, Negative Control, Control

Journal: Experimental & Molecular Medicine

Article Title: CCL12 induces trabecular bone loss by stimulating RANKL production in BMSCs during acute lung injury

doi: 10.1038/s12276-023-00970-w

Figure Lengend Snippet: a Phosphorylated Jak2 protein levels in BMSCs in response to 0, 50, and 100 ng/ml recombinant CCL12 treatment for 6 h were determined by western blotting. b Phosphorylated STAT3 protein levels in BMSCs in response to 0, 50, and 100 ng/ml recombinant CCL12 treatment for 6 h were determined by western blotting. c Phosphorylated STAT4 protein levels in BMSCs in response to 0, 50, and 100 ng/ml recombinant CCL12 treatment for 6 h were determined by western blotting. d Phosphorylated STAT3 protein levels in the nuclear fraction of BMSCs in response to 0, 50, and 100 ng/ml recombinant CCL12 treatment for 6 h were determined by western blotting. e Phosphorylated STAT4 protein levels in the nuclear fraction of BMSCs in response to 0, 50, and 100 ng/ml recombinant CCL12 treatment for 6 h were determined by western blotting. f RANKL mRNA expression in BMSCs in response to recombinant CCL12 (100 ng/ml) and/or AG490 (50 μM) treatment for 48 h ( n = 8). g RANKL mRNA expression in BMSCs in response to recombinant CCL12 (100 ng/ml) treatment for 48 h and/or STAT3/4 knockdown ( n = 8). h Luciferase activity of RANKL promoter deletion mutant-driven luciferase reporter gene vectors, including Luc (empty vector as negative control), −1005 bp/+156 bp (containing putative STAT3 and STAT4 binding sites), −454 bp/+156 bp (containing putative STAT4 binding site), and −1005 bp/−429 bp (containing putative STAT3 binding site), in BMSCs treated with recombinant CCL12 for 48 h ( n = 8). i Luciferase activity of RANKL promoter deletion mutant-driven luciferase reporter gene vectors, including Luc, STAT3 + STAT4 binding site, 3×STAT3 + STAT4 binding site, and STAT3 + 3×STAT4 binding site, in BMSCs treated with recombinant CCL12 for 48 h ( n = 8). j Luciferase activity of RANKL promoter deletion mutant-driven luciferase reporter gene vectors with and without pcDNA3.1-STAT3/4 vector cotransfection in BMSCs treated with recombinant CCL12 for 48 h ( n = 8). α-Tubulin was used as an internal control for qPCR and western blot analysis of total proteins. Lamin B1 was used as a loading control for western blot analysis of nuclear proteins. The Renilla luciferase vector was used as a control for transfection efficiency. The data are representative of three independent experiments. The data are shown as the means ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001, ns no significance.

Article Snippet: BMSCs were treated in vitro with 50 and 100 ng/ml recombinant mouse CCL12 (R&D Systems, Minneapolis, MN).

Techniques: Recombinant, Western Blot, Expressing, Knockdown, Luciferase, Activity Assay, Mutagenesis, Plasmid Preparation, Negative Control, Binding Assay, Cotransfection, Control, Transfection

Journal: Experimental & Molecular Medicine

Article Title: CCL12 induces trabecular bone loss by stimulating RANKL production in BMSCs during acute lung injury

doi: 10.1038/s12276-023-00970-w

Figure Lengend Snippet: LPS induces acute lung injury, and CCL12 levels are significantly elevated in the peripheral blood ( a ) and bone marrow ( b ) in response to inflammation. In bone marrow, CCL12 binds to CCR2 on the cell membrane of BMSCs to activate RANKL transcription through the Jak2/STAT4 axis ( b ). RANKL promotes osteoclastic differentiation in bone marrow monocytes ( c ), which results in enhanced bone resorption activity and trabecular bone loss in acute lung injury.

Article Snippet: BMSCs were treated in vitro with 50 and 100 ng/ml recombinant mouse CCL12 (R&D Systems, Minneapolis, MN).

Techniques: Membrane, Activity Assay

Journal: Journal of Inflammation Research

Article Title: Neuroinflammation and Microglial Activation at Rostral Ventrolateral Medulla Underpin Cadmium-Induced Cardiovascular Dysregulation in Rats

doi: 10.2147/JIR.S325528

Figure Lengend Snippet: Top 20 Cytokine and Chemokine Gene Changes in Rostral Ventrolateral Medulla (RVLM) of Rats

Article Snippet: Rn01464638_m1 , Ccl12 , 22.4.

Techniques: Control