Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: Sequences of the primers used for reverse transcription polymerase chain reaction.

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Reverse Transcription, Sequencing

Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: The expression of CCL3 and its receptors in cornea after alkali injury. A : Semi-quantitative RT–PCR was performed to assess mRNA expression of CCL3 and its receptors, CCR1 and CCR5 , and the ratios of target gene expression to β-actin were determined. All values represent the mean±SEM of three to five independent measurements. B : Whole eyes were obtained at 0, 2, 4, and 7 days after alkali injury and processed for immunohistochemical analysis using anti-CCL3 (upper panels) or anti-CCR1 antibodies (lower panels). Representative results from five individual animals are shown. Original magnifications, 400X. Scale bar, 50 μm.

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Quantitative RT-PCR, Targeted Gene Expression, Immunohistochemical staining

Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: Alkali-induced corneal neovascularization and macrophage infiltration. A : The macroscopic appearances of WT, CCL3-KO, CCR1-KO, and CCR5-KO mouse eyes two weeks after alkali injury are illustrated. Representative results from at least 10 animals in each group are shown here. B : Corneal tissues were obtained from WT, CCR1-KO, and CCL3-KO mice two weeks after the injury. Tissues were stained with hematoxylin and eosin (left panels) or immunostained with anti-CD31 antibodies (right panels), and representative results from five individual animals are shown. Original magnifications, 400X. Scale bar, 50 μm. C : CNV numbers per mm 2 in hot spots (upper panel) and % CNV areas in hot spots (lower panel) were determined on corneas obtained from WT or KO mice two weeks after the injury. Each value represents the mean and SEM (n=5 animals). An asterisk represents a p<0.05 and that the value was obtained comparing WT and CCL3-KO mice. D : The numbers of infiltrated F4/80 positive macrophages were determined two and four days after the injury. Each value represents the mean and SEM (n=5). The double asterisk indicates a p<0.01 and that the value was obtained comparing WT and CCL3-KO mice.

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Staining

Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: Angiogenic factor expression. A : RT–PCR analysis of pro-angiogenic and anti-angiogenic gene expressions in the injured corneas of WT and CCL3-KO mice. RT–PCR analysis was performed on total RNAs extracted from eyes 0, 2, 4, and 7 days after alkali injury, and then the ratios of VEGF to β-actin, bFGF to β-actin, and TSP-1 to β-actin of WT (black bars) and CCL3-KO mice (open bars) were determined. All values represent the mean and SEM (n=3-5 animals). The asterisk denotes a p<0.05; the hash mark denotes a p<0.01 and that the value was obtained comparing WT and KO mice. The effects of CCL3 on VEGF expression by murine peritoneal macrophages is shown in B and C . B : RT–PCR was performed on macrophages incubated with the indicated concentrations of CCL3 for 12 h, and the ratio of VEGF to β-actin was calculated. Each value represents the mean and SEM (n=3). C : Murine macrophages were stimulated with either 0, 10, or 100 ng/ml of CCL3 for 24 h. VEGF concentrations in the supernatants were determined with ELISA as described in Methods. The representative results from three independent experiments are shown. The asterisk denotes a p<0.05 and the double asterisk denotes a p<0.01 when compared to untreated.

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: Intracorneal CCR5 positive cell infiltration. A : A double-color immunofluorescence analysis of CCR5-expressing cells is illustrated. Corneas were obtained from WT mice 0 and 4 days after the injury. The samples were immunostained with a combination of anti-CCR5 and anti-CCR2 antibodies as described in Methods and observed with fluorescence microscopy (original magnification, 400X). Signals were digitally merged in the right panels. Arrows indicate the double, positively stained cells. Representative results from three independent experiments are shown. B : Corneal tissues from WT mice (left panel) or CCL3-KO mice (right panel) obtained four days after the injury were stained with anti-CCR5 Ab. Scale bar, 100 μm. C : The numbers of intracorneal CCR5 positive cells four days after the injury were determined as described in Methods, and the mean and SEM are shown here (n=5).

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Immunofluorescence, Expressing, Fluorescence, Microscopy, Staining

Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: The effects of topical CCL3 application on corneal neovascularization. A : Macroscopic appearances of WT, CCL3-KO mice, and CCL3-KO mice topically applied with CCL3 two weeks after alkali injury are shown. Representative results from five animals from each group are shown here. B : Corneal tissues were obtained two weeks after the injury from WT, CCL3-KO, and CCL3-KO mice topically applied with CCL3 and were immunostained with anti-CD31 antibodies. Representative results from five individual mice from each group are shown. Original magnification, 400X. Scale bar, 50 μm. C : The CNV numbers per mm 2 in hot spots (left panel) and % CNV areas in hot spots (right panel) were determined. Each value represents the mean and SEM (n=5 animals). D : The number of infiltrated F4/80 positive macrophages was determined on WT, CCL3-KO, and CCL3-KO, which were all treated with CCL3, two days after the injury. Each value mean represents both the mean and SEM (n=5). The asterisk denotes a p<0.05, and the double asterisk means a p<0.01 when compared with CCL3-KO (this applies to both C and D ).

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques:

Journal: PLoS pathogens

Article Title: Myeloid derived hypoxia inducible factor 1-alpha is required for protection against pulmonary Aspergillus fumigatus infection.

doi: 10.1371/journal.ppat.1004378

Figure Lengend Snippet: Figure 6. Decreased neutrophil levels in HIFC mice correlates to decreased production of CXCL1 and not migration defects. Migration of littermate (WT) and HIFC BMDNs in the presence of (A) RPMI media containing FBS or nothing or (B) 12 hr BALF from HIFC or WT mice inoculated with 76107 conidia (Af) was measured using a 3 mm transwell chamber. (C) In vivo CXCL1 protein was measured in BALFs from 4 and 8 hr infections using an ELISA from AssayBiotech. (D) WT and HIFC BMDMs were incubated with A. fumigatus conidia in a 10:1 ratio for 8 hrs. mRNA abundance of cxcl1 was determined using quantitative RT-PCR, normalized to rpl13a, and relative to the HIFC sample (3 biological and 3 technical replicates). (E) In vivo CXCL2 and CXCL5 protein was measured in BALFs from 8 hr infections. (F) In vivo cytokine protein production was measured in BALFs from the same model in (B) using a cytokine luminex bead array from BioRad. (C,E) Data represent 3–4 biological replicates and depicted as mean plus SEM (N.D. = not detectable). * indicates a P value of ,0.03 and ** indicates a P value of ,0.05 (unpaired Students t test). doi:10.1371/journal.ppat.1004378.g006

Article Snippet: Commercially available ELISA kits for CXCL1 (Assay Biotech, OK-0189), CXCL2 (R&D systems, DY452), and CXCL5 (R&D Systems, DY443) were used according to the manufactures’ instructions.

Techniques: Migration, In Vivo, Enzyme-linked Immunosorbent Assay, Incubation, Quantitative RT-PCR, Luminex

Journal: Cell reports

Article Title: Identification of CCL3 as a Schwann cell chemotactic factor essential for nerve regeneration.

doi: 10.1016/j.celrep.2025.115322

Figure Lengend Snippet: Figure 2. CCL3 is a chemoattractant for Schwann cells (A) Rat SC migration in Boyden chambers in response to no factors (control), 3% serum (serum), or 10 or 50 ng/ml CCL3 diluted in 0.1% bovine serum albumin/ PBS (n = 8 independent experiments).

Article Snippet: Bridge fibroblasts, bridge macrophages, bridge CM, mouse macrophage cell line CM (from cells cultured at 20% O2,1.5% O2 alone or treated with scrambled short interfering RNA (Scr siRNA) or CCL3 siRNAs 1–3), recombinant rat CCL3 at 10 and 50 ng/ml (R&D systems) and recombinant human CCL3 at 50 ng/ml (R&D systems) were used to test the role of macrophage derived CCL3 in SCmigration.

Techniques: Migration, Control

Journal: Cell reports

Article Title: Identification of CCL3 as a Schwann cell chemotactic factor essential for nerve regeneration.

doi: 10.1016/j.celrep.2025.115322

Figure Lengend Snippet: Figure 4. CCL3 ablation impairs Schwann cell migration and axonal regrowth following injury (A and B) Representative confocal images of immunostained longitudinal sections of control and CCL3 KO mouse sciatic nerves at Day 7 after transection to detect SCs (eGFP, green, PLP- eGFP mice), axons (NF, white), and nuclei (Hoechst, blue). Dashed lines indicate the region of the nerve bridge. Scale bars, 300 mm. (C and D) Quantification of SC area (C) and axonal area (D) (n = 7 control mice, n = 8 CCL3 KO mice). (E and F) (E) Representative images of SC cords (eGFP, green) from the bridge showing organized, aligned cords of SCs in control mice, which are more disorganized in CCL3 KO animals. Scale bar, 100 mm, quantified in (F) (n = 169 SC cords from 7 control mice; n = 251 SC cords from 8 CCL3 KO mice). (G) Representative confocal images of longitudinal sections of control and CCL3 KO injured sciatic nerves at Day 5 following injury, immunostained for blood vessels (CD31, magenta). Scale bar, 300 mm. (H) Quantification of blood vessel (BV) area in the nerve bridge (n = 7 control mice, n = 7 CCL3 KO mice). Data are presented as mean ± SEM. For (C), (D), and (H), an unpaired two-tailed Student’s t test was used. For (F), an unpaired two-tailed Welch’s t test was used. *p < 0.05; **p < 0.01; ns, not significant. See also Figure S4.

Article Snippet: Bridge fibroblasts, bridge macrophages, bridge CM, mouse macrophage cell line CM (from cells cultured at 20% O2,1.5% O2 alone or treated with scrambled short interfering RNA (Scr siRNA) or CCL3 siRNAs 1–3), recombinant rat CCL3 at 10 and 50 ng/ml (R&D systems) and recombinant human CCL3 at 50 ng/ml (R&D systems) were used to test the role of macrophage derived CCL3 in SCmigration.

Techniques: Migration, Control, Two Tailed Test