Journal: Respiratory Research

Article Title: Gene profile of fibroblasts identify relation of CCL8 with idiopathic pulmonary fibrosis

doi: 10.1186/s12931-016-0493-6

Figure Lengend Snippet: Genes showing 10-fold or greater changes in gene expression between the IPF and control groups

Article Snippet: In addition, we measured the CCL8 protein amount in supernatants and cell lysates of the cultured fibroblasts (1 × 10 6 ) using CCL8 ELISA kit (Abnova, Taipei, Taiwan).

Techniques: Gene Expression, Control

Journal: Respiratory Research

Article Title: Gene profile of fibroblasts identify relation of CCL8 with idiopathic pulmonary fibrosis

doi: 10.1186/s12931-016-0493-6

Figure Lengend Snippet: CCL8 mRNA and protein levels in lung tissue-derived fibroblasts from 14 IPF patients and 10 controls. ( a ) RT-PCR, ( b ) densitometry of the CCL8 RT-PCR band intensity normalized to that of β-actin, ( c ) real-time PCR, and ( d ) correlations of the CCL8 mRNA levels of 12 subjects determined by the transcriptome chip with those by real-time PCR. e , f CCL8 protein level of Culture media and cell lysate, and ( g ) correlations of the CCL8 protein levels and CCL8 mRNA levels of 24 subjects determined by the ELISA with those by real-time PCR. The data were presented as median values with 25 and 75% quartiles

Article Snippet: In addition, we measured the CCL8 protein amount in supernatants and cell lysates of the cultured fibroblasts (1 × 10 6 ) using CCL8 ELISA kit (Abnova, Taipei, Taiwan).

Techniques: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Respiratory Research

Article Title: Gene profile of fibroblasts identify relation of CCL8 with idiopathic pulmonary fibrosis

doi: 10.1186/s12931-016-0493-6

Figure Lengend Snippet: CCL8 protein concentrations in BAL fluids and ROC curves. a CCL8 protein was detected in 25 of 41 normal controls, 80 of 86 IPF patients, 11 of 22 NSIP patients, 8 of 20 HP patients and 13 of 19 sarcoidosis patients. Open and closed circles indicate CCL8 protein levels detected (>1.5pg/mL) and those below the lower limit of detection, respectively. The data were presented as median values with 25 and 75% quartiles. b ROC curve of the CCL8 protein concentration between the two groups. A cut-off value of 2.17pg/mL had 80.2% accuracy, 86.0% specificity, and 65.7% sensitivity for differentiating IPF patients from controls. c ROC curve of the CCL8 protein concentration between the other interstitial lung diseases groups. A cut-off value 0.53pg/mL had 70.7% accuracy, 91.9% specificity, 57.4% sensitivity between the IPF patients and the other interstitial lung diseases group. d A Kaplan-Meier plot of 69 subjects with IPF followed up for 1to 8 years. The percent survival rate was markedly lower in the group with a CCL8 level of >28.61 pg/mL (red line) compared with that in the group with a CCL8 level of <28.61 pg/mL (black line) (hazard ratio = 3.93, CI: 1.25–12.39, p = 0.012)

Article Snippet: In addition, we measured the CCL8 protein amount in supernatants and cell lysates of the cultured fibroblasts (1 × 10 6 ) using CCL8 ELISA kit (Abnova, Taipei, Taiwan).

Techniques: Protein Concentration

Journal: Respiratory Research

Article Title: Gene profile of fibroblasts identify relation of CCL8 with idiopathic pulmonary fibrosis

doi: 10.1186/s12931-016-0493-6

Figure Lengend Snippet: Representative double immunofluorescence-stained images of IPF and control lung tissues. CCL8 and α-smooth muscle actin (α-SMA) were stained using PE- (red) and FITC-conjugated antibodies (green), respectively. A proportion of interstitial fibroblasts (IT) and the peribronchial and vascular area (VS) showed staining for both CCL8 and α-SMA (magnification, 200×)

Article Snippet: In addition, we measured the CCL8 protein amount in supernatants and cell lysates of the cultured fibroblasts (1 × 10 6 ) using CCL8 ELISA kit (Abnova, Taipei, Taiwan).

Techniques: Immunofluorescence, Staining, Control

Journal: Respiratory Research

Article Title: Gene profile of fibroblasts identify relation of CCL8 with idiopathic pulmonary fibrosis

doi: 10.1186/s12931-016-0493-6

Figure Lengend Snippet: Genes showing 10-fold or greater changes in gene expression between the IPF and control groups

Article Snippet: The sections were incubated in Fc receptor blocker (InnovexBiosciences, Richmond, CA, USA) for 30 min, incubated in TBS with 5% BSA for 1 h to block non-specific binding, and then incubated with monoclonal anti–human CCL8 antibody (1:100, Origene, Rockville, MD, USA) or polyclonal anti-human α-SMA antibody (1:200, Abcam, Cambridge, MA, USA) in 5% BSA overnight at 4 °C.

Techniques: Gene Expression, Control

Journal: Respiratory Research

Article Title: Gene profile of fibroblasts identify relation of CCL8 with idiopathic pulmonary fibrosis

doi: 10.1186/s12931-016-0493-6

Figure Lengend Snippet: CCL8 mRNA and protein levels in lung tissue-derived fibroblasts from 14 IPF patients and 10 controls. ( a ) RT-PCR, ( b ) densitometry of the CCL8 RT-PCR band intensity normalized to that of β-actin, ( c ) real-time PCR, and ( d ) correlations of the CCL8 mRNA levels of 12 subjects determined by the transcriptome chip with those by real-time PCR. e , f CCL8 protein level of Culture media and cell lysate, and ( g ) correlations of the CCL8 protein levels and CCL8 mRNA levels of 24 subjects determined by the ELISA with those by real-time PCR. The data were presented as median values with 25 and 75% quartiles

Article Snippet: The sections were incubated in Fc receptor blocker (InnovexBiosciences, Richmond, CA, USA) for 30 min, incubated in TBS with 5% BSA for 1 h to block non-specific binding, and then incubated with monoclonal anti–human CCL8 antibody (1:100, Origene, Rockville, MD, USA) or polyclonal anti-human α-SMA antibody (1:200, Abcam, Cambridge, MA, USA) in 5% BSA overnight at 4 °C.

Techniques: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Respiratory Research

Article Title: Gene profile of fibroblasts identify relation of CCL8 with idiopathic pulmonary fibrosis

doi: 10.1186/s12931-016-0493-6

Figure Lengend Snippet: CCL8 protein concentrations in BAL fluids and ROC curves. a CCL8 protein was detected in 25 of 41 normal controls, 80 of 86 IPF patients, 11 of 22 NSIP patients, 8 of 20 HP patients and 13 of 19 sarcoidosis patients. Open and closed circles indicate CCL8 protein levels detected (>1.5pg/mL) and those below the lower limit of detection, respectively. The data were presented as median values with 25 and 75% quartiles. b ROC curve of the CCL8 protein concentration between the two groups. A cut-off value of 2.17pg/mL had 80.2% accuracy, 86.0% specificity, and 65.7% sensitivity for differentiating IPF patients from controls. c ROC curve of the CCL8 protein concentration between the other interstitial lung diseases groups. A cut-off value 0.53pg/mL had 70.7% accuracy, 91.9% specificity, 57.4% sensitivity between the IPF patients and the other interstitial lung diseases group. d A Kaplan-Meier plot of 69 subjects with IPF followed up for 1to 8 years. The percent survival rate was markedly lower in the group with a CCL8 level of >28.61 pg/mL (red line) compared with that in the group with a CCL8 level of <28.61 pg/mL (black line) (hazard ratio = 3.93, CI: 1.25–12.39, p = 0.012)

Article Snippet: The sections were incubated in Fc receptor blocker (InnovexBiosciences, Richmond, CA, USA) for 30 min, incubated in TBS with 5% BSA for 1 h to block non-specific binding, and then incubated with monoclonal anti–human CCL8 antibody (1:100, Origene, Rockville, MD, USA) or polyclonal anti-human α-SMA antibody (1:200, Abcam, Cambridge, MA, USA) in 5% BSA overnight at 4 °C.

Techniques: Protein Concentration

Journal: Respiratory Research

Article Title: Gene profile of fibroblasts identify relation of CCL8 with idiopathic pulmonary fibrosis

doi: 10.1186/s12931-016-0493-6

Figure Lengend Snippet: Representative double immunofluorescence-stained images of IPF and control lung tissues. CCL8 and α-smooth muscle actin (α-SMA) were stained using PE- (red) and FITC-conjugated antibodies (green), respectively. A proportion of interstitial fibroblasts (IT) and the peribronchial and vascular area (VS) showed staining for both CCL8 and α-SMA (magnification, 200×)

Article Snippet: The sections were incubated in Fc receptor blocker (InnovexBiosciences, Richmond, CA, USA) for 30 min, incubated in TBS with 5% BSA for 1 h to block non-specific binding, and then incubated with monoclonal anti–human CCL8 antibody (1:100, Origene, Rockville, MD, USA) or polyclonal anti-human α-SMA antibody (1:200, Abcam, Cambridge, MA, USA) in 5% BSA overnight at 4 °C.

Techniques: Immunofluorescence, Staining, Control

Journal: Respiratory Research

Article Title: USP18 confers protection against allergic asthma by suppressing CCL8 production in alveolar type II epithelial cells

doi: 10.1186/s12931-025-03433-0

Figure Lengend Snippet: CCL8 is upregulated in asthma. a and b Gene Ontology (GO) enrichment analysis of molecular function based on RNA-seq data from AT2 cells in the OVA- ( a ) or HDM-induced ( b ) mice, compared with the AT2 cells from the PBS-treated controls. c Venn diagram showing the overlap between DEGs in the OVA and HDM groups, DEGs in the public GSE74075 dataset of patients with asthma, and genes encoding secreted proteins from the GeneCards database. d Heatmap illustrating the mRNA expression of asthma-related inflammatory factors in the AT2 cells isolated from the lung tissues of the indicated mice ( n = 2 or 3). e CCL8 mRNA levels in the induced sputum of patients with asthma compared with the healthy controls in the public GSE74075 dataset. f Correlation between levels of USP18 expression and CCL8 expression in asthma ( GSE69497 , n = 51). g Heatmap depicting Ccl8 mRNA expression in the lung leukocytes, mesenchymal cells, endothelial cells, airway epithelial cells, and AT2 cells from PBS, OVA, and HDM mice ( n = 3). h The BALF CCL8 concentrations in mice treated with PBS, OVA or HDM were measured by ELISA. i Immunofluorescence examination of CCL8 expression in the lung tissues. CCL8 (red), AT2 cell marker SFTPC (green), cell nucleus (DAPI, blue). Scale bars, 50 μm and 20 μm. j Immunohistochemical analysis of CCL8 expression in the lung tissues from OVA- or HDM-induced mice. Scale bars, 250 μm and 50 μm. Data shown are from one representative replicate. Quantitative data are presented as the mean ± SD, with the number of biological replicates per group and the P values indicated (unpaired, two-tailed Student’s t -test; g)

Article Snippet: Imject Alum adjuvant (77161), Lipofectamine 3000 (L3000015), and the mouse IgE ELISA kit (88–50460-22) were acquired from Thermo Fisher Scientific, the mouse CCL8 ELISA kit (DY790) was purchased from R&D Systems, and recombinant murine CCL8 (CS45) was sourced from NovoProtein.

Techniques: RNA Sequencing, Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Marker, Immunohistochemical staining, Two Tailed Test

Journal: Respiratory Research

Article Title: USP18 confers protection against allergic asthma by suppressing CCL8 production in alveolar type II epithelial cells

doi: 10.1186/s12931-025-03433-0

Figure Lengend Snippet: USP18 deficiency aggravates asthmatic inflammation via increasing CCL8 production. a Immunofluorescence analysis of CCL8 in AT2 cells in the lung tissues. CCL8 is depicted in red, the AT2 cell marker SFTPC in green, and the cell nucleus stained with DAPI in blue. Scale bars, 50 μm and 20 μm. b The serum IgE concentrations were measured by ELISA. c Flow cytometric analysis of eosinophils (Eos, CD11c − Siglec-F + ), and alveolar macrophages (AMs, CD11c − Siglec-F + ) in the BALF of OVA model mice. d and e The percentages ( d ) and absolute numbers ( e ) of eosinophils and AMs from the panel c . f The percentages of Th2 cells from the panel g . g Flow cytometric analysis of Th2 cells (CD45 + CD3 + CD4 + CD8 − GATA3 + ) in the lung tissues from the OVA and HDM models. h Immunofluorescence analysis of MUC5AC expression in the lung tissues. Scale bar, 50 μm. i The lung tissues from the indicated mice were stained with Hematoxylin and eosin (H&E), Periodic acid-Schiff (PAS), and Masson’s trichrome. Arrowheads highlight inflammatory infiltration in H&E staining, goblet cells containing mucus (magenta) in PAS staining, and collagen fibers in Masson’s trichrome staining. Scale bars, 2000 μm and 100 μm. Data shown are from one representative replicate. Quantitative data are presented as the mean ± SD, with the number of biological replicates per group and the P values indicated (two-way ANOVA with Sidak’s post hoc analysis; b and d - f )

Article Snippet: Imject Alum adjuvant (77161), Lipofectamine 3000 (L3000015), and the mouse IgE ELISA kit (88–50460-22) were acquired from Thermo Fisher Scientific, the mouse CCL8 ELISA kit (DY790) was purchased from R&D Systems, and recombinant murine CCL8 (CS45) was sourced from NovoProtein.

Techniques: Immunofluorescence, Marker, Staining, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Respiratory Research

Article Title: USP18 confers protection against allergic asthma by suppressing CCL8 production in alveolar type II epithelial cells

doi: 10.1186/s12931-025-03433-0

Figure Lengend Snippet: CCL8 drives asthma development. a Schematic overview of experimental design for CCL8 treatment. b Immunofluorescence analysis of CCL8 in AT2 cells in the lung tissues. CCL8 (red), AT2 cell marker SFTPC (green), cell nucleus (DAPI, blue). Scale bars, 50 μm and 20 μm. c Schematic diagram for flow cytometry analysis of the lung tissue cells for the panels d and j . d Flow analysis of alveolar macrophages (AMs, CD45 + CD64 + CD11c + Siglec-F + ), neutrophils (Neu, CD45 + CD64 − Ly6G + CD11b + ), and eosinophils (Eos, CD45 + CD64 − CD11c − Siglec-F + ) ( d ) from the lung tissues of PBS and CCL8 mice. The percentages are indicated in the flow plots. Data are representative of three independent experiments. e – g Quantification of the percentages of AMs e , neutrophils ( f ), and eosinophils ( g ) in the lung tissues as shown in d . h and i Total cell numbers ( h ) and total protein concentrations ( i ) in the bronchoalveolar lavage fluid (BALF). j Flow analysis of Th2 cells (CD45 + CD3 + CD4 + CD8 − GATA3 + ) in the lung tissues of PBS and CCL8 mice. The percentages are indicated in the flow plots. Data are representative of three independent experiments. k Quantification of the percentages of Th2 cells in the lung tissues as shown in j . l Immunofluorescence analysis of MUC5AC expression in the lung tissues. Scale bar, 50 μm. m Hematoxylin and eosin (H&E), Periodic acid-Schiff (PAS), and Masson’s trichrome-stained the lung tissues from the indicated mice. Arrowheads highlight inflammatory infiltration in H&E staining, goblet cells with mucus (magenta) in PAS staining, and collagen fibers in Masson’s trichrome staining. Scale bars, 2000 μm and 100 μm. Data shown are from one representative replicate. Quantitative data are presented as the mean ± SD, with the number of biological replicates per group and the P values indicated (unpaired, two-tailed Student’s t -test; e-i, and k)

Article Snippet: Imject Alum adjuvant (77161), Lipofectamine 3000 (L3000015), and the mouse IgE ELISA kit (88–50460-22) were acquired from Thermo Fisher Scientific, the mouse CCL8 ELISA kit (DY790) was purchased from R&D Systems, and recombinant murine CCL8 (CS45) was sourced from NovoProtein.

Techniques: Immunofluorescence, Marker, Flow Cytometry, Expressing, Staining, Two Tailed Test

Journal: Respiratory Research

Article Title: USP18 confers protection against allergic asthma by suppressing CCL8 production in alveolar type II epithelial cells

doi: 10.1186/s12931-025-03433-0

Figure Lengend Snippet: Knockdown CCL8 alleviates Th2 inflammation and maintains epithelial integrity in the USP18-knockout mice. a Immunofluorescence analysis of E-cadherin, claudin1, and ZO-1 expression in AT2 cells in the lung tissues. AT2 cell marker SFTPC (green), cell nucleus (DAPI, blue). Scale bar, 100 μm. b Immunofluorescence analysis of vimentin and fibronectin expression in AT2 cells in the lung tissues. AT2 cell marker SFTPC (green), cell nucleus (DAPI, blue). Scale bar, 100 μm. c Schematic diagram illustrating AT2 cell-specific CCL8 knockdown in USP18-deficient mice within the OVA-induced asthma model. d Immunofluorescence analysis of CCL8 in AT2 cells in the lung tissues. CCL8 is shown in red, the AT2 cell marker SFTPC is green, and cell nuclei are stained with DAPI (blue). Scale bars, 50 μm and 20 μm. e Flow cytometry analysis of eosinophils (Eos, CD11c − Siglec-F + ), and alveolar macrophages (AMs, CD11c − Siglec-F + ) in the BALF. f and g Relative frequencies ( f ) and total cell numbers ( g ) of eosinophils and AMs from e . h Flow cytometry analysis of and Th2 cells (CD45 + CD3 + CD4 + CD8 − GATA3 + ) in the lung tissues. i The percentages of Th2 cells from h . j ELISA measurements of serum IgE levels. k MUC5AC expression in the lung tissues is assessed by immunofluorescence. Scale bar, 50 μm. l Immunofluorescence analysis of E-cadherin, claudin1, and ZO-1 expression in AT2 cells in the lung tissues. AT2 cell marker SFTPC (green), cell nucleus (DAPI, blue). Scale bar, 100 μm. m Immunofluorescence analysis of vimentin and fibronectin expression in the lung tissues. AT2 cells in the lung tissues. AT2 cell marker SFTPC (green), cell nucleus (DAPI, blue). Scale bar, 100 μm. n Hematoxylin and eosin (H&E), Periodic acid-Schiff (PAS), and Masson’s trichrome-stained lung tissues from the depicted mice. Arrowheads indicate areas of inflammatory infiltration in H&E staining, goblet cells containing mucus (magenta) in PAS staining, and collagen fibers in Masson staining. Scale bars, 2000 μm and 100 μm. Data shown are from one representative replicate. Quantitative data are presented as the mean ± SD, with the number of biological replicates per group and the P values indicated (unpaired, two-tailed Student’s t -test; f, g, i, and j)

Article Snippet: Imject Alum adjuvant (77161), Lipofectamine 3000 (L3000015), and the mouse IgE ELISA kit (88–50460-22) were acquired from Thermo Fisher Scientific, the mouse CCL8 ELISA kit (DY790) was purchased from R&D Systems, and recombinant murine CCL8 (CS45) was sourced from NovoProtein.

Techniques: Knockdown, Knock-Out, Immunofluorescence, Expressing, Marker, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: iScience

Article Title: Behenic acid alleviates inflammation and insulin resistance in gestational diabetes mellitus by regulating TLR4/NF-κB signaling pathway

doi: 10.1016/j.isci.2024.111019

Figure Lengend Snippet:

Article Snippet: Mouse MCP2 (Monocyte Chemotactic Protein 2 ELISA Kit , ELK Biotechnology, China , Cat#ELK8888.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, RNA Extraction, SYBR Green Assay, CCK-8 Assay, Software