Journal: Nature Communications

Article Title: The CCL20–integrin α5β1 interaction enhances TGF-β/Smad signaling to promote fibroblast activation in pulmonary fibrosis

doi: 10.1038/s41467-025-64211-6

Figure Lengend Snippet: a List of the 10 proteins that interact with CCL20 identified by MS analyses. b Volcano plot of enriched proteins that interact with CCL20 (n = 3 technical replicates per group). Significantly altered proteins are shown in red (false discovery rate (FDR) < 0.05, log 2 FC > 1) and blue (FDR < 0.05, log 2 FC < −1). c Identification of integrin α5 and integrin β1 from CCL20 immunoprecipitates in fibroblasts by MS analysis. d Surface plasmon resonance (SPR) analysis of the kinetic interaction between CCL20 and integrin α5β1. CoIP analysis of the interaction between CCL20 and integrin α5 ( e ) or integrin β1 ( f ) in HEK293T cells. IF images showing the colocalization of CCL20 with integrin α5 ( g ) or integrin β1 ( h ) in MRC-5 cells. Scale bars, 10 μm. i Expression of Col1a1, Col3a1, Fibronectin and Acta2 in primary fibroblasts treated with or without ATN-161 in the presence of CCL20. j Immunoblots showing COL1 and α-SMA levels in primary fibroblasts treated with or without ATN-161 in the presence of CCL20. k Migratory ability of primary fibroblasts treated with or without ATN-161 in the presence of CCL20. Scale bars, 100 μm. l Expression of ACTA2 in MRC-5 cells treated with or without ATN-161 in the presence of CCL20. m Immunoblots showing the levels of COL1 and α-SMA in MRC-5 cells treated with or without ATN-161 in the presence of CCL20. n Migratory ability of MRC-5 cells treated with or without ATN-161 in the presence of CCL20. Scale bars, 100 μm. The data are presented as the means ± SEM. For ( i–n ), n = 3 technical replicates per group. The data in i–n were analyzed by one-way ANOVA. *p < 0.05 ; **p < 0.01; ***p < 0.001. Source data are provided as a file.

Article Snippet: Myc-DDK-tagged integrin α5 was obtained from Origene, Myc-tagged integrin β1 and GFP-tagged human CCL20 were obtained from YouBio (Changsha, China), and HA-tagged CCR6 was purchased from Sino Biological, Inc. (Beijing, China).

Techniques: SPR Assay, Expressing, Western Blot

Journal: Nature Communications

Article Title: The CCL20–integrin α5β1 interaction enhances TGF-β/Smad signaling to promote fibroblast activation in pulmonary fibrosis

doi: 10.1038/s41467-025-64211-6

Figure Lengend Snippet: a CoIP analysis of the interaction between LAP and integrin α5. b Ratio of active TGF-β to total TGF-β in the culture medium of CCL20-treated MRC-5 cells, as determined by ELISA (n = 3 technical replicates per group). c TGF-β-reporter luciferase activity in HEK293T cells in response to the cell supernatant of fibroblasts treated with or without CCL20 (n = 4 technical replicates per group). d Immunoblots showing the levels of phosphorylated and total Smad3 or Smad2 in primary fibroblasts following CCL20 stimulation (n = 3 technical replicates per group). e Immunoblots showing the levels of phosphorylated and total Smad3 or Smad2 in MRC-5 cells following CCL20 stimulation (n = 3 technical replicates per group). f IF staining for phosphorylated Smad3 in primary fibroblasts stimulated with CCL20. Scale bars, 20 μm. g IF staining for phosphorylated Smad3 in MRC-5 cells stimulated with CCL20. Scale bars, 20 μm. h Immunoblots showing the levels of phosphorylated and total Smad3 or Smad2 in primary fibroblasts treated with or without ATN-161 in the presence of CCL20 (n = 3 technical replicates per group). i IF staining for phosphorylated Smad3 in primary fibroblasts treated with or without ATN-161 in the presence of CCL20. Scale bars, 20 μm. j Immunoblots showing the levels of phosphorylated and total Smad3 or Smad2 in MRC-5 cells treated with or without ATN-161 in the presence of CCL20 (n = 3 technical replicates per group). k IF staining for phosphorylated Smad3 in MRC-5 cells treated with or without ATN-161 in the presence of CCL20. Scale bars, 20 μm. l IF staining for α-SMA in primary fibroblasts treated with or without SIS3 in the presence of CCL20. Scale bars, 20 μm. m IF staining for α-SMA in MRC-5 cells treated with or without SIS3 in the presence of CCL20. Scale bars, 20 μm. n Expression of Acta2 in primary fibroblasts treated with or without SIS3 in the presence of CCL20 (n = 3 technical replicates per group). o Expression of ACTA2 in MRC-5 cells treated with or without SIS3 in the presence of CCL20 (n = 3 technical replicates per group). p Schematic diagram of the method used to evaluate the therapeutic effects of ATN-161 on the PF model. q Hydroxyproline levels in lung tissues from the indicated mice after BLM exposure (n = 7 samples for BLM + PBS, BLM + PBS + ATN161, BLM + CCL20 + ATN161, n = 6 samples for BLM + CCL20 + PBS). r Masson’s trichrome staining of lung tissues from the indicated mice after BLM exposure. Scale bar, 100 μm. s The expression levels of Fibronectin and Acta2 in lung tissues from the indicated mice after BLM exposure (n = 6 mice per group). t Immunoblots showing the levels of phosphorylated and total Smad3 or Smad2 in primary lung fibroblasts isolated from the indicated mice (n = 3 samples per group). The data are presented as the means ± SEM. The data in b–e , s , t were analyzed via two-tailed Student’s t -test. The data in h–j , n–o , q were analyzed via one-way ANOVA. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001. Source data are provided as a file.

Article Snippet: Myc-DDK-tagged integrin α5 was obtained from Origene, Myc-tagged integrin β1 and GFP-tagged human CCL20 were obtained from YouBio (Changsha, China), and HA-tagged CCR6 was purchased from Sino Biological, Inc. (Beijing, China).

Techniques: Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Western Blot, Staining, Expressing, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: The CCL20–integrin α5β1 interaction enhances TGF-β/Smad signaling to promote fibroblast activation in pulmonary fibrosis

doi: 10.1038/s41467-025-64211-6

Figure Lengend Snippet: a SPR analysis of the kinetic interaction of Pep-CCL20 and integrin α5β1. b IP analysis of the interaction between CCL20 and integrin α5 or integrin β1 in the presence of Pep-CCL20. c TGF-β-reporter luciferase activity in HEK293T cells exposed to the supernatant of fibroblasts subjected to the indicated treatments. d Expression of ACTA2 in primary fibroblasts treated with or without Pep-CCL20 in response to CCL20 stimulation. e Immunoblot showing the levels of COL1 in primary fibroblasts treated with or without Pep-CCL20 in response to CCL20 stimulation. f Migration analysis of primary fibroblasts treated with or without Pep-CCL20 in response to CCL20 stimulation. Scale bars, 100 μm. g Expression of ACTA2 in MRC-5 cells treated with or without Pep-CCL20 in response to CCL20 stimulation. h Immunoblot showing the levels of COL1 in MRC-5 cells treated with or without Pep-CCL20 in response to CCL20 stimulation. i Migration analysis of fibroblasts treated with or without Pep-CCL20 in response to CCL20 stimulation. Scale bars, 100 μm. j Schematic diagram of the method for evaluating the therapeutic effects of Pep-CCL20 on the PF model. Masson’s trichrome staining ( k ), hydroxyproline content ( l ) (n = 6 samples per group), protein and mRNA expression of genes associated with fibrosis ( m , n ), and IHC staining for phosphorylated Smad3 ( o ) in lung tissues from PBS- or Pep-CCL20-treated PF mice (n = 6 samples per group). Scale bars, 100 μm ( k ) and 50 μm ( m, o ). The data are presented as the means ± SEM. For ( c–i ), n = 3 technical replicates per group. For ( m–o ), n = 3 samples per group. The data in m , o were analyzed via two-tailed Student’s t -test. The data in c–i , l were analyzed by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001. Source data are provided as a file.

Article Snippet: Myc-DDK-tagged integrin α5 was obtained from Origene, Myc-tagged integrin β1 and GFP-tagged human CCL20 were obtained from YouBio (Changsha, China), and HA-tagged CCR6 was purchased from Sino Biological, Inc. (Beijing, China).

Techniques: Luciferase, Activity Assay, Expressing, Western Blot, Migration, Staining, Immunohistochemistry, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: Integrin VLA-5 and FAK are Good Targets to Improve Treatment Response in the Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

doi: 10.3389/fonc.2014.00112

Figure Lengend Snippet: The expression level of integrin α5 subunit in Ph+ leukemia cell line SUP-B15 is significantly increased after serum starvation . (A) The percentages of cells expressing integrin α4, α5, and β1 subunits before and after serum starvation for 7 h detected by flow cytometry. (B) The mean fluorescent intensity (MFI) of integrin α4, α5, and β1 subunits from whole alive cell population before and after serum starvation for 7 h detected by flow cytometry.

Article Snippet: Human integrin α5 shRNA lentiviral particles (Catalog # sc-29372-V, Santa Cruz) were thawed at room temperature and added to leukemia cell suspension in 15 ml conical tubes and were spun at 800 × g (2500 rpm) for 90 min at 37°C.

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: Integrin VLA-5 and FAK are Good Targets to Improve Treatment Response in the Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

doi: 10.3389/fonc.2014.00112

Figure Lengend Snippet: Integrin α5 subunit antibody inhibits the adhesion of Ph+ leukemia cells to human fibronectin and enhances the killing of imatinib . (A) Cell adhesion assay showed that α5 subunit inhibitory antibody (CD49e, clone IIA1 BD Biosciences) was the only tested integrin antibody that significantly inhibited the adhesion of Ph+ leukemia cells to fibronectin with adhesion percentage of 6.6 ± 3.8% compared with control IgG of 44.8 ± 7.9%, p < 0.01. (B) . Annexin-V plus PI by flow cytometry. (i) P2 gate identifies the CD38 positive cell population representing SUP-B15 Ph+ leukemia cells. (ii) Ph+ leukemia cells grown on HS-5 stromal cells. (iii) Ph+ leukemia cells treated with 10 μM imatinib (IM). (iv) SUP-B15 cultured with stromal cells treated with 10 μM imatinib. (v) SUP-B15 cultured with stromal cells treated with 10 μM imatinib and 10 μg/ml anti-α5 Ab. (C) Quantitative analysis for apoptosis rate of different conditions. *After culture on HS-5 cells for 24 h, the apoptosis rate of leukemia cells SUP-B15 decreased from 29.4 ± 2.3 to 16.7 ± 3%, p < 0.05. # When inhibitory antibody to integrin α5 was combined with imatinib to treat Ph+ leukemia cells cultured on stromal cells, the apoptosis rate 38.0 ± 8.0% was significantly increased compared with imatinib by itself 25.7 ± 3.3%, p < 0.05.

Article Snippet: Human integrin α5 shRNA lentiviral particles (Catalog # sc-29372-V, Santa Cruz) were thawed at room temperature and added to leukemia cell suspension in 15 ml conical tubes and were spun at 800 × g (2500 rpm) for 90 min at 37°C.

Techniques: Cell Adhesion Assay, Control, Flow Cytometry, Cell Culture

Journal: Frontiers in Oncology

Article Title: Integrin VLA-5 and FAK are Good Targets to Improve Treatment Response in the Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

doi: 10.3389/fonc.2014.00112

Figure Lengend Snippet: Blocking integrin α5 affects the engraftment of Ph+ leukemia cells in immunodeficient mice . (A) The incubation for 1 h of Ph+ leukemia cells with disintegrin, a peptide inhibitor of integrins, impaired the engraftment of leukemia in NSG mice. Representative figures of n = 2 showed bioluminescence imaging from day 7 to 28 after inoculation of leukemia cells. (B) Animal total body bioluminescence was measured using the Xenogen IVIS Imaging System 200 Series with total imaging time of 2 min and compared with control animals that received cells that were not treated with Echistatin. (C) Anti-integrin α5 inhibitory antibodies clone IIA1 (BD Biosciences) and clone P1D6 (Millipore) decreased the engraftment of Ph+ leukemia cells in the bone marrow of NOD/SCID mice ( n = 2).

Article Snippet: Human integrin α5 shRNA lentiviral particles (Catalog # sc-29372-V, Santa Cruz) were thawed at room temperature and added to leukemia cell suspension in 15 ml conical tubes and were spun at 800 × g (2500 rpm) for 90 min at 37°C.

Techniques: Blocking Assay, Incubation, Imaging, Control

Journal: Frontiers in Oncology

Article Title: Integrin VLA-5 and FAK are Good Targets to Improve Treatment Response in the Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

doi: 10.3389/fonc.2014.00112

Figure Lengend Snippet: Knocking down integrin α5 delayed the engraftment of Ph+ leukemia cells in immunodeficient NOD/SCID mice . (A) Real-time QPCR assay showed that we had successfully knocked down the expression level of the integrin α5 knocking in clone 10. The cycle threshold (Ct) of the target gene (integrin α5) was normalized to the chosen reference gene GAPHD. Relative quantification, R = 2 −(ΔCt sample − ΔCt control) . Result shows the fold change normalized to SUP-LUC2 cells. (B) Western blot showed a reduced protein expression level of integrin α5 in clone 10. (C) Flow cytometry showed reduced mean fluorescent intensity of integrin α5 in clone 10. (D) Representative figures showed bioluminescent imaging at 2 weeks, and 5–8 weeks post leukemia cells inoculation. (E) Animal total body bioluminescence was quantified to compare with control. **The levels of bioluminescence became most significantly different 2 months post inoculation between the α5 knock-down group and control group [mean (SD) radiance vs. control, 5.3 (0.1) vs. 2.1 (0.2) × 10 6 p/s/cm 2 /sr, p < 0.01 at 2 months, n = 3.].

Article Snippet: Human integrin α5 shRNA lentiviral particles (Catalog # sc-29372-V, Santa Cruz) were thawed at room temperature and added to leukemia cell suspension in 15 ml conical tubes and were spun at 800 × g (2500 rpm) for 90 min at 37°C.

Techniques: Expressing, Quantitative Proteomics, Control, Western Blot, Flow Cytometry, Imaging, Knockdown

Journal: Cell Reports

Article Title: Importin α5 Regulates Anxiety through MeCP2 and Sphingosine Kinase 1

doi: 10.1016/j.celrep.2018.11.066

Figure Lengend Snippet:

Article Snippet: Briefly, two different short hairpin RNA (shRNA) target sequences from the open reading frame of the mouse importin α5 gene (Kpna1) were cloned into shRNA expression cassettes using BamHI/PacI restriction sites driven by the u6 promoter in a modified third-generation lentiviral plasmid with hUBC-driven EGFP (FUGW was from the Baltimore lab at Caltech, Addgene plasmid #14883) with 3x-NLS RFP fluorescent reporter driven under human synapsin (hSyn) promoter.

Techniques: Plasmid Preparation, Recombinant, RNA Extraction, Purification, Expressing, Next-Generation Sequencing, shRNA, Software, Binding Assay