Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin

doi: 10.1152/ajplung.00087.2017

Figure Lengend Snippet: Cadmium (Cd) induces peribronchiolar fibrosis. A: mice (n = 5) were treated intratracheally with saline or CdCl2 (0.009 mg/kg body wt) every other day for 8 wk. Cryosections from weeks 1, 2, 4, and 8 mice lungs were stained with hematoxylin-eosin, α-smooth muscle actin (α-SMA), vimentin, collagen-1, or picro-sirius red. Representative photomicrographs from 5 animals on week 4 are shown, and peribronchiolar fibrosis is indicated by arrows. Bar = 100 μm. B: the peribronchiolar subepithelial layer in mice (n = 5) was measured and comparable to the saline control. Each symbol represents an individual subjects, and horizontal bars indicate the mean values. C: respiratory mechanics demonstrating increased airway resistance following methacholine challenge. ##P < 0.01 vs. saline mice with methacholine at 20 or 40 mg/ml. D: mice were treated as shown in A, and collagen content in the right lung at different time points was assessed using Sircol collagen assay. **P < 0.01 vs. saline control.

Article Snippet: Vimentin concentrations in mice plasma and BAL were quantified using a commercially available mouse vimentin enzyme-linked immunosorbent assays (ELISA) kit (MyBioSource, San Diego, CA) according to the manufacturer’s recommendation.

Techniques: Saline, Staining, Control, Sircol Collagen Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin

doi: 10.1152/ajplung.00087.2017

Figure Lengend Snippet: Cd induces phosphorylation of AKT and cdc2 as well as vimentin phosphorylation at Ser39 and Ser55. A and B: after 24 h of serum starvation, primary lung fibroblasts were treated with CdCl2 (20 μM) for the indicated times. Immunoblotting were performed using antibodies shown above. The results shown in A and B are representative of those from 3 independent experiments. Arrows represent specific band.

Article Snippet: Vimentin concentrations in mice plasma and BAL were quantified using a commercially available mouse vimentin enzyme-linked immunosorbent assays (ELISA) kit (MyBioSource, San Diego, CA) according to the manufacturer’s recommendation.

Techniques: Phospho-proteomics, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin

doi: 10.1152/ajplung.00087.2017

Figure Lengend Snippet: Both AKT and cdc2 inhibitors inhibit Cd-induced vimentin phosphorylation at Ser39 and Ser55, α-SMA activation, ECM accumulation, and collagen secretion. A; after 24 h of serum starvation, primary lung fibroblasts (n = 5) were pretreated with the MEK 1 inhibitor PD 985059 (PD; 20 μM), the PKC inhibitor G06983 (G0; 250 nM), the AKT inhibitor LY294002 (LY; 20 μM), the cdc2 inhibitor Roscovitine (RO; 10 μM) only, or combination of AKT and cdc2 inhibitors (LY + RO) 1 h before 20 μM CdCl2 treatment for 3 h and then allowed to recover for 48 h. The soluble collagen levels were evaluated by Sircol collagen assay. Horizontal bars indicate the mean values. **P < 0.01 vs. control. Cells were also treated with TGF-β (2 ng/ml) for 48 h as a positive control. B and C: cells were pretreated with inhibitor shown as above before 20 μM CdCl2 treatment for 2 h (B) or 3 h and then allowed to recover for 48 h (C), followed by immunoblot analysis for indicated antibodies. The results shown in A–C are representative of those from 3 independent experiments.

Article Snippet: Vimentin concentrations in mice plasma and BAL were quantified using a commercially available mouse vimentin enzyme-linked immunosorbent assays (ELISA) kit (MyBioSource, San Diego, CA) according to the manufacturer’s recommendation.

Techniques: Phospho-proteomics, Activation Assay, Sircol Collagen Assay, Control, Positive Control, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin

doi: 10.1152/ajplung.00087.2017

Figure Lengend Snippet: Cd-induced vimentin fragmentation and secretion can be inhibited by both AKT and cdc2 inhibitors. A: mice (n = 5) were treated intratracheally with saline or CdCl2 (0.009 or 0.018 mg/kg) as shown in Fig. 1. Plasma and bronchoalveolar lavage (BAL) were collected at week 4 and the vimentin levels were examined by ELISA. Horizontal bars indicate the mean values. B: primary lung fibroblasts were incubated with or without CdCl2 (20 μM) for 3 h and then allowed to recover for the indicated periods of time, and the supernatants were collected, concentrated by ultrafiltration and equal aliquots were analyzed by Western blotting using an anti-vimentin antibody. C: cells were pretreated with the AKT inhibitor (LY; 20 μM), the cdc2 inhibitor (RO; 10 μM), or a combination of AKT and cdc2 inhibitors (LY + RO) for 1 h, followed by 20 μM of CdCl2 treatment for 3 h and then allowed to recover for 48 h. Supernatants was analyzed as above. *P < 0.05, **P < 0.01. Arrows represent positions for the indicated molecular weight. The data are representative of 3 independent experiments.

Article Snippet: Vimentin concentrations in mice plasma and BAL were quantified using a commercially available mouse vimentin enzyme-linked immunosorbent assays (ELISA) kit (MyBioSource, San Diego, CA) according to the manufacturer’s recommendation.

Techniques: Saline, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Molecular Weight

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin

doi: 10.1152/ajplung.00087.2017

Figure Lengend Snippet: Vimentin is required for Cd-induced peribronchiolar fibrosis. A and B: IMR-90 cells were transfected with vimentin siRNA or NT-siRNA and followed by 20 μM of CdCl2 treatment for 2 h (A) or 3 h and then allowed to recover for 48 h (B). Western blots were performed using antibodies shown above. C: the soluble collagen levels were evaluated by Sircol collagen assay (n = 5). **P < 0.01. D: wild-type (WT) and Vimentin−/− mice (n = 5 of each group) were treated intratracheally with saline or CdCl2 (0.009 mg/kg body wt) every other day for 8 wk. Cryosections from weeks 1, 2, 4, and 8 mice lungs were stained with collagen-1 or picro-sirius red. Bar = 100 μm. Representative photomicrographs from 5 animals on week 4 are shown. E: 3,3′-diaminobenzidine staining density was quantified and the average positive area density was calculated from 10 random areas. Densities are denoted in arbitrary units. **P < 0.01. F: WT and Vimentin−/− mice (n = 5 of each group) were treated as above and airway resistance following methacholine challenge was measured. ##P < 0.01 vs. WT Cd mice with methacholine at 20 or 40 mg/ml. Horizontal bars indicate the mean values.

Article Snippet: Vimentin concentrations in mice plasma and BAL were quantified using a commercially available mouse vimentin enzyme-linked immunosorbent assays (ELISA) kit (MyBioSource, San Diego, CA) according to the manufacturer’s recommendation.

Techniques: Transfection, Western Blot, Sircol Collagen Assay, Saline, Staining

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin

doi: 10.1152/ajplung.00087.2017

Figure Lengend Snippet: Cd induces nuclear localization and association of Yes-associated protein 1 (YAP1) and Smad2/3. A: primary lung fibroblasts were incubated with or without CdCl2 (10 or 20 μM) or TGF-β (2 ng/ml) for 2 h and followed by immunoblot analysis with anti-P-Smad2/3 or anti-Smad2/3. B: cells were subjected to subcellular fractionation to obtain nuclear and cytoplasm fractions. Equivalent protein amounts of each fraction were separated by SDS-PAGE, and immunoblotted with anti-Smad2/3, YAP1, 14-3-3, or anti-vimentin. Tubulin, lamin, and β-actin were used as loading controls. C: cells were pretreated with AKT inhibitor (LY; 20 μM) and cdc2 inhibitor (RO; 10 μM) or phosphatase inhibitor [calf intestinal phosphatase (CIP); 10 units/ml] for 1 h and then stimulated with CdCl2 for 2 h, lysed, and subjected to YAP1 or vimentin immunoprecipitation followed by Smad2/3, 14-3-3, vimentin, or YAP1 Western analysis. The densities of protein bands were determined by densitometry and the data represent a 1-fold increase from the control density. **P < 0.01. Horizontal bars indicate the mean values. The results shown in A–C are representative of those from 3 independent experiments.

Article Snippet: Vimentin concentrations in mice plasma and BAL were quantified using a commercially available mouse vimentin enzyme-linked immunosorbent assays (ELISA) kit (MyBioSource, San Diego, CA) according to the manufacturer’s recommendation.

Techniques: Incubation, Western Blot, Fractionation, SDS Page, Immunoprecipitation, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin

doi: 10.1152/ajplung.00087.2017

Figure Lengend Snippet: Proposed model for the role of vimentin phosphorylation and YAP1 in Cd-induced peribronchiolar fibrosis, airway remodeling, and subsequent COPD. Prolonged exposure to Cd results in fibrosis around the airways. Cd-induced phosphorylation of AKT and cdc2 kinase is required for the vimentin phosphorylation at Ser39 and Ser55, respectively. Such phosphorylated vimentin will bind 14-3-3 and facilitate the nuclear translocation of YAP1 and Smad2/3 and complex formation of YAP1 and Smad2/3 in the nucleus, which activates myofibroblast and induces ECM protein deposition.

Article Snippet: Vimentin concentrations in mice plasma and BAL were quantified using a commercially available mouse vimentin enzyme-linked immunosorbent assays (ELISA) kit (MyBioSource, San Diego, CA) according to the manufacturer’s recommendation.

Techniques: Phospho-proteomics, Translocation Assay

Journal: Frontiers in Microbiology

Article Title: MLKL Mediated Necroptosis Accelerates JEV-Induced Neuroinflammation in Mice

doi: 10.3389/fmicb.2017.00303

Figure Lengend Snippet: MLKL deleted mice show decreased inflammatory cytokines during JEV infection . The serum and brain from each mice in MLKL −/− and wild group were collected at 5 dpi, and the main inflammatory cytokines in serum and brain were tested. (A) The level of CCL-2, IL-1β, IFN-γ, TNF-α in the serum of WT or MLKL −/− mice was detected by ELISA assay kit (WT-PBS = 2, MLKL −/− -PBS = 2, WT-JEV = 9, MLKL −/− -PBS = 9). (B) The expression of CCL-2, IL-1β, IFN-γ, TNF-α in the brain of WT or MLKL −/− mice was detected by qRT-PCR (WT-PBS = 2, MLKL −/− -PBS = 2, WT-JEV = 9, MLKL −/− -PBS = 9, * P < 0.05, *** P < 0.001).

Article Snippet: The level of IL-1β, CCL-2, IFN-γ, and TNF-α was tested with Mouse ELISA Kit (AMEKO, China) and the absorbance was measured at 450 nm.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: Induction of proinflammatory mediators by CHI3L1 is reduced by chitin treatment: decreased tumor metastasis in a breast cancer model

doi: 10.1002/ijc.26379

Figure Lengend Snippet: CHI3L1 increases the expression of CCL2, CXCL2 and MMP-9. Macrophages from normal mice were cultured overnight with either 1 ng/mL or 5 ng/mL rmCHI3L1 alone (A,C,E) or in combination with LPS (1 μg/mL) (B,D,F) and cell-free supernatants were analyzed by ELISA for CCL2 (A–B) ; CXCL2 (C–D) or MMP-9 (E–F). N= 10 mice/group, (A–B) *p<0.0015, **p<0.0001, (CD)*p<0.0005, ** p<0.0002, and (E–F) *p<0.004, **p<0.0016.

Article Snippet: Cell culture supernatants and sera from control and mammary tumor bearers were analyzed for protein expression by ELISA for CCL2, CXCL2 and IFN-γ (BD Biosciences, San Jose, CA), MMP-9 and CHI3L1 (R&D Systems) according to the manufacturer's instructions.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: Induction of proinflammatory mediators by CHI3L1 is reduced by chitin treatment: decreased tumor metastasis in a breast cancer model

doi: 10.1002/ijc.26379

Figure Lengend Snippet: Silencing CHI3L1 decreases secretion of proinflammatory molecules by macrophages. (A) In vitro treatment of LPS (1 μg/mL) stimulated macrophages from 5-week DA-3 tumor bearers with 50 nM CHI3L1 siRNA analyzed by qRT-PCR for CHI3L1 gene expression and protein expression as determined by ELISA (B). (C–E) Purified macrophages from 5-week DA-3 tumor bearers were cultured with 50 nM CHI3L1 siRNA or 50 NM nontarget siRNA in the presence of LPS and cell-free culture supernatants analyzed for CCL2 (C), CXCL2 (D) and MMP-9 (E). N = 5 mice/group, *p < 0.05, Student's t-test

Article Snippet: Cell culture supernatants and sera from control and mammary tumor bearers were analyzed for protein expression by ELISA for CCL2, CXCL2 and IFN-γ (BD Biosciences, San Jose, CA), MMP-9 and CHI3L1 (R&D Systems) according to the manufacturer's instructions.

Techniques: In Vitro, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Purification, Cell Culture

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: Induction of proinflammatory mediators by CHI3L1 is reduced by chitin treatment: decreased tumor metastasis in a breast cancer model

doi: 10.1002/ijc.26379

Figure Lengend Snippet: In vivo treatment with chitin decreases CHI3L1, CCL2, CXCL2 and MMP-9 expression in DA-3 tumor-bearing mice. (A) Serum from 2- and 5-week DA-3 tumor bearers either untreated or chitin treated (1 mg/mouse) was analyzed for CHI3L1 expression by ELISA. (B–C) Splenocytes (B) and splenic macrophages (C) from untreated or chitin treated 5-week DA-3 tumor-bearing mice were cultured overnight in the presence or absence of LPS (1 μg/mL) and cell-free supernatants were analyzed for CHI3L1 by ELISA. (D–F) Purified macrophages and T cells from spleens of 5-week DA-3 tumor-bearing mice, untreated, or chitin treated were cultured overnight with mitogens as described, and cell-free supernatants were analyzed by ELISA for CCL2 (D), CXCL2 (E) and MMP-9 (F). Data shown are the results of three independent experiments with N= 4 mice/group; *p<0.003, **p<0.002, Student's t-test

Article Snippet: Cell culture supernatants and sera from control and mammary tumor bearers were analyzed for protein expression by ELISA for CCL2, CXCL2 and IFN-γ (BD Biosciences, San Jose, CA), MMP-9 and CHI3L1 (R&D Systems) according to the manufacturer's instructions.

Techniques: In Vivo, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Purification