Journal: Antioxidants (Basel, Switzerland)

Article Title: Inhibition of Oxidative Stress and ALOX12 and NF-κB Pathways Contribute to the Protective Effect of Baicalein on Carbon Tetrachloride-Induced Acute Liver Injury.

doi: 10.3390/antiox10060976

Figure Lengend Snippet: Figure 2. Protein expression levels involved ferroptosis were detected in the liver tissues of mice challenged with CCl4. (A) Protein expression levels of ALOX12, HO-1, COX-2, p21 and Nrf2 at 6 and 24 h identified by Western blotting analysis of liver tissues of mice treated with CCl4; the quantitative analysis is showed in (B) (n = 4). 6 h vs. ctrl, * p < 0.05, ** p < 0.01; 24 h vs. ctrl, # p < 0.05, ## p < 0.01. Ctrl: control.

Article Snippet: The following primary antibodies were used to probe the membranes: primary rabbit antibodies against p21 (1:1000), COX2 (1:1000), Nrf2 (1:1000), and HO-1 (1:1000) (ProteinTech Group, Inc., Chicago, IL, USA), mouse monoclonal antibody against ALOX12 (1:1000) (Abcam, Cabridge, MA, USA) and β-actin (1:1000) (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Western Blot, Control

Journal: Antioxidants (Basel, Switzerland)

Article Title: Inhibition of Oxidative Stress and ALOX12 and NF-κB Pathways Contribute to the Protective Effect of Baicalein on Carbon Tetrachloride-Induced Acute Liver Injury.

doi: 10.3390/antiox10060976

Figure Lengend Snippet: Figure 6. Effects of baicalein supplementation on apoptotic pathway in the liver tissues of mice challenged with CCl4. (A) Representative TUNEL-stained sections showing apoptosis in the liver tissue of mice. (B) Quantitative analysis of TUNEL positive rates (n = 4). (C,D) Activities of (C) caspases-3 and -9 (D) (n = 6). (E) The relative expression of Bax, GADD45a and p21 mRNAs in the liver tissues (n = 5). ** p < 0.01, compared to the control group; # p < 0.05 or ## p < 0.01, compared to the CCl4 only group. Bar = 100 µm. Bai: baicalein.

Article Snippet: The following primary antibodies were used to probe the membranes: primary rabbit antibodies against p21 (1:1000), COX2 (1:1000), Nrf2 (1:1000), and HO-1 (1:1000) (ProteinTech Group, Inc., Chicago, IL, USA), mouse monoclonal antibody against ALOX12 (1:1000) (Abcam, Cabridge, MA, USA) and β-actin (1:1000) (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: TUNEL Assay, Staining, Expressing, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: MicroRNA-150 relieves vascular remodeling and fibrosis in hypoxia-induced pulmonary hypertension.

doi: 10.1016/j.biopha.2018.11.058

Figure Lengend Snippet: Fig. 4. Effect of miR-150 on pulmonary fibrosis of pulmonary hypertension rats. (A) Pulmonary fibrosis was detected by Masson’s staining (200× magnification). Scale bars, 100 μm. (B) The area of pulmonary fibrosis was calculated and shown. The mRNA expressions of TGF-β1 (C) and collagen I (D) in lung tissues were evaluated by qPCR. (E) The protein levels of TGF-β1 and collagen I in lung tissues were measured by western blot assay. (F)&(G) Relative grey values of the protein bands were shown. (H) The expressions of TGF-β1 and collagen I in lung tissues were detected by immunohistochemical staining (400× magnification). Scale bars, 50 μm. Data were presented as mean ± SD. **P < 0.01, ***P < 0.001 versus the indicated group.

Article Snippet: The membranes were blocked in 5% nonfat milk for 1 h at room temperature, then incubated with primary antibodies against TGF-β1 (1:400, BOSTER, China), Collagen I (1:300, BOSTER, China), p-AKTser473 (1:500, KeyGen, China), AKT (1:500, KeyGen, China), p-mTORser2481 (1:500, Sangon Biotech, China), mTOR (1:1000, Cell signaling Technology, USA), and β-actin (1:500, Bioss, China) at 4°C overnight, followed by incubation with HRP-labeled Goat AntiRabbit or Goat Anti-Mouse IgG (1:5000, Beyotime, China) at 37°C for 45min.

Techniques: Staining, Western Blot, Immunohistochemical staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: MicroRNA-150 relieves vascular remodeling and fibrosis in hypoxia-induced pulmonary hypertension.

doi: 10.1016/j.biopha.2018.11.058

Figure Lengend Snippet: Fig. 6. Effect of miR-150 on the expressions of fibrosis-related molecules. The mRNA expressions of TGF-β1 (A) and collagen I (B) in PASMCs were measured by qPCR. (C) The protein levels of TGF-β1 and collagen I in PASMCs were detected by western blot assay. (D)&(E) Relative grey values of the protein bands were shown. Data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 versus the indicated group.

Article Snippet: The membranes were blocked in 5% nonfat milk for 1 h at room temperature, then incubated with primary antibodies against TGF-β1 (1:400, BOSTER, China), Collagen I (1:300, BOSTER, China), p-AKTser473 (1:500, KeyGen, China), AKT (1:500, KeyGen, China), p-mTORser2481 (1:500, Sangon Biotech, China), mTOR (1:1000, Cell signaling Technology, USA), and β-actin (1:500, Bioss, China) at 4°C overnight, followed by incubation with HRP-labeled Goat AntiRabbit or Goat Anti-Mouse IgG (1:5000, Beyotime, China) at 37°C for 45min.

Techniques: Western Blot

Journal: Arthritis research & therapy

Article Title: The circadian regulator Bmal1 in joint mesenchymal cells regulates both joint development and inflammatory arthritis.

doi: 10.1186/s13075-018-1770-1

Figure Lengend Snippet: Fig. 1 Targeting Bmal1 for deletion in joint mesenchymal cells. a The basic helix-loop-helix–encoding exon of Bmal1 was targeted for deletion in cells expressing Col6a1. b Fibroblast-like synoviocytes (FLS) were cultured from mouse hind limbs to passage 3, and assessed by flow cytometry for the absence of leukocytes (CD45−) and presence of the FLS marker CD90.2. CD90.2+ cells were then selected for further culture. c qPCR confirmed significant reduction in Bmal1 transcript in targeted cells (n = 3; unpaired t test), which corresponded with (d) loss of BMAL1 protein

Article Snippet: Membranes were incubated with primary antibody against BMAL1 (D2L7G; Cell Signaling Technology, Danvers, MA, USA) and β-ACTIN (ab8226; Abcam, Cambridge, UK), followed by horseradish peroxidaseconjugated secondary antibody, and imaged using enhanced chemiluminescence (Clarity; Bio-Rad Laboratories, Hercules, CA, USA).

Techniques: Expressing, Cell Culture, Flow Cytometry, Marker

Journal: Arthritis research & therapy

Article Title: The circadian regulator Bmal1 in joint mesenchymal cells regulates both joint development and inflammatory arthritis.

doi: 10.1186/s13075-018-1770-1

Figure Lengend Snippet: Fig. 2 Deletion of Bmal1 in joint mesenchymal cells renders fibroblast-like synoviocytes (FLS) and chondrocytes arrhythmic. a Cultured and purified FLS and femoral head tissue from Col6a1-Bmal1−/−and wild-type animals (on a PER2::luc background) were placed under photomultiplier tubes to record bioluminescence (representative of n = 3/genotype). b Cultured FLS were harvested every 4 h for RNA extraction, and clock gene expression was profiled. Values are made relative to expression in wild-type cells at time point 0. c Clock gene expression in FLS (CD45−CD90.2+) sorted from the limbs of male and female mice (9–13 weeks) at ZT6 and ZT18 (n = 4–6). Two-way analysis of variance and post hoc Bonferroni correction were performed

Article Snippet: Membranes were incubated with primary antibody against BMAL1 (D2L7G; Cell Signaling Technology, Danvers, MA, USA) and β-ACTIN (ab8226; Abcam, Cambridge, UK), followed by horseradish peroxidaseconjugated secondary antibody, and imaged using enhanced chemiluminescence (Clarity; Bio-Rad Laboratories, Hercules, CA, USA).

Techniques: Cell Culture, Purification, RNA Extraction, Gene Expression, Expressing

Journal: Arthritis research & therapy

Article Title: The circadian regulator Bmal1 in joint mesenchymal cells regulates both joint development and inflammatory arthritis.

doi: 10.1186/s13075-018-1770-1

Figure Lengend Snippet: Fig. 3 Effect of Bmal1 deletion in joint mesenchymal cells on joint development. a Hind paw thickness (in mm) plotted against animal weight (in g) measured in male (7 wild-type, 8 Col6a1-Bmal1−/−) and female (18 wild-type, 7 Col6a1-Bmal1−/−) animals. b X-ray images at 9 months. c Whole-mount staining of the hind limbs from 9-month-old wild-type and Col6a1-Bmal1−/−mice. d Quantification of pixel intensity from x-rays of 9-month-old mice in an ROI superior to the calcaneus (n = 4–6 limbs). Analysis was by unpaired t test. e Safranin O staining of ankle joints from 14- to 17-week-old mice where cartilage is stained red. Scale bar represents 500 μm. f H&E staining of ankle joints from 14- to 17 week old mice. Scale bar represents 500 μm. Col6a1-Bmal1−/−mice show thickened synovium (S); the enthesis (N) is eroded (dashed line delimits the interface between eroded bone and repair tissue), and the erosion is filled with exuberant chondro-osseous repair tissue (C) with the formation of a spur (P); there is chondroid metaplasia (M) at the junction of the synovium, joint capsule and enthesis repair tissue. The chondroid metaplasia differs from the chondro-osseous repair tissue in its morphology and tinctorial reaction with Safranin O. The synovium is fibrotic (B) and contains a mixture of spindle and round connective tissue cells. At its interfaces with connective tissues other than metaplastic chondroid tissue, there is a marked increase in adipocytes (A)

Article Snippet: Membranes were incubated with primary antibody against BMAL1 (D2L7G; Cell Signaling Technology, Danvers, MA, USA) and β-ACTIN (ab8226; Abcam, Cambridge, UK), followed by horseradish peroxidaseconjugated secondary antibody, and imaged using enhanced chemiluminescence (Clarity; Bio-Rad Laboratories, Hercules, CA, USA).

Techniques: Staining

Journal: Arthritis research & therapy

Article Title: The circadian regulator Bmal1 in joint mesenchymal cells regulates both joint development and inflammatory arthritis.

doi: 10.1186/s13075-018-1770-1

Figure Lengend Snippet: Fig. 4 Bmal1 deletion alters the balance of fibroblasts and chondrocytes within joints. a Flow cytometric analysis of the cellular composition of the joints from wild-type (n = 9) and Col6a1-Bmal1−/−(n = 12) mice (aged 9–18 weeks) after exclusion of doublets and dead cells. Analysis by unpaired t test. b Expression of chondrocyte-associated genes in the hind limbs of mice, aged 10 weeks (n = 4). Analysis by unpaired t test

Article Snippet: Membranes were incubated with primary antibody against BMAL1 (D2L7G; Cell Signaling Technology, Danvers, MA, USA) and β-ACTIN (ab8226; Abcam, Cambridge, UK), followed by horseradish peroxidaseconjugated secondary antibody, and imaged using enhanced chemiluminescence (Clarity; Bio-Rad Laboratories, Hercules, CA, USA).

Techniques: Expressing

Journal: Arthritis research & therapy

Article Title: The circadian regulator Bmal1 in joint mesenchymal cells regulates both joint development and inflammatory arthritis.

doi: 10.1186/s13075-018-1770-1

Figure Lengend Snippet: Fig. 5 Enhanced response of Col6a1-Bma1−/−mice to collagen antibody-induced arthritis. a Hind paw thickness and disease score after initiation of collagen antibody-induced arthritis (CAIA) in wild-type (n = 4) and Col6a1-Bmal1−/−(n = 5) mice. One-way analysis of variance (ANOVA) and Bonferroni multiple comparisons. b Serum IL-6 levels in CAIA animals at the time they were killed on day 7. Analysis by t test. c Quantification of leukocytes within hind paws of arthritic (n = 4–5/group) and non-arthritic (n = 4/group) animals. Two-way ANOVA and post hoc Bonferroni correction. Stars directly above CAIA bars indicate significant difference compared with control counterparts. d Representative plots showing increased presence of LycChi monocytes in inflamed limbs of Col6a1-Bmal1−/−mice, and quantification of Ly6Chi and Ly6Clo monocytes within hind paws of arthritic (n = 4–5/group) and non-arthritic (n = 4/group) animals. Two-way ANOVA and post hoc Bonferroni correction. Stars directly above CAIA bars indicate significant differences compared with control counterparts. e Transcripts of inflammatory cytokines in hind limbs harvested from control (n = 4/group) and CAIA mice (n = 4–5/group). Two-way ANOVA and post hoc Bonferroni correction. Stars directly above CAIA bars indicate significant difference compared with control counterparts

Article Snippet: Membranes were incubated with primary antibody against BMAL1 (D2L7G; Cell Signaling Technology, Danvers, MA, USA) and β-ACTIN (ab8226; Abcam, Cambridge, UK), followed by horseradish peroxidaseconjugated secondary antibody, and imaged using enhanced chemiluminescence (Clarity; Bio-Rad Laboratories, Hercules, CA, USA).

Techniques: Control

Journal: Arthritis research & therapy

Article Title: The circadian regulator Bmal1 in joint mesenchymal cells regulates both joint development and inflammatory arthritis.

doi: 10.1186/s13075-018-1770-1

Figure Lengend Snippet: Fig. 6 Altered response of fibroblast-like synoviocytes (FLS) lacking Bma1−/−to inflammatory stimuli. FLS were cultured from Col6a1-Bmal1−/−and wild-type littermates to passage 3 and purified for CD90.2 expression before stimulation for 8 h with tumour necrosis factor (TNF)-α. Expression of inflammatory cytokines in cell supernatants was assessed by Bioplex and enzyme-linked immunosorbent assay (CXCL5 only) (n = 5–6). One-way analysis of variance and post hoc Bonferroni correction. Stars directly above TNF-α bars indicate significant difference compared with control counterparts

Article Snippet: Membranes were incubated with primary antibody against BMAL1 (D2L7G; Cell Signaling Technology, Danvers, MA, USA) and β-ACTIN (ab8226; Abcam, Cambridge, UK), followed by horseradish peroxidaseconjugated secondary antibody, and imaged using enhanced chemiluminescence (Clarity; Bio-Rad Laboratories, Hercules, CA, USA).

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