Journal: Journal of experimental & clinical cancer research : CR

Article Title: Inhibition of EZH2 via activation of SAPK/JNK and reduction of p65 and DNMT1 as a novel mechanism in inhibition of human lung cancer cells by polyphyllin I.

doi: 10.1186/s13046-016-0388-x

Figure Lengend Snippet: Fig. 3 PPI decreased protein expression of DNMT1 and EZH2 through SAPK/JNK pathway. a PC9 and A549 cells were exposed to increased concentration of PPI for 24 h. Afterwards, the expression of EZH2 and DNMT1 protein were detected by Western blot. b PC9 and A549 cells were exposed to PPI (1.6 μM) for 24 h, followed by measuring the mRNA levels by qRT-PCR. c PC9 and A549 cells were transfected with a wild type human EZH2 promoter reporter construct ligated to luciferase reporter gene and internal control for 24 h, followed by treating with PPI for an additional 24 h. Afterwards, the promoter activities were determined using the Secrete-Pair Dual Luminescence Assay Kit as described in the Materials and Methods Section. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. d PC9 and A549 cells were treated with SP600125 (20 μM) for 2 h before exposure of the cells to PPI (1.6 μM) for an additional 24 h. Afterwards, the expression of EZH2 and DNMT1 protein were detected by Western blot using antibodies against EZH2 and DNMT1. The bar graphs represent the mean ± SD of EZH2 or DNMT1/GAPDH of three independent experiments. *Indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from PPI treated alone (P < 0.05)

Article Snippet: In the separated experiment, 2 μg of the control (pCMV6) and expression constructs containing Myc-DDK-tagged- or Myc/FLAG-tagged ORFs of human DNMT1 or EZH2 obtained from OriGene Technologies, Inc. (Rockville, MD, USA), the control (pCMV4) and p65 overexpression vector (pCMV4-p65) obtained from the Addgene (Plasmid #21966, Cambridge, MA, USA) [34] at a final concentration of 2 μg/mL, were transfected into the cells with the Lipofectamine 3000 reagent.

Techniques: Expressing, Concentration Assay, Western Blot, Quantitative RT-PCR, Transfection, Construct, Luciferase, Control, Luminescence Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Inhibition of EZH2 via activation of SAPK/JNK and reduction of p65 and DNMT1 as a novel mechanism in inhibition of human lung cancer cells by polyphyllin I.

doi: 10.1186/s13046-016-0388-x

Figure Lengend Snippet: Fig. 4 Activation of SAPK/JNK pathway-mediated inhibition of p65 expression contributed to the PPI-decreased protein expression of DNMT1. a PC9 and A549 cells were exposed to increased concentration of PPI for 24 h, followed by measuring the protein expression of p65 and p50 by Western blot. The bar graphs represent the mean ± SD of p65/GAPDH of three independent experiments. b PC9 and A549 cells were treated with SP600125 (20 μM) for 2 h before exposure of the cells to PPI (1.6 μM) for an additional 24 h. Afterwards, the expression of phosphorylation of SAPK/JNK and p65 protein were detected by Western blot. c PC9 and A549 cells were transfected with control and p65 overexpression vector for 24 h before exposing the cells to PPI for an additional 24 h. Afterwards, p65 and DNMT1 protein expressions were determined using Western blot. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *Indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from PPI treated alone (P < 0.05)

Article Snippet: In the separated experiment, 2 μg of the control (pCMV6) and expression constructs containing Myc-DDK-tagged- or Myc/FLAG-tagged ORFs of human DNMT1 or EZH2 obtained from OriGene Technologies, Inc. (Rockville, MD, USA), the control (pCMV4) and p65 overexpression vector (pCMV4-p65) obtained from the Addgene (Plasmid #21966, Cambridge, MA, USA) [34] at a final concentration of 2 μg/mL, were transfected into the cells with the Lipofectamine 3000 reagent.

Techniques: Activation Assay, Inhibition, Expressing, Concentration Assay, Western Blot, Phospho-proteomics, Transfection, Control, Over Expression, Plasmid Preparation

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Inhibition of EZH2 via activation of SAPK/JNK and reduction of p65 and DNMT1 as a novel mechanism in inhibition of human lung cancer cells by polyphyllin I.

doi: 10.1186/s13046-016-0388-x

Figure Lengend Snippet: Fig. 5 Exogenously expressed EZH2 not only restored cell growth, but also feedback antagonized PPI increased SAPK/JNK signaling. a PC9 and A549 cells were transfected with the control or expression constructs of DNMT1 for 24 h before exposing the cells to PPI for an additional 24 h. Afterwards, EZH2 and DNMT1 protein expression were determined using Western blot. b–c PC9 and A549 cells were transfected with the control or expression constructs of EZH2 for 24 h before exposing the cells to PPI for an additional 24 h. Afterwards, EZH2 and DNMT1 protein expression (b) and cell viability (c) were determined using Western blot and MTT assays, respectively. d PC9 and A549 cells were transfected with the control or expression constructs of EZH2 for 24 h before exposing the cells to PPI for an additional 24 h. Afterwards, EZH2, phosphor-SAPK/JNK were determined using Western blot. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *Indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from PPI treated alone (P < 0.05)

Article Snippet: In the separated experiment, 2 μg of the control (pCMV6) and expression constructs containing Myc-DDK-tagged- or Myc/FLAG-tagged ORFs of human DNMT1 or EZH2 obtained from OriGene Technologies, Inc. (Rockville, MD, USA), the control (pCMV4) and p65 overexpression vector (pCMV4-p65) obtained from the Addgene (Plasmid #21966, Cambridge, MA, USA) [34] at a final concentration of 2 μg/mL, were transfected into the cells with the Lipofectamine 3000 reagent.

Techniques: Transfection, Control, Expressing, Construct, Western Blot

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Inhibition of EZH2 via activation of SAPK/JNK and reduction of p65 and DNMT1 as a novel mechanism in inhibition of human lung cancer cells by polyphyllin I.

doi: 10.1186/s13046-016-0388-x

Figure Lengend Snippet: Fig. 6 Effects of PPI in subcutaneous xenograft model. Mice (n = 12/group) were divided to 3 groups [Con (saline), Low (L, 1 mg/kg) and High (H, 3 mg/kg)], and PPI was given by intraperitoneal injection for up to 27 days. a The xenografts were assessed by in vivo bioluminescence imaging at the sixth and end of the experiments (on day 6 and 27). The tumor growth was monitored by injecting luciferin in the mice followed by measuring bioluminescence and analyzed as described in the Materials and Methods section. Representative images are shown. b and c The xenografts were harvested on day 27, and the size and weight of tumors were determined. The bar graphs represented the tumor weight and size of mice results of as mean ± SD. d–e At the end of the experiments, xenografted tumors were isolated from individual animals and the corresponding lysates were detected p-SAPK/JNK, DNMT1, p65 and EZH2 proteins by Western blot and Immunohistochemistry as described in the Materials and Methods sections. Scale bar 50 μM. GAPDH was used as loading control for Western blot. Values in bar graphs were given as the mean ± SD from three independent Western blot experiments. *Indicates the significant difference from untreated control (p < 0.05). f The diagram shows that PPI inhibits growth of NSCLC cells through SAPK/JNK-mediated inhibition of p65 and DNMT1, subsequently; this results in the reduction of EZH2 gene expression. The interactions among p65, DNMT1 and EZH2, and feedback regulation of SAPK/JNK by EZH2 converge on the overall responses of PPI

Article Snippet: In the separated experiment, 2 μg of the control (pCMV6) and expression constructs containing Myc-DDK-tagged- or Myc/FLAG-tagged ORFs of human DNMT1 or EZH2 obtained from OriGene Technologies, Inc. (Rockville, MD, USA), the control (pCMV4) and p65 overexpression vector (pCMV4-p65) obtained from the Addgene (Plasmid #21966, Cambridge, MA, USA) [34] at a final concentration of 2 μg/mL, were transfected into the cells with the Lipofectamine 3000 reagent.

Techniques: Saline, Injection, In Vivo, Imaging, Isolation, Western Blot, Immunohistochemistry, Control, Inhibition, Gene Expression

Journal: Nature Communications

Article Title: Macrophage lineage cells-derived migrasomes activate complement-dependent blood-brain barrier damage in cerebral amyloid angiopathy mouse model

doi: 10.1038/s41467-023-39693-x

Figure Lengend Snippet: a Plasma CD5L concentration of CAA patients ( N = 12) and HC ( N = 12) as well as CAA models ( N = 5 in each group) and WT C57/BL6 mice ( N = 5). Data are presented as mean values ± SEM with the indicated significance (by two-tailed Student’s t test). b Left: CD5L deposition in mice brain was evaluated with immunostaining. Right: Immunostaining of CD31 (red), CD5L (green), C5b-9 (blue) and TSPAN4 (purple). Data are representative of 4 biologically independent experiments. c Plasma C5-9 concentration of mice. N = 6 in PBS-M, Aβ40-M and CD5L KO Aβ40-M transferred mice and N = 5 in CD5L KO PBS-M treated group. Data are presented as mean values ± SEM with the indicated significance (by one-way ANOVA followed by Tukey’s post-test). d C5b-9 deposition in brain as assessed with immunostaining. Data are representative of 3 biologically independent experiments. e Fluorescent intensity of 3kDa-Dextran in the plasma and eyeballs of the recipients. N = 6 in PBS-M, Aβ40-M and CD5L KO Aβ40-M transferred mice and N = 5 in CD5L KO PBS-M treated group. Data are presented as mean values ± SEM with the indicated significance (by one-way ANOVA followed by Tukey’s post-test). f Leakage of 3kDa-Dextran in brain parenchyma was assessed with fluorescent microscopy. Data are representative of 3 biologically independent experiments. g Coronal brain sections of the recipients were subjected to immunostaining of CD31 (green) and ZO-1 (red). Data are representative of 3 biologically independent experiments. h Skin autopsy sample of a CAA patient (Left) and brain tissue of WT and Tg-SwDI/B +/+ mice (24w of age) (Right) were subjected to IEM with CD5L staining. Blue dash lines outline micro-vessel wall. Red stars emphasize blood vessel lumen. Red arrow heads emphasize peri-vessel macrophages. Red arrows emphasize CD5L deposition in blood vessels within migrasome-like structures. Data are representative of 3 biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: Concentration of mouse NEFL (FineTest, EM1688), mouse C5b-9 (FineTest, EM1392), mouse CD5L (Boster, EK1414), human CD5L (MEIMIAN, MM-50587H1), human Aβ40 (CUSABIO, CSB-E08299h), human Aβ42 (MAI Bio, LM-2685H), and human C5b-9 (FineTest, EH3858) in plasma was assessed with commercial kits according to instructions of manufacturers.

Techniques: Clinical Proteomics, Concentration Assay, Two Tailed Test, Immunostaining, Microscopy, Staining

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 4. The effects of milk-derived extracellular vesicles (MEVs) supplementation on baseline homeostatic human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148-5P transcript levels (a), DNMT1 protein abundance (b) and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (d), and DNMT enzymatic activity and DNMT1 protein level (e). * p < 0.05, ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 6. The effects of milk-derived extracellular vesicles (MEVs) on IFN-γ primed human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (p < 0.05) (d), and DNMT enzymatic activity and DNMT1 protein level (p < 0.05) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, Control

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 7. The effects of milk-derived extracellular vesicles (MEVs) on primed human microglia clone 3 (HMC3) cells 9 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels relative to miR-148a-5P levels (p ≤ 0.05) (d), and DNMT enzymatic activity relative to DNMT1 protein level (A.U) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, Control

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: Serum proteins separated by two dimensional gel electrophoresis in early and advanced NAFLD, with and without HCC . These are representative blots comparing immune depleted and desalted serum from a patient with NAFLD and no fibrosis, a patient with NAFLD cirrhosis, and a patient with NAFLD cirrhosis and cancer. Serum profiles separated by 2D gel electrophoresis from 5 patients in each group, run in duplicate, were compared using Progenesis software. 5 spots were characterised by MS after excision. Spots 1,2 and 3 were isoforms of apolipoprotein A1, spot 4 was apolipoprotein A4, and spot 5 was identified as CD5 antigen like, or CD5L.

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Software

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: CD5L discriminates poorly between cirrhotic patients with and without HCC . Serum levels of CD5L were measured by ELISA assay in cirrhotic patients with (218 ± 221; n = 45) and without (194 ± 166; n = 49) HCC, as shown in 2A. Characteristics of the individuals are shown in Table 1. There was no significant difference between the two groups. ROC analyses is shown in 2B. The area under the curve is 0.495 (95% confidence intervals 0.376 and 0.614). A level > 400 ng/ml has a sensitivity of only 20%, but a specificity of 88%. Levels > 500 ng/ml have a specificity of 96%, > 600 ng/ml of 98% and > 700 ng/ml of 100%. For comparison, the mean CD5L serum level from patients without cirrhosis (detailed in figure 3) is also shown.

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: CD5L discriminates between steatohepatitis patients with and without cirrhosis . Serum levels of CD5L were measured in a total of 113 patients with either ALD or NAFLD. Fibrosis was scored histologically on liver biopsy as per the Brunt scoring system. The numbers of individuals in each group are shown within the boxes of chart 3A. The difference in CD5L levels between the different stages of fibrosis is statistically significant by univariate analysis (p = 0.004) controlled for both age and sex. This difference The ROC analyses for the identification of those individuals with stage 4 fibrosis (cirrhosis) is depicted in 3B. The area under the curve is 0.719 (95% confidence intervals 0.623 and 0.816), p < 0.0001. A level of CD5L 50 ng/ml has a sensitivity of 78% and a specificity of 46%, while a level of 100 ng/ml has a sensitivity of 63% and a specificity of 72%. A level greater than 200 ng/ml has a specificity of 95% (sensitivity 41%), and greater than 300 ng/ml of 97% (sensitivity 27%).

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques:

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: CD5L mRNA expression is not altered in association with either fat, inflammation or fibrosis scores in pre-cirrhotic NAFLD liver tissues . mRNA CD5L expression was quantified by real-time PCR, relative to GAPDH and a normal liver sample, in 21 pre-cirrhotic NAFLD biopsy tissues, 13 normal liver samples taken at the time of liver resection, one cirrhotic liver and one HCC. As shown in 4A-C, there was no difference in any pre-cirrhotic NAFLD biopsy tissues in association with the degree of fat, inflammation or fibrosis. There was a significant increase in the NAFLD tissues as a group (n = 21), compared with normal liver tissues (n = 13) as represented in 4D (6.945 ± 0.722 versus1.68 ± 0.269; p = 0.000, ***). The elevated CD5L mRNA expression in one cirrhotic and HCC tissue pair, obtained at the time of laparoscopic radiofrequency ablation, is also presented in 4C and is in keeping with the elevated serum CD5L levels identified in the larger cohort of patients studied.

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: The identification of the differentiating protein spots on 2D gels.

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques: Sequencing