Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: The expression of SMARCAD1 is significantly higher in pancreatic cancer and positively correlated with poor prognosis. A. The mRNA expression level of SMARCAD1 in pancreatic cancer is much higher than that of normal tissues from GSE16515, GSE11838 and GSE15471. B-C. Expression levels of SMARCAD1 by immunohistochemistry performed with tissue microarray of PC (n=69) and adjacent normal tissues (n=68). Representative images showed positive expression of SMARCAD1 in PC and negative expression in paired normal tissues, respectively. Scale bars=50μm. D. Kaplan-Meier analysis shows the correlation between SMARCAD1 expression and overall survival in patients. Patients with high SMARCAD1 expression had poorer overall survival than those with low expression. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Expressing, Immunohistochemistry, Microarray

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 enhances proliferation of PANC-1 cells. A-B. The efficiency of SMARCAD1 knockdown (A) or overexpression (B) in PANC-1 cells was detected by western blotting. β-actin was used as an internal control. C-D. CCK8 assay was performed to determine the proliferation of PANC-1 cells with SMARCAD1 knockdown (C) or overexpression (D) at the indicated time points after plated. Cell viability was measured at 450nm. E-F. The effect of SMARCAD1 knockdown (E) or overexpression (F) on Colony-forming of PANC-1 cells was shown in the top panels. Number of foci was counted as shown in the bottom panels. All data were presented as mean ±SEM. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Knockdown, Over Expression, Western Blot, Control, CCK-8 Assay

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 promotes migration and invasion of PANC-1 cells. A-B. Effect of SMARCAD1 knockdown (A) or SMARCAD1 overexpression (B) on cell migration was detected by wound healing at indicated time points after scratching. The wound healing was measured by ImageJ software. C-D. Motility ability of PANC-1 cells with SMARCAD1 depletion (C) or overexpression (D) was assessed by transwell assay at 24h. Representative images of migration were photographed at 24h (Top panel). The number of migrated cells was counted from 5 randomly selected fields under microscope (Bottom panel). E-F. Invasion ability of PANC-1 cells with SMARCAD1 depletion (E) or overexpression (F) was assessed by transwell assay at 48h. Representative images of invasion were photographed at 48h (Top panel). The number of invaded cells was counted from 5 randomly selected fields under microscope (Bottom panel). Scale bars=150um. Data were presented as mean ±SEM. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Migration, Knockdown, Over Expression, Software, Transwell Assay, Microscopy

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 induces EMT in PANC-1 cells. A-B. The morphology changes of PANC-1 cells: cells lose contact with each other with SMARCAD1 depletion (A) or gain more contact with SMARCAD1 overexpression (B), Scale bars=250μm. C-D. Changes in mRNA level of EMT relative markers were tested by Quantitative real-time PCR in SMARCAD1 knockdown (C) or overexpression (D) cells. The results were presented as mean ±SEM. All values were normalized to the level (=1) in NC or control cells. *p<.05, **p<.01. (D). E-F. The protein levels of EMT relative markers in SMARCAD1 knockdown (E) or overexpression (F) cells were assessed by western blotting. β-actin was used as an internal control.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Knockdown, Control, Western Blot

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1-induced EMT was regulated by Wnt/beta-catenin signaling pathway. A-B. The mRNA level of β-catenin was detected by Quantitative real-time PCR in PANC-1 cells with SMARCAD1 knockdown (A) or overexpression (B) respectively. The data were presented as mean ±SEM. All values were normalized to the level (=1) in NC or control cells. *p<.05, **p<.01. C-D. β-catenin, cyclin-D1, c-Myc and survivin protein levels were assayed by western blotting in PANC-1 cells with SMARCAD1 knockdown (C) or overexpression (D) respectively. E. PANC-1 cells with SMARCAD1 depletion were treated with CHIR99021 (6μM/ml) for 24h. The protein levels of EMT markers and Wnt/β-catenin target genes (β-catenin, cyclin-D1, c-Myc and survivin) were detected by western blotting. β-actin was used as an internal control.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Real-time Polymerase Chain Reaction, Knockdown, Over Expression, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: qRT-PCR primers

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: ID1 expression was induced by etoposide in ESCC cell lines. A, up-regulated ID1 mRNA level was detected in 34 tumors compared with normal adjacent epithelia by qRT-PCR (paired t test). B, an example case showed the expression of ID1 in ESCC tumors and normal counterparts by immunohistochemistry staining on the tissue microarray (upper panels). Quantitative analysis of the ID1 staining between ESCC tissues and the matched normal esophageal epithelia is shown in the lower panel (paired t test). C, mRNA and protein level of endogenous ID1 was detected in ESCC cell lines by qRT-PCR (left panel) and Western blot (right panel). D, KYSE140, KYSE150, and KYSE450 cells were treated with 10 μm etoposide for the indicated time and harvested. ID1 expression was determined by qRT-PCR (upper panels) and Western blot (lower panels). β-Actin was used as a loading control. The data are shown as means ± S.E. from multiple independent experiments, one-way analysis of variance test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining, Microarray, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: Overexpression of ID1 enhances cellular resistance to etoposide. A, KYSE150, KYSE140, KYSE450, and KYSE180 cells were treated with increasing concentrations of etoposide for 48 h, and then cell viability was examined by MTS assay. B, KYSE150 and KYSE450 cells stably transfected with empty vector (pLVX) or ID1 (pLVX-ID1) were incubated with DMSO (control) or etoposide (10 μm) for the indicated time, and cell growth was detected using MTS assay. The values are the means ± S.D. of absorbance at 490 nm for three independent experiments. C and D, ID1 transfectants and empty vector controls were treated with 10 μm etoposide for 48 h and then subjected to annexin V-FITC and propidium iodide (PI) staining. The values are expressed as percentages of annexin V-positive versus total cells (C). The expression levels of ID1, p53, cleaved caspase 3, and PARP were examined by Western blot (D). β-Actin was used as a loading control. E and F, KYSE450 cells were transiently transfected with negative control (NC) or ID1 siRNA, followed by 10 μm etoposide treatment. The cells were labeled with annexin V-FITC and propidium iodide and analyzed by flow cytometry. The values are expressed as percentages of annexin V-positive versus total cells (E). The expression levels of ID1, p53, cleaved caspase 3, and PARP were examined by Western blot (F). β-Actin was used as a loading control. The data are expressed as means ± S.D. *, p < 0.05; **, p < 0.01, one-way analysis of variance test.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Over Expression, MTS Assay, Stable Transfection, Transfection, Plasmid Preparation, Incubation, Control, Staining, Expressing, Western Blot, Negative Control, Labeling, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: Up-regulating ID1 upon etoposide activation is mediated through AP-1 binding sites. A, KYSE450 cells were transiently transfected with the promoter construct of ID1 for 24 h and treated with 10 or 20 μm etoposide. After 24 h, the luciferase activity was determined and normalized to an internal cytomegalovirus Renilla luciferase control. B, comparison of nucleotide sequences among seven different species. The AP-1 DNA binding site is represented with a shaded box. * indicates the same nucleotide sequence. C, schematic representation depicts the location of the mutant variant in the 2-kb ID1 promoter. TSS stands for transcription start site (upper panel). KYSE450 cells were cotransfected with ID1 wild-type or mutant luciferase reporters, together with c-Jun/c-Fos or control vector for 24 h. Then luciferase activity was determined and normalized to an internal cytomegalovirus Renilla luciferase control. The data are shown as means ± S.E. from multiple independent experiments (lower panel). D, KYSE450 cells were transfected with ID1 promoter reporter construct containing either wild-type or mutant putative AP-1 binding site and treated with or without 10 μm etoposide, and then the luciferase activity was determined. E, KYSE450 cells were treated with 10 μm etoposide for 4 h, and then ChIP assays were carried out with antibody against c-Jun, c-Fos, or IgG. The percentages of input of coprecipitating DNAs were calculated by qRT-PCR. The data represent the means ± S.D. of triplicate experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001, one-way analysis of variance test.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Activation Assay, Binding Assay, Transfection, Construct, Luciferase, Activity Assay, Control, Comparison, Sequencing, Mutagenesis, Variant Assay, Plasmid Preparation, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: The activation of ID1 required c-Jun/c-Fos in response to etoposide. A and B, KYSE450 cells were treated with 10 μm etoposide for the indicated time, and the expression of c-Jun/c-Fos and ID1 was determined by qRT-PCR (A) and Western blot (B). C, KYSE450 cells were transiently transfected with c-Jun, c-Fos, c-Jun/c-Fos, and TAM67 as described under “Experimental Procedures.” After 24 h, the expression of c-Jun, c-Fos, and ID1 was determined by qRT-PCR and Western blot. D, KYSE450 and KYSE150 cells were transiently transfected with c-Jun/c-Fos siRNA as described under “Experimental Procedures.” After 24 h, the expression of c-Jun, c-Fos, and ID1 was determined by Western blot. The data represent the means ± S.D. of triplicate experiments. NC, negative control. **, p < 0.01; ***, p < 0.001, one-way analysis of variance test.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: ID1 inhibits etoposide-induced cell apoptosis in a c-Jun/c-Fos-dependent manner. A, KYSE450 cells were transiently transfected with either negative control (NC) or c-Jun/c-Fos siRNA as indicated. After 24 h, cells were incubated with DMSO or 10 μm etoposide. The expression of c-Jun, c-Fos, ID1, p53, cleaved caspase 3, and PARP was determined by Western blot. β-Actin was used as a loading control. B, KYSE450 cells were transiently transfected with c-Jun/c-Fos siRNA and rescued ID1 with pLVX-ID1 compared with pLVX for 24 h. After that, cells were treated with DMSO or 10 μm etoposide and then subjected to Annexin V-FITC and propidium iodide (PI) staining. The values are expressed as a percentage of annexin V-positive versus total cells. The data are expressed as means ± S.D. *, p < 0.05, one-way analysis of variance test.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Transfection, Negative Control, Incubation, Expressing, Western Blot, Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: Positive correlation between c-Jun/c-Fos and ID1 in human cancers and prognostic value of high c-Jun/c-Fos and ID1 expression for cancer patients survive. A, a statistically significant positive correlation between c-Jun/c-Fos and ID1 mRNA was observed by Pearson's method in ESCC and patients in three independent published data sets including acute myeloid leukemia (GSE12417), ovarian cancer (GSE49997), and colorectal cancer (GSE24551), Pearson correlation analysis. B, clinical outcome data were analyzed by using PROGgeneV2 from published studies for correlations between c-Jun/c-Fos-ID1 expression levels and survival of cancer patients.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: Up-regulated p53 target gene in MEG3 microarray.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Microarray

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: Down-regulated p53 target gene in MEG3 microarray.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Microarray

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: (A) Reporter assays detected stimulation of p53-mediatd transactivation by MEG3 deletion mutants M1, M1+M2, M3, M2+M3 in HepG2 cells. The value are means of three independent experiments ±S.D, * P<0.05. (B) Cropped blots show the increased level of p53 protein 48h after transfection of the pcDNA-MEG3 in HepG2 cells. (C) Analysis of the effect of MEG3 overexpression on the half-life of p53. Cropped blots show the relative abundance of p53 after treated with translation inhibitor CHX for various amount of time in HepG2 cells (0, 0.5,1, 2h) overexpressing MEG3. (D) Analysis of the effect of MEG3 overexpression on the half-life of p53. Cropped blots show the relative abundance of p53 after treated with translation inhibitor CHX for various amount of time in doxo-induced HepG2 cells (0, 2,4, 6h)overexpressing MEG3.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Transfection, Over Expression

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: (A) Western blot of p53 protein bound to in vitro transcribed biotinylated MEG3 incubated with HEK293 cell extract transfected with Flag-p53 vector. (B) Western blot of p53 protein bound to in vitro transcribed biotinylated MEG3 and deletion mutations RNA incubated with doxo-induced HepG2 cell extract (MEG3 deletion mutants M1, M1+M2, M3, M2+M3 are shown in ). (C) In vitro transcribed biotinylated MEG3 retrieved purified GST-p53 but not GST. (D) RIP experiments were performed using an antibody against the p53 on extracts from doxo-induced HepG2 cells. The purified RNA was used for qRT-PCR, and the enrichment of the lncRNA MEG3 was normalized to GAPDH (upper, western blot of p53 protein after immunoprecipitation). Data was relative to mock-IP (IgG). (E) A series of p53 deletion mutants which were flag-tagged was treated as in (A), and association was detected by anti-Flag. Up represents successful expression of p53 deletion mutants. Down represents in vitro transcribed biotinylated MEG3 RNA was incubated with HEK293 cell extract which was transfected with p53 deletion mutants and associated proteins were detected by anti-Flag.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Western Blot, In Vitro, Incubation, Transfection, Plasmid Preparation, Purification, Quantitative RT-PCR, Immunoprecipitation, Expressing

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: (A) SK-Hep–1 hepatocellular carcinoma cell lines with stably expression of MEG3 were determined by qRT-PCR. The value are means of three independent experiments ±SD, *** P<0.001. (B) Expression of GADD45A, EGR1, SESN2 and TGFA was examined by qRT-PCR and normalized to GAPDH. The bars represent the relative fold change of these genes in SK-Hep–1 after transfection with pcDNA3.0-MEG3 compared with blank vector pcDNA3.0. (C) Expression of GADD45A, EGR1, SESN2 and TGFA was examined by qRT-PCR and normalized to GAPDH after knockdown p53 level in HepG2 cells. The bars represent the relative fold change of these genes in HepG2 cells after co-transfection of pcDNA3.0 and NC, pcDNA3.0-MEG3 and NC, pcDNA3.0-MEG3 and sip53, pcDNA3.0 and sip53, respectively. (D) A schematic diagram illustrating how MEG3 can function as tumor suppressor through interactions with p53.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Stable Transfection, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Knockdown, Cotransfection

Journal: Science Progress

Article Title: Role of thrombus-derived exosomal lncRNA LOC101928697 in regulating endothelial function via FUS protein interaction in myocardial infarction

doi: 10.1177/00368504251372111

Figure Lengend Snippet: Analysis of the correlation between FUS protein and myocardial infarction. (a) Enrichment Analysis Bar Plot based on differential gene expression profiles in lncRNA microarray analysis.(b) Detection information about lncRNA LOC101928697 binding to FUS proteins in AnnoLnc2 database. (c) Detection information about lncRNA LOC101928697 binding to FUS protein in RBPDP database. (d) Scores in the RPISeq database on the model of lncRNA LOC101928697 binding to FUS protein. (e-g) Prediction information about lncRNA LOC101928697 binding to FUS protein in catRAPID website, (e) Statistical map information about protein and RNA binding sites, (f) Total scoring information, and (g) Interaction map showing the interaction region between protein and RNA. (h-i) Analyses about bioinformatics techniques based on GSE163772 in the GEO database, where (h) is a statistical map of FUS gene expression in endothelial cells of a mouse model of myocardial infarction, and (i) A scatter plot about the correlation between the level of FUS gene expression and the disease state (control vs. myocardial infarction).

Article Snippet: After extensive washing, the bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blot using anti-FUS antibody (Proteintech, Cat No. 11570-1-AP, dilution 1:5000) to detect the enrichment of FUS protein.

Techniques: Gene Expression, Microarray, Binding Assay, RNA Binding Assay, Control

Journal: Science Progress

Article Title: Role of thrombus-derived exosomal lncRNA LOC101928697 in regulating endothelial function via FUS protein interaction in myocardial infarction

doi: 10.1177/00368504251372111

Figure Lengend Snippet: Interaction of exosomal lncRNA LOC101928697 with FUS proteins. (a and b) The western blot detection of FUS protein expression in each group of cells and the statistical graph. (c) Statistical graph of RT-qPCR to detect the expression of FUS at the mRNA level in each group of cells. (d) The fluorescence graph of fluorescence in situ hybridization (FISH) experiment. In which FUS was labeled with green fluorescence, lncRNA LOC101928697 was labeled with red fluorescence, and the nucleus was labeled with blue fluorescence (20×). (e) Western blot detection of FUS protein following RNA pull-down using sense or antisense LOC101928697 transcripts. (f) Quantification of FUS protein enrichment in sense RNA pull-down versus antisense control, based on densitometric analysis. (g-h) Western blot detection of FUS protein expression in each group of cells after knockdown or overexpression of lncRNA LOC101928697 and the statistical graphs. (i) Statistical graph of mRNA level expression of FUS in each group of cells after knockdown or overexpression of lncRNA LOC101928697 by RT-qPCR assay. a p < 0.05 compared to control group. b p < 0.05 compared to exosome group. c p < 0.05 compared to siRNA + exosome group.

Article Snippet: After extensive washing, the bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blot using anti-FUS antibody (Proteintech, Cat No. 11570-1-AP, dilution 1:5000) to detect the enrichment of FUS protein.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Fluorescence, In Situ Hybridization, Labeling, Protein Enrichment, Control, Knockdown, Over Expression

Journal: Molecular Oncology

Article Title: JNK‐mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation‐dependent activation of Snail

doi: 10.1002/1878-0261.13143

Figure Lengend Snippet: Overexpression of P‐SIRT1 Ser27 in CRC tissues and its implications for SIRT1 stability and proliferation and migration of colon cancer cells. (A) SW480 cells (5 × 10 6 ) stably expressing mock or SIRT1 vector were injected subcutaneously into the flank of BALB/c nude mice. Photographs show the xenograft tumors resected from mice at the time of harvest (day 50). The tumor volume and the tumor weight of the mice at the time of harvest were measured. Comparison of the means between mock and SIRT1 vector group was determined by Student's t ‐test. The results are shown as the mean ± S.D. of four xenografts for each group: * P < 0.05. Scale bar represents 1 cm. (B) Histological structures in sections from excised xenograft tumors were analyzed by H&E staining (first column). The yellow arrow indicates neovascularization. The expression of SIRT1, IL‐6, and 8 in excised xenograft tumors were examined by immunohistochemical analysis (second to last columns). Each scale bar represents 100 µm. The data are presented as the mean ± S.D. of three independent samples for each group in Fig. S1H: * P < 0.05; ** P < 0.01. (C) Kaplan–Meier survival curve of overall survival in colon cancer patients with high ( n = 497) and low ( n = 506) levels of SIRT1 based on the data collected from the GENT2 database ( http://gent2.appex.kr ). (D) Kaplan–Meier survival curve of disease‐specific survival in colon cancer patients with high ( n = 238) and low ( n = 238) levels of SIRT1 based on the data obtained from the GENT2 database ( http://gent2.appex.kr ) (E) Immunofluorescence staining of tissue microarray containing thirty‐five pairs of colon adenocarcinoma and matched adjacent normal colon tissues was conducted using anti‐SIRT1 and P‐SIRT1 Ser27 antibodies. The adjacent normal area is 1.5 cm distant from the tumor, which was taken by a histologist. Images of H&E‐stained tissue sections were offered by US Biomax Inc. Each scale bar represents 200 µm. (F) The fluorescence intensity was measured by image analysis software ImageJ, and results are shown as a violin plot. Statistics were calculated using both paired t ‐test and Wilcoxon matched‐pairs test. (G) The protein levels of SIRT1 and P‐SIRT1 Ser27 in colon cancer and corresponding normal tissues were measured by Western blot analysis. The data are presented as the mean ± S.D. of eleven pairs of human tissue specimens in Fig. D: * P < 0.05.

Article Snippet: The membranes were then incubated with primary antibodies against SIRT1, Snail, actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA); ubiquitin (Life Technologies by Thermo Fisher Scientific Inc.; Waltham, MA, USA); P‐SIRT1 Ser27 , P‐SIRT1 Ser47 , P‐JNK, JNK, acetylated‐lysine, and acetylated‐p53 Lys382 (Cell Signaling Technology; Danvers, MA, USA).

Techniques: Over Expression, Migration, Stable Transfection, Expressing, Plasmid Preparation, Injection, Comparison, Staining, Immunohistochemical staining, Immunofluorescence, Microarray, Fluorescence, Software, Western Blot

Journal: Molecular Oncology

Article Title: JNK‐mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation‐dependent activation of Snail

doi: 10.1002/1878-0261.13143

Figure Lengend Snippet: Comparison of the effects of SIRT1‐WT and SIRT1‐S27A on the tumorigenicity of HCT‐116 cells. (A) HCT‐116 cells (5 × 10 6 ) transfected with mock, SIRT1‐WT, or SIRT1‐S27A vector were inoculated subcutaneously into the flank of BALB/c nude mice. Photographs show the xenograft tumors collected from mice at the end of the experiment (day 20). The tumor volume and the tumor weight of the mice at the time of harvest were measured, and the one‐way ANOVA with Tukey's multiple comparisons test was performed for the statistical comparison. The results are shown as the mean ± S.D. of six xenografts for each group: * P < 0.05; ** P < 0.01. Scale bar represents 1 cm. (B) H&E staining and immunohistochemical analysis of SIRT1, P‐SIRT1 Ser27 , IL‐6, and IL‐8 were performed on sections from resected xenograft tumor. Each scale bar represents 100 µm. For quantification of each target, the DAB intensity was measured by image analysis software FIJI (the enhanced version of imagej2 ). Statistics were calculated using the one‐way ANOVA with Tukey's multiple comparisons test, and results are presented as the mean ± S.D. ( n = 4 per group): ** P < 0.01; *** P < 0.001. (C) Identification of high‐confidence kinase candidates contributing phosphorylation of SIRT1 at serine 27 by gps 5.0 ( http://gps.biocuckoo.cn/ ). (D) Multiple sequence alignment using clustalw software ( https://www.genome.jp/tools‐bin/clustalw ) showing an evolutionarily conserved phosphorylation site of SIRT1 at serine 27 (arrow) between different species. The positions which have a fully conserved residue are indicated by asterisks. (E) HCT‐116 cells were treated with two different concentrations (10 and 20 µ m ) of a JNK inhibitor (SP600125), an ERK inhibitor (U0126) or a p38 inhibitor (SB203580) for 3 h. The protein levels of P‐SIRT1 Ser27 and SIRT1 were measured by Western blot analysis. One‐way ANOVA with Tukey's multiple comparisons test was used to determine the significance. The results are shown as the mean ± S.D. ( n = 3): ** P < 0.01; *** P < 0.001. (F) HCT‐116 cells were treated with CHX (20 μg·mL −1 ) for indicated periods in the absence or presence of SP600125 (20 μ m ). Comparison of the means between two groups was determined by Student's t ‐test. The values are presented as the mean ± S.D. of three independent experiments: *** P < 0.001.

Article Snippet: The membranes were then incubated with primary antibodies against SIRT1, Snail, actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA); ubiquitin (Life Technologies by Thermo Fisher Scientific Inc.; Waltham, MA, USA); P‐SIRT1 Ser27 , P‐SIRT1 Ser47 , P‐JNK, JNK, acetylated‐lysine, and acetylated‐p53 Lys382 (Cell Signaling Technology; Danvers, MA, USA).

Techniques: Comparison, Transfection, Plasmid Preparation, Staining, Immunohistochemical staining, Software, Phospho-proteomics, Sequencing, Residue, Western Blot

Journal: Molecular Oncology

Article Title: JNK‐mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation‐dependent activation of Snail

doi: 10.1002/1878-0261.13143

Figure Lengend Snippet: Strong inactivation of Snail in HCT‐116 cells expressing SIRT1‐S27A. (A) Nuclear extracts prepared from HCT‐116 cells transfected with SIRT1‐WT or SIRT1‐S27A were subjected to the transcription factor activation profiling array to analyze the activity of ninety‐six different transcription factors. The top 10 hits are listed in the Table in an ascending order of the SIRT1‐S27A to SIRT1‐WT ratio. The red arrow denotes the top hit whose activity was most reduced in HCT‐116 cells harboring the SIRT1‐S27A mutation. (B) Following transfection of HCT‐116 cells with mock, SIRT1‐WT, or SIRT1‐S27A vector for 48 h, the mRNA (upper panels) and protein (lower panels) levels of Snail were measured by RT‐PCR and immunoblotting analyses, respectively. Differences in the means among the groups were assessed by one‐way ANOVA with Tukey's multiple comparisons test. Data are presented as the mean ± S.D. ( n = 3 per group): ** P < 0.01; *** P < 0.001. NS, not significant. (C) Immunohistochemical (IHC) analysis of Snail was carried out on sections from resected xenograft tumor corresponding to the Fig. . Each scale bar represents 100 µm. The DAB intensity was assessed by image analysis software fiji (the enhanced version of imagej2 ). Statistics were calculated using one‐way ANOVA with Tukey's multiple comparisons test, and the data are presented as the mean ± S.D. ( n = 4 per group): *** P < 0.001. (D) IHC analysis of Snail and P‐SIRT1 Ser27 was performed on sections from resected xenograft tumor corresponding to the Fig. . Each scale bar represents 100 µm. The results are shown as the mean ± S.D. of three independent samples for each group in Fig. B: * P < 0.05; ** P < 0.01.

Article Snippet: The membranes were then incubated with primary antibodies against SIRT1, Snail, actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA); ubiquitin (Life Technologies by Thermo Fisher Scientific Inc.; Waltham, MA, USA); P‐SIRT1 Ser27 , P‐SIRT1 Ser47 , P‐JNK, JNK, acetylated‐lysine, and acetylated‐p53 Lys382 (Cell Signaling Technology; Danvers, MA, USA).

Techniques: Expressing, Transfection, Activation Assay, Activity Assay, Mutagenesis, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemical staining, Software

Journal: Molecular Oncology

Article Title: JNK‐mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation‐dependent activation of Snail

doi: 10.1002/1878-0261.13143

Figure Lengend Snippet: A proposed mechanism underlying contribution of aberrantly stabilized SIRT1 to oncogenicity of human colon cancer cells. JNK‐dependent phosphorylation of SIRT1 at Ser27 contributes to its stabilization and deacetylation of Snail to enhance the production of IL‐6 and IL‐8, thereby promoting proliferation and migration of human colon cancer cells.

Article Snippet: The membranes were then incubated with primary antibodies against SIRT1, Snail, actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA); ubiquitin (Life Technologies by Thermo Fisher Scientific Inc.; Waltham, MA, USA); P‐SIRT1 Ser27 , P‐SIRT1 Ser47 , P‐JNK, JNK, acetylated‐lysine, and acetylated‐p53 Lys382 (Cell Signaling Technology; Danvers, MA, USA).

Techniques: Phospho-proteomics, Migration

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) Joint swelling and clinical scores in wild-type (WT), Fas lpr/lpr , Fasl gld/gld , and Fas –/– mice (n = 6 per group). ( B ) Joint swelling and clinical scores in WT, Fasl Δm/Δm , Fasl Δs/Δs , and Fasl Δs/Δs mice injected with sFasL (n = 6 per group). ( C , D ) Gross and microscopic examination of arthritis (magnified 10× in the upper panel and 200× in the lower panel). Scale bars: 1 cm ( C ), 200 μm ( D , upper panel), and 100 μm ( D , lower panel). ( E ) Tandem mass spectra of unique DR5 peptides. ( F ) Transcript levels of Tnfrsf10b in synovial CD45 + immune cells and CD45 – non-immune cells from WT mice with or without AIA. ( G ) Immunohistochemistry of DR5 expression in joint tissue from a healthy control subject and a patient with rheumatoid arthritis (n = 3; magnified 400×, scale bar: 50 μm). ( H ) Flow cytometric analysis of biotinylated protein binding to EL4 cells transfected with human WT TNFRSF10B preincubated with recombinant hTRAIL, or simultaneously incubated with anti-FasL, or anti-DR5 antibodies. ( I ) Flow cytometric analysis of biotinylated FasL binding on hFLSCs with FAS and/or TNFRSF10B knockout, and TNFRSF10B and/or FAS overexpression in FAS and TNFRSF10B double knockout (DKO) cells. ( J ) hLFSCs were preincubated with TNF-α (as a negative control), FasL, or TRAIL and cross–linked with BS 3 . Lysates from these cells were immunoprecipitated with anti–DR5 or control IgG antibody and immunoblotted with anti-DR5, TNF-α, FasL, or TRAIL antibodies. ( K ) Flow cytometric analysis of DR5–Fc binding on EL4 cells transfected with human WT FASLG in the presence of recombinant hTRAIL, anti-DR5, or FasL antibodies. Data were pooled from three ( A , B , and D–G ) or four ( H, K ) independent experiments and are presented as mean ± standard error of the mean (SEM). *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way analysis of variance (ANOVA). Figure 1—source data 1. Numerical data obtained during experiments represented in , , and .

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Injection, Immunohistochemistry, Expressing, Control, Protein Binding, Transfection, Recombinant, Incubation, Binding Assay, Knock-Out, Over Expression, Double Knockout, Negative Control, Immunoprecipitation

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) Schematic diagram showing AP–MS analyses. ( B ) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of biotinylated Fc (control) or sFasL–Fc cross-linked protein complexes. The fractions used in in-gel digestion are separated by red lines. ( C ) Expression of DR5 in CD45 + and CD45 – cells from the joint tissues of WT mice with arthritis. ( D ) Flow cytometry analyses of the expression of Fas and DR5 in human (h) fibroblast-like synovial cells (FLSCs). ( E ) Flow cytometry analyses of FasL–Fc binding to hFLSCs in the presence of anti-DR5 and/or anti-Fas antibodies. ( F , G ) Flow cytometry analyses of human DR5 ( F ) and Fas ( G ) expression in EL4 cells transfected with human WT tumor necrosis factor receptor superfamily (TNFRSF)10B ( F ) and FAS ( G ). ( H ) Expression of TNFRSF10B and FAS in EL4 mouse T cells transfected with various human genes. ( I ) Flow cytometry analyses of biotinylated protein binding to EL4 cells transfected with human WT FAS preincubated with recombinant human (h) TNF-related apoptosis-inducing ligand (TRAIL) or treated simultaneously with anti-Fas and DR5 antibodies. ( J ) Flow cytometry analyses of FasL–Fc binding to hFLSCs after preincubation with recombinant sTRAIL or sFasL. All experiments were performed four times independently.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Protein-Protein interactions, Polyacrylamide Gel Electrophoresis, Control, Expressing, Flow Cytometry, Binding Assay, Transfection, Protein Binding, Recombinant

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) hFLSCs and mouse synovial fibroblasts were stimulated with human or mouse sFasL in the presence or absence of anti-mouse Fas or anti-DR5 antibodies, as well as anti-human Fas or anti-DR5 antibodies for 24 hr. CX3CL1 levels in culture supernatants were measured using ELISA. ( B , C ) Jurkat cells ( B ) or mouse splenocytes ( C ) were incubated for 24 hr with recombinant FasL and TRAIL in the presence or absence of human or mouse anti-DR5 or anti-Fas antibodies. Jurkat and gated splenic TCRβ + CD4 + T cell death was measured using flow cytometry. ( D ) Jurkat cell death was measured using flow cytometry after FasL or FasL–Fc treatment in the presence or absence of anti-Fas or anti-DR5 antibodies for 24 hr. Data are presented as mean ± SEM. All experiments were performed three times independently. *p<0.05; **p<0.01. Data were analyzed using one-way ANOVA.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Recombinant, Flow Cytometry

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) Flow cytometry analyses of FasL–Fc binding to hFLSCs that were knocked down with FAS , TNFRSF10B , TNFRSF1A , TNFRSF10A , or TNFRSF12 . ( B ) Expression of FAS , TNFRSF10B , TNFRSF10A , TNFRSF1A , and TNFRSF12 in siRNA-transfected hFLSCs. ( C ) Expression of FAS and TNFRSF10B in FAS - and/or TNFRSF10B -knockout (KO) cells and KO cells transfected with TNFRSF10B and/or FAS overexpression vector. ( D , E ) Expression of Fas and DR5 ( D ) and biotinylated TRAIL binding ( E ) in FAS - and/or TNFRSF10B -KO hFLSCs and DKO (DR5 and Fas gene double knockout) cells transfected with TNFRSF10B and/or FAS in expression vectors. Biotinylated TRAIL binding was quantified by streptavidin (sAv)–fluorescein isothiocyanate staining intensity using flow cytometry. ( F, G ) Surface plasmon resonance assays for DR5–FasL ( F ) and DR5–TRAIL ( G ) interactions. ( H ) hFLSCs were incubated with PBS (control), 6× His-tagged FasL, or 6× His-tagged TRAIL and cross-linked using BS 3 . Cell lysates were immunoprecipitated with anti-His or control IgG antibodies and then immunoblotted with anti-Fas, DR5, TRAIL, and FasL antibodies. ( I ) FASLG , TNFSF10 , and TNF expression in EL4 mouse T cells transfected with human genes. ( J ) Flow cytometry analyses of DR5–Fc and Fas–Fc binding to EL4 cells transfected with human WT FASLG in the presence of anti-FasL antibodies. ( K, L ) Flow cytometry analyses of human FasL, TNF-α, and TRAIL expression ( K ), as well as DR5–Fc and Fas–Fc binding to EL4 cells transfected with human WT FASLG , TNFA , or TRAIL ( L ) and gated on transfected cells expressing the target proteins. Data were pooled from three independent experiments and are presented as mean ± SEM (n = 4 in B ). **p<0.01; ****p<0.0005. Data were analyzed using one-way ANOVA.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Flow Cytometry, Binding Assay, Expressing, Transfection, Knock-Out, Over Expression, Plasmid Preparation, Double Knockout, Staining, SPR Assay, Incubation, Control, Immunoprecipitation

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A , B ) FasL–Fc binding to hFLSCs or EL4 cells transfected with human FAS , or TNFRSF10B after preincubation with human sTRAIL or sFasL. ( C ) Model of FasL and DR5 derived from the crystal structure of the FasL/DcR3 complex (Protein Data Bank: 4 MSV) and TRAIL/DR5 complex (Protein Data Bank: 1D4V). ( D , E ) Flow cytometric analysis of FasL–Fc or DR5–Fc binding to EL4 cells transfected with human WT or mutated TNFRSF10B or FASLG . ( F , G ) Comparison of the effects of sFasL and sTRAIL on ( F ) apoptosis and ( G ) necroptosis in hFLSCs. ( H ) Joint swelling and clinical scores in Fasl gld/gld mice injected with Z–VAD–FMK and/or sFasL (n = 6 per group). Data were pooled from four ( A , B , and D–G ) or three ( H ) independent experiments and are presented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way ANOVA. Figure 2—source data 1. Numerical data obtained during experiments represented in , .

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Binding Assay, Transfection, Derivative Assay, Comparison, Injection

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) hLFSCs were preincubated with FasL and TRAIL using an excess of TRAIL in lane 3 (TRAIL [4 μg/mL]+FasL) and an excess of FasL in lane 6 (FasL [4 μg/mL]+TRAIL) before cross-linking with BS 3 . Lysates from these cells were immunoprecipitated with anti-DR5 (lanes 2, 3, 5, and 6) or control IgG (lanes 1 and 4) antibodies and immunoblotted with anti-DR5, FasL, or TRAIL antibodies. ( B ) Crystal structures of the TRAIL/DR5 (Protein Data Bank: 1D4V) and FasL/DcR3 (Protein Data Bank: 4 MSV) complexes. ( C ) Alignment of the human DcR3 and DR5 as well as the human FasL and TRAIL sequences. The point mutations in the mutant huDR5–cysteine-rich domains (CRD)two and CRD3, the mutant FasL–CRD2 interacting domain, and the mutant FasL–CRD3 interacting domain are indicated by asterisks (*). ( D ) Flow cytometry analyses of human DR5 in EL4 cells transfected with human WT or mutant TNFRSF10B . ( E ) Flow cytometry analyses of human FasL in EL4 cells transfected with human WT or mutant FASLG . Experiments ( A , D , and E ) were performed three times independently.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Immunoprecipitation, Control, Mutagenesis, Flow Cytometry, Transfection

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A, B ) Microarray assay using joint tissues from WT, Fas lpr/lpr , and Fasl gld/gld mice with arthritis. ( C ) Cx3cl1 transcript levels estimated in joint tissues from WT, Fas lpr/lpr , Fas –/– , Fasl gld/gld , Fasl Δs/Δs , and Tnfrsf10b KO mice with arthritis. ( D ) Cx3cl1 expression in CD45 + immune and CD45 – non–immune cells from the joints of WT mice with arthritis after sFasL treatment. ( E, F ) CX3CL1 transcript levels estimated in hFLSCs in the presence of anti-Fas and/or anti-DR5 antibodies ( E ) and FAS (Fas), TNFSF10B (DR5), or FAS , and TNFRSF10B DKO, or TNFRSF10B and FAS overexpression in DKO hFLSCs ( F ). ( G ) Cx3cl1 expression in synovial fibroblasts from WT, Fas lpr/lpr , Fas –/– , or Tnfrsf10b KO mice in the presence or absence of sFasL. ( H, I ) CX3CL1 transcript levels estimated after sFasL stimulation in hFLSCs in the presence of MEK (U0126), ERK (PD980259), p38 kinase (SB203580), and NF-κB (MG132 and BMS345541) inhibitors ( H ) or transfection with control, RELA , CHUK (IKKa), or IKBKB (IKKb) siRNA ( I ). ( J ) Synovial fibroblasts obtained from WT mice with arthritis were incubated with sFasL or sTRAIL and CX3CL1 levels were measured using ELISA. ( K ) hFLSCs were stimulated with sFasL after preincubation with various concentrations of sTRAIL for 30 min and CX3CL1 levels were measured in the culture supernatant. Data were pooled from three ( C–G and K ) or four ( H–J ) independent experiments and are presented as mean ± SEM (n = 4 for C–K ). *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way ANOVA. Figure 3—source data 1. Numerical data obtained during experiments represented in , and .

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Microarray, Expressing, Over Expression, Transfection, Control, Incubation, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) CX3CL1 was measured in culture supernatants of CD45 + immune and CD45 – non-immune cells from the joints of WT mice with arthritis after sFasL stimulation. ( B–H ) Cx3cl1 transcripts and CX3CL1 protein in culture supernatants were estimated in human ( B–F ) and mouse ( G, H ) FLSCs after stimulation with sFasL or FasL–Fc in the presence of anti-Fas or anti-DR5 antibodies. ( I ) CX3CL1 transcript of hFLSCs transfected with control, TNFRSF1A , FAS , TNFRSF12 , TNFRSF10A , and TNFRSF10B siRNA was measured after stimulation with sFasL. ( J ) FAS and/or TNFRSF10B KO hFLSCs, and DKO hFLSCs were transfected with TNFRSF10B. FAS in the expression vector was stimulated with sFasL. CX3CL1 levels were measured in the culture supernatants. ( K ) Levels of CX3CL1 in culture supernatants of synovial fibroblasts obtained from WT, Fas lpr/lpr , Fas –/– , or Tnfrsf10b KO mice with arthritis after incubation with sFasL. ( L , M ) Levels of CX3CL1 in culture supernatants of hFLSCs after sFasL stimulation for 2 hr in the presence of MEK (U0126), ERK (PD980259), p38 kinase (SB203580), and NF-κB (MG132, and BMS345541) inhibitors ( L ) or transfection with control, RELA , CHUK (IKKa), or IKBKB (IKKb) siRNA ( M ). ( N ) Blotting assay for components of the NF-κB signaling pathway in hFLSCs stimulated with sFasL for the durations indicated, all preincubated with anti-Fas antibodies. ( O , P ) CX3CL1 transcript ( O ) and CX3CL1 protein ( P ) levels in culture supernatants from hFLSCs after stimulation with sFasL in the presence of 50 μM Z–VAD–FMK. Data were pooled from four ( A–F , L , and M ) or three ( G–K , O , and P ) independent experiments and are presented as mean ± SEM (n = 5; A–M , O , and P ). NS, not significant; *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way ANOVA.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Incubation

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) Joint swelling and clinical scores in WT mice injected with anti-DR5 or anti-Fas antibodies to measure AIA (n = 5 per group). ( B, C ) Joint swelling and clinical scores in WT and Tnfrsf10b KO mice injected with sFasL or phosphate-buffered saline (PBS) to measure AIA (n = 5 per group). ( D ) Cx3cl1 transcript levels in the joints were estimated in WT, Tnfrsf10b KO, Fasl Δs/Δs , and Fasl Δm/Δm mice injected with sFasL or PBS to measure AIA (n = 5). ( E, F ) Joint swelling and clinical scores ( E ), and transcript levels of various cytokines and chemokines in joint tissues of Tnfrsf10b KO mice injected with CX3CL1 or PBS to measure AIA ( F ) (n = 6 per group). ( G ) Joint swelling and clinical scores of WT and Cx3cr1 KO mice in the presence or absence of sFasL to measure AIA (n = 6 per group). Data were pooled from three independent experiments and are presented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way ANOVA. Figure 4—source data 1. Numerical data obtained during experiments represented in , .

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Injection, Saline

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A, B ) Joint swelling and clinical scores together with transcript levels of various cytokines and chemokines in joint tissues from Fasl gld/gld mice injected with sFasL, as well as anti-Fas, or anti-DR5 antibodies (n = 6 per group). ( C, D ) Joint swelling and clinical scores in Fasl Δs/Δs ( C ) and Fasl gld/gld ( D ) mice injected with CX3CL1 or PBS. ( E, F ) Transcript levels of various cytokines and chemokines in joint tissues from Fasl Δs/Δs ( E ) or Fasl gld/gld ( F ) mice injected with CX3CL1 or PBS to measure AIA (n = 6 per group). Data were pooled from four ( A, B ) or three ( C–F ) independent experiments and are presented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way ANOVA.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Injection

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet:

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Immunohistochemistry, Cytometry, Purification, Recombinant, In Vitro, Western Blot, In Vivo, Injection, Neutralization, Control, Cell Isolation, Enzyme-linked Immunosorbent Assay, Gene Expression, Negative Control, Staining, Sequencing, Software

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a) Distribution of annotated single hits over MEG3 gene, with statistically filtered EZH2-FLASH reads from two biological replicates in HUVECs. b) The occupancy of EZH2 hits over MEG3 features. Total reads per feature are given with exons being mostly occupies vs introns. c) Proportion of overlapping features over MEG3. The occupancy of EZH2 over each MEG3 exon is shown for two constitutively expressed transcripts. For both given transcripts there is high occupancy of exon 3. d) RNA immunoprecipitation (RIP) for EZH2 and H3K27me3 (repressive chromatin) followed by qPCR analysis. RIP-purified RNA from UV crosslinked HUVECs was used to prepare cDNA for qPCR analysis with primers against MEG3 (exon 3 region). Primers against U1snRNA gene serves as a negative control. Side diagram of EHZ2-MEG3 interacting region is charted as per FLASH hits and sequence. e) Distribution of EZH2 hybrids hits over MEG3 gene. Intermolecular MEG3-RNA interactions found in chimeras are captured by EZH2-FLASH-seq. Hits represent MEG3:MEG3 hybrids (black). IgG hybrids are plotted but are <1. f) Total MEG3:MEG3 hybrid count against predicted free energy of hybridization (dG) for MEG3 interactions ( red lncRNA:MEG3, blue mRNA:MEG3, green MEG3:antisense, purple snoRNA:MEG3) with free hybridization energy cutoff at dG<-10 kcal mol -1 , as captured by EZH2-FLASH-seq ( i ) vs. IgG control ( ii ) .

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: RNA Immunoprecipitation, Purification, Negative Control, Sequencing, Hybridization, Control

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. Overview of the critical steps to obtain MEG3-bound genomic loci and intersections with EZH2 and H3K27me3 signals (obtained from GEO databases for HUVECs). In addition, enhancer regions were mapped within the genomic tracks. The intersection between GEO EZH2 ChIP data, GEO H3K27me3 ChIP data and statistically filtered MEG3-ChIRP data from two biological replicates was performed. The number of genes and degree of overlap is obtained between MEG3 and PRC2-dependent genes. The p-values are a result of hypergeometric test. b. Distribution of MEG3 peaks overlapping EZH2-ChIP peaks or H3K27me3-peaks with intersecting reads in relation to (i) gene regions and (ii) gene-type. c. Maximum peak score of ChIP signal for EZH2 and H3K27me3 intersecting the top enriched MEG3 peaks associated with nearest genes. Highest EZH2 peak score is over ITGA4, whereas H3K27me3 was detected in ITGA4, ITGA7, ITGA8 and ITGA9, members of ITGA family. d. Normalized reads from RNA-seq de novo analysis of GEO: GSE71164 dataset on Hg38, and expression of ITGA4 gene between Scr and siEZH2 depleted HUVECs, showing that ITGA4 is targeted by EZH2. Dataset in d and e is compared using Student’s t-test. e. ITGA4 expression from microarray analysis in C2C12 cells depleted of MEG3 (10nM, LNA GapMer) as per GEO dataset: GSE73524. The data shows that ITGA4 is a direct target of MEG3. f. (i) Total number of representable peaks (mRNA, antisense and lncRNA genes) from ChIP-seq analysis of Scr vs. MEG3 KD HUVECs. (ii ) Depletion of MEG3 gene in HUVECs (10nM LNA gapmers) was achieved with relative expression showing ∼70% reduction compared with Scr control. g. (i) Heat map showing distribution of reads and EZH2 densities at all unique RefSeq genes within TSSs ± 3 kb, sorted by EZH2 occupancy, in Control vs. MEG3 deficient (10nM) HUVECs. (ii) Overlap of ChIP-results between MEG3 and EZH2-dependent genes, with overlapped genes belonging to the biological pathway regulating cell adhesion. The common targets had lost or reduced EZH2 ChIP-signal.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: RNA Sequencing, Expressing, Microarray, ChIP-sequencing, Control

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a) Computational analysis pipeline used to obtain orthologous peaks in human and intersect regions and genes enriched in repressive chromatin (H3K27me3) from ChIP-seq public dataset GSE114283. Up- and down-regulated genes were obtained associated with the peak region within 2000bp, and relevant function and biological pathway were associated using GREAT and DAVID analysis b) Overlap of the GEO datasets from a (Microarray GSE73524 ) and b (RNA-seq GSE71164 ) and the GSE114283 ChIP-seq reads of H3K27me 3 distribution in mouse MN cells depleted of MEG3 vs. control. ChIP extracted peaks unique to Ctrl vs. MEG3 KD were obtained, and associated mouse gene list composed based on reduction in H3K27me 3 signal. Using gene orthologous analysis in gProfiler we obtained human orthologous targets that was used for data intersection. c) Maximum peak scores of the overlapping signal over ITGA4 promoter, obtained by intersection of EZH2 ChIP signal with MEG3-ChIRP signal at this region. Upon depletion of MEG3 the EZH2 signal is significantly reduced whereby no overlap with MEG3 ChIRP signal is seen. d) Relative expression of ITGA4 in HUVEC measuring the levels of ITGA4 following addition of siRNA (50nM).

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: ChIP-sequencing, Microarray, RNA Sequencing, Control, Expressing

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. Venn diagram showing the intersection between statistically filtered FLASH data from two biological replicates of our MEG3-ChIRP-seq-data (green), de novo hg38 analysed GEO RNA-seq data from siEZH2 deficient HUVECs (GSE71164, blue), and EZH2 ChIP-seq following MEG3 KD (yellow) and FLASH-seq transcriptome following EZH2 IP (pink). b. Correlation between gene expression levels and FLASH signal. Gray, expressed RefSeq genes with reproducible FLASH signal consistently detected in RNA-seq. Blue, genes with the highest RNA-seq signals and no reproducible FLASH signal belonging to integrin cell surface interaction pathway. Red , expressed ITGA4 gene, and green, ITGB1 gene, without reproducible FLASH signals. Data are from two biological replicates of each EZH2 FLASH sample and three biological replicates of EZH2 RNA-seq samples (Scr vs. siEZH2, GSE71164). c. Genomic tracks showing ChIRP-seq signal (MEG3 Odd, Even and LacZ) in HUVECs over ITGA4 gene only. The MEG3 binding site is located upstream of the ITGA4 gene in the promoter region, and it overlaps with the H3K27me3 signal and EZH2; as well as downstream within the ITGA4 gene body, where it overlaps with within the EZH2 signal in the intronic region of the gene. d. MEG3-ChIRP followed by qPCR, analysis of MEG3 binding region on ITGA4 in HUVECs. The crosslinked cell lysates were incubated with combined biotinylated probes against MEG3 lncRNA and the binding complexes recovered by magnetic streptavidin-conjugated beads. The qPCR was performed to detect the enrichment of specific region that associated with MEG3, peaks were related to input control and compared vs. the non-biotynilated control. e. ChIP-QPCR enrichment for EZH2 and H3K27me3 over ITGA4 promoter region in HUVECs depleted of MEG3 vs. Control.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: RNA Sequencing, ChIP-sequencing, Gene Expression, Binding Assay, Incubation, Control, ChIP-qPCR

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. ChIP signal enrichment vs . 1% input for EZH2 and H3K27me3 mark over ITGA4 promoter regions in HUVECs treated with A-395 (5µM, 24h) inhibitor of PRC2 vs. Control (DMSO). The expression was measured using two sets of primers against the same promoter region of ITGA4. Representative graphs are average of three qPCR datasets ± SEM. b. ITGA4 expression in the presence of A-395 vs . DMSO control, N=6 independent experiments compared using t -test. c. Measuring the expression levels of ITGA4 upon depletion of MEG3 using LNA GapmeRs (10nM, 48h), data is mean of N=5 independent experiments (biological replicates). d. Representative image of immunofluorescence staining for ITGA4 protein levels in ECs treated with A-395 vs . DMSO, or upon MEG3 depletion like in b . e. Intra-cellular localisation of MEG3 (chromatin associated lncRNA) between different cellular compartments in HUVECs treated with A-395 vs. DMSO, whereby the distribution of MEG3 has shifted upon PRC2 inhibition with A-395; from the nucleus (where it was highly chromatin bound) into the cytoplasm. Representative bars were compared by t-test and on-way Anova. f. MEG3-ChIRP followed by qPCR, N =3, analysis of MEG3 binding over ITGA4 promoter region in HUVECs treated with A-395 (5µM, 24h) vs. DMSO. MEG3-ChIRP HUVEC lysates treated with A-395 resulted in reduced engagement of MEG3 with ITGA4 site compared with either DMSO control or ChIRP with non-biotinylated probes. The non-biotin probes served as a negative control, and we detected the background level <1.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: Control, Expressing, Immunofluorescence, Staining, Inhibition, Binding Assay, Negative Control

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. Measure of cell migratory capacity using ECIS functional analysis in ECs treated with control or A-395 (5µM, 24h) inhibitor. Experiments were performed in duplicates (technical replicates) and four experiments were run for migration assay and six for adhesion (biological replicates). The data showing ECIS trace (left hand side) is mean ±SD as calculated by the ECIS. The graph on the right is mean±SEM with N =6, data was compared using ordinary one-way ANOVA with Dunnett’s multiple comparisons tests. b. Adhesion to Fibronectin, FN (20µg/ml) was used to coat the culture plates and assess adhesion of endothelial cells within 3h of ECIS assay, following cell pre-treatment with A-395, 24h. The difference in resistance change was calculated over 3h. c. Subcutaneous Matrigel plug injection (200µl) into mice ( N =5) treated with DMSO (control, left flange) and A-395 (1mg/ml, right flange) was done for 2 weeks. Matrigel plugs were collected and processed for histology. Staining for H3K27me3 was done, displaying nuclear positivity with strong intensity in control (<0.02% DMSO in water) and the A-395 treatment decreased total H3K27me3 staining, as compared by t-test. d. Staining for arterioles was performed to assess vessel growth as angiogenesis and data was compared using Student’s t-test. The data shows increased area of staining for Isolectin B4 (Iso-B4) dye in A-395 vs. DMSO treated Matrigel plugs with increased neovascularization, P<0.05. e. A-395 has increased the percentage of vessels positive for ITGA4 (red) within the Isolectin B4 positive cells, compared with the DMSO using t -test. f. Graphical abstract. 1 Maternally Expressed Gene–MEG3 is highly expressed with hypoxia and bound to EZH2 in endothelial cells (EC) affected by ischaemic insult. 2 Such MEG3:EZH2 complex assembles onto the target genes to 3 direct the EZH2 activity to “write” H3K27me3 trimethylation repressive mark and block expression of target gene i.e. integrin alpha 4 (ITGA4) and its ability to dimerise with integrin beta 1 (ITGB1), leading to 4 reduced EC function as measured by adhesion and migration. Hence 5 targeted disruptions of MEG3:EZH2 interaction, or inhibition of EZH2 activity could increase EC function under ischaemia.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: Functional Assay, Control, Migration, Injection, Staining, Activity Assay, Blocking Assay, Expressing, Inhibition

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: HDAC1, HDAC2, and HDAC3 were upregulated in breast cancer tissues and correlated with worse prognosis in breast cancer patients. ( a ) Representative immunohistochemical (IHC) staining photos of HDAC1, HDAC2, and HDAC3 in breast specimens. HDAC1, HDAC2, and HDAC3 levels were obviously elevated in the tumor tissues compared to the non‐neoplastic adjacent tissues of patients with invasive ductal carcinoma (IDC). Yellow boxes indicated mammary ducts. Scale bars, 100 μm. ( b ) Representative IHC photos of three different kinds of HDAC3 subcellular localization. Scale bars, 100 μm. ( c – f ) Overall survival (OS) curves of 139 IDC patients with different HDAC1 ( c ) ( ) HDAC1 Low ( ) HDAC1 High ( ) HDAC1 low‐censored ( ) HDAC1 high‐censored, HDAC2 ( d ) ( ) HDAC2 Low ( ) HDAC2 High ( ) HDAC2 low‐censored ( ) HDAC2 high‐censored, cytoplasmic HDAC3 ( e ) ( ) HDAC3 C‐low ( ) HDAC3 C‐high ( ) HDAC3 C‐low‐censored ( ) HDAC3 C‐high‐censored, and nuclear HDAC3 ( f ) levels ( ) HDAC3 N‐low ( ) HDAC3 N‐high ( ) HDAC3 N‐low‐censored ( ) HDAC3 N‐high‐censored. According to another set of criteria in which cases with either high nuclear or cytoplasmic expression were classified into the C‐high/N‐high group and other cases were classified into the Others group, the overall survival curve of the 139 IDC patients was reproduced as Figure 1g ( ) HDAC3 Others ( ) HDAC3 C‐high/N‐high ( ) HDAC3 Others‐censored ( ) HDAC3 C‐high/N‐high‐censored. P values of the Kaplan‐Meier plots in (c‐g) were calculated by log‐rank test in IBM SPSS Statistics 19 software. ( h – j ) Kaplan‐Meier survival curves with log‐rank analysis were used to assess the correlation between HDAC1 ( h ) ( ) HDAC1 Low (≤ 75th percentile) ( ) HDAC1 High (> 75th percentile), HDAC2 ( i ) ( ) HDAC2 Low (≤ 75th percentile) ( ) HDAC2 High (> 75th percentile), and HDAC3 ( j ) ( ) HDAC3 Low (≤ 80th percentile) ( ) HDAC3 High (> 80th percentile) expression and overall survival of 4903 breast cancer patients in the bc‐GenExMiner platform (website: http://bcgenex.centregauducheau.fr ; all DNA microarray data, node mixed, ER mixed; optimized split for HDAC1 and 2, an 80th percentile customized cutoff for HDAC3).

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Software, Microarray

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: HDACs expression exhibited different roles in overall survival of IDC patients ( n = 16l)

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Expressing

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: Relationship between clinicopathological characteristics and HDACs expression in IDC patients ( n = 139)

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Expressing, Over Expression

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: HDACs expression exhibited different roles in the onset of brain metastasis of IDC patients ( n = 161)

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Expressing

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: The roles of HDACs expression and other clinicopathological characteristics played in the prognosis of breast cancer patients after brain metastasis ( n = 63)

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Expressing

Journal: Integrative zoology

Article Title: cba-miR-222-3p involved in photoperiod-induced apoptosis in testes of striped hamsters by targeting TRAF7.

doi: 10.1111/1749-4877.12918

Figure Lengend Snippet: Figure 3 Validation of cba-miR-222-3p targeting TRAF7 and TRAF7 expression in the testes of striped hamsters. (a) Sequences and peak maps of cba-miR-222-3p, TRAF7-WT, and TRAF7-MT. (b) Relative luciferase activity detected by Dual-Luciferase Reporter Assay. (c) Immunohistochemistry (IHC, tissue microarray [TMA]) of TRAF7 in testes. (d) Integrated density of TRAF7 detected by IHC (TMA; n = 4). (e) Protein expression levels of TRAF7 in the testes detected by western blot (n = 4). (f) Pearson correlation analysis of cba-miR-222-3p and TRAF7. LD, long daylength; MD, moderate daylength; SD, short daylength; ∗, P < 0.05; ∗∗, P < 0.01.

Article Snippet: After performing electrophoresis, the proteins were transferred to PVDF membranes, which were then incubated with TRAF7 antibodies (11780-1-AP, Proteintech, China, RRID:AB_2877793) at a 1:1000 dilution and β-actin antibodies (20536-1-AP, Proteintech, China, RRID:AB_10700003) at a 1:1000 dilution, and subsequently incubated with IRDye 800 CW goat anti-rabbit secondary antibodies (31 460, Thermo Fisher, USA).

Techniques: Biomarker Discovery, Expressing, Luciferase, Activity Assay, Reporter Assay, Immunohistochemistry, Microarray, Western Blot

Journal: Integrative zoology

Article Title: cba-miR-222-3p involved in photoperiod-induced apoptosis in testes of striped hamsters by targeting TRAF7.

doi: 10.1111/1749-4877.12918

Figure Lengend Snippet: Figure 4 Expression of cba-miR-222-3p and TRAF7 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis after in vivo injection in the testes of striped hamsters. (a) Schematic diagram of in vivo injection experiment of miRNA mimics. (b) Fluorescence in situ hybridization (FISH, tissue microarray [TMA]) of cba-miR-222-3p in testes after in vivo injection. (c) Fluorescence intensity of cba-miR-222-3p detected by FISH (TMA; n = 4). (d) Immunohistochemistry (IHC, TMA) of TRAF7 in testes after in vivo injection. (e) Integrated density of TRAF7 detected by IHC (TMA; n = 4). (f) TUNEL (TMA) staining of the testes after in vivo injection. (g) The proportion of TUNEL (TMA) staining with apoptotic activity in the testes (n = 4). (h) Relative expression of MEKK3 (n = 6). (i) Relative expression of p38 (n = 6). (j) Relative expression of p53 (n = 6). AG, agomir injection; NC, agomir negative control injection; DAPI, 4′6′-diamidino-2-phenylindole. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: After performing electrophoresis, the proteins were transferred to PVDF membranes, which were then incubated with TRAF7 antibodies (11780-1-AP, Proteintech, China, RRID:AB_2877793) at a 1:1000 dilution and β-actin antibodies (20536-1-AP, Proteintech, China, RRID:AB_10700003) at a 1:1000 dilution, and subsequently incubated with IRDye 800 CW goat anti-rabbit secondary antibodies (31 460, Thermo Fisher, USA).

Techniques: Expressing, TUNEL Assay, In Vivo, Injection, Fluorescence, In Situ Hybridization, Microarray, Immunohistochemistry, Staining, Activity Assay, Negative Control

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: RT-PCR primer list

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques:

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Absence of TRKB receptors result in reduced expression of Jagged1 and NOTCH target genes in the mouse ovary. Panel A, Decrease in Jagged1 mRNA content in the ovary of 7-d-old TrkB−/− mice detected using cDNA microarrays. Changes in mRNA content are expressed as fold-decrease with respect to mRNA values in TrkB+/+ mice of the same age. Filled squares represent the values detected in independent microarray determinations. Panel B, Jagged1 mRNA content was reduced in the ovary of 7-d-old TrkB−/− mice, as assessed by real-time PCR. Inset, Jagged1 mRNA content increased in 7-d-old WT ovaries treated in vitro with BDNF (100 ng/ml, 8 h) compared to control (C) ovaries incubated with vehicle. Relative mRNA values are expressed as arbitrary units (AU), normalized using 18s RNA or Ppia mRNA values as the normalizing unit. Panel C, Hes1 and Hey2, but not Notch, mRNA abundance, was also reduced in TrkB-null ovaries. Panel D, JAGGED1 immunoreactive material (green color) mostly localizes to oocytes in the ovary from 7-d-old TrkB+/+ mice. Panel E, JAGGED1 immunoreactivity was noticeably decreased in oocytes of TrkB−/− mice. Panel F, Section incubated without JAGGED1 antibodies. Cell nuclei stained with the DNA-binding dye Hoechst are shown in blue. White arrows point to examples of oocytes showing JAGGED1 staining. Columns in B and C represent means from four to five animals per group, and vertical lines are sem. *, P < 0.05; **, P < 0.01 vs. WT controls. Scale bars, 50 μm.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Microarray, Real-time Polymerase Chain Reaction, In Vitro, Incubation, Staining, Binding Assay

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Changes in ovarian content of Jagged1, Notch2, Hes1, and Hey2 mRNA during the first postnatal week of life of the mouse, as assessed by real-time PCR. A, Jagged1 mRNA. B, Notch2 mRNA. C, Hes1 mRNA. D, Hey2 mRNA. Relative mRNA values are expressed as arbitrary units (AU), normalized using 18s RNA values as the normalizing unit. E–J, In situ hybridization, using a mouse-specific 35S-uridine triphosphate-labeled Jagged1 cRNA probe, shows that Jagged1 mRNA is exclusively expressed in oocytes and that the abundance of Jagged1 mRNA increases during the first 12 d of postnatal life. Bright field images are shown in E–G and dark field images in H–J. Black and white arrows point to examples of Jagged1 mRNA-containing oocytes. Bars represent the mean of four to five mice per group, and vertical lines are sem. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. 0-d-old group. Scale bar, 50 μm.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Real-time Polymerase Chain Reaction, In Situ Hybridization, Labeling

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Lentiviral-mediated delivery of Jagged1, using the Gdf9 promoter to target expression of a JAGGED1-HA fusion protein to oocytes, correctly targets JAGGED1 to the cell membrane of oocytes. A, Map of the lentiviral delivery construct (LV-Jagged1) used in this study. The lentiviral vector employed has been previously described (79). The 3′LTR of this vector contains a 400-bp deletion that results in the self-inactivation (SIN) of the vector. The other components include the packaging signal (ψ), the Rev response element binding site (RRE), the central polypurine tract (cPPT), and the woodchuck-hepatitis-virus posttranslational regulatory element (wPRE). The LV-Jagged1 construct contains a bicistronic transgene cassette in which expression of a Jagged1-HA cDNA is driven by the rat Gdf9 promoter (Gdf9p). The Jagged1-HA cDNA is linked to an enhanced green fluorescent protein (eGFP) cDNA via an internal ribosome entry site (IRES). A construct lacking Jagged1-HA (LV-no Jagged1) was used as a negative control. B–D, Immunohistofluorescent images of sections from 3-d-old mouse ovaries cultured for 4 d in the presence of LV-Jagged1 and stained with monoclonal antibodies against the HA epitope. E, Section from an ovary not infected with LV. F, Section from an ovary infected with LV-Jagged1 and incubated without HA antibodies. G, Section from an ovary infected with LV-no Jagged1. JAGGED1 immunoreactive cells are seen in red, and cell nuclei stained with the DNA-binding dye Hoechst are shown in blue. Scale bar, 50 μm.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Construct, Plasmid Preparation, Binding Assay, Negative Control, Cell Culture, Staining, Infection, Incubation

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Oocyte-specific restoration of JAGGED1 expression, via lentiviral-mediated gene transfer, rescues the deficit in follicle growth of TrkB−/− ovaries. A, Increased number of secondary follicles in TrkB−/− ovaries incubated for 4 d with LV-Jagged1 in comparison with TrkB−/− ovaries infected with LV-no Jagged1. B, Section from a TrkB−/− ovary infected with LV-no Jagged1. C, Section from a TrkB−/− ovary infected with LV-Jagged1. Arrows point to secondary follicles, which contain an oocyte surrounded by two layers of GC. Scale bar, 50 μm. Columns represent the mean of four mice per group, and vertical lines are sem. One ovary from each animal was infected with LV-Jagged1 and the contralateral ovary from the same animal with LV-no Jagged1. *, P < 0.05.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Incubation, Infection

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Oocyte-specific restoration of JAGGED1 expression, via lentiviral-mediated gene transfer, rescues the deficit in GC proliferation of TrkB−/− ovaries. A, Percent of follicles showing at least one PCNA-positive GC. B, Number of PCNA-positive GC per follicle (primary and secondary). C, Image of a section from a TrkB−/− ovary incubated for 4 d with LV-no Jagged1. D, A section from a TrkB−/− ovary incubated for 4 d with a LV-Jagged1. E, Ovarian section immunostained in absence of primary antibodies. Columns represent the mean of four mice per group, and vertical lines are sem. In each group, four sections per ovary were used for quantification. One ovary from each animal was infected with LV-Jagged1 and the contralateral ovary from the same animal with LV-no Jagged1. Scale bar, 50 μm. ***, P < 0.001 vs. LV-no Jagged1.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Incubation, Infection

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: TrkB signaling sustains c-Myc and Odc1 expression but not the expression of core regulatory components of the cell cycle. Panel A, c-Myc mRNA content was reduced in 7-d-old TrkB−/− ovaries as compared with WT animals. Inset, In vitro exposure of WT ovaries to NT4/5 (100 ng/ml, 8 h) increased c-Myc mRNA abundance as compared to control (C) ovaries incubated with vehicle. Panel B, Odc1 mRNA abundance was also decreased in TrkB−/− ovaries. Inset, NT4/5 increased Odc1 mRNA abundance in WT ovaries. Panels C and D, The content of mRNA encoding cyclins (CycD2 and CycE1), CDK (Cdk2 and Cdk4), and the CKI of the INK4 family (p15INK4b, p16INK4a, p18INK4c, and p19INK4d) and CIP/KIP family (p21Cip1, p27Kip1, and p57Kip2) remain unaltered in TrkB−/− ovaries as compared with TrkB+/+ mice. Panel E, c-Myc mRNA levels were increased in TrkB−/− ovaries after oocyte-specific restoration of JAGGED1 synthesis. The ovaries from 3-d-old mice were incubated for 4 d with a lentiviral construct carrying the Jagged1-coding region under the control of the Gdf9 promoter. Panel F, Neither p19INK4D nor p27Kip1 mRNA levels changed after lentiviral-mediated restoration of JAGGED1 synthesis. Control ovaries were infected with a LV lacking Jagged1 cDNA (LV-no Jagged1). Each column represents the mean of four to five mice per group, and vertical lines are sem. One ovary from each animal was infected with LV-Jagged1 and the contralateral ovary with LV-no Jagged1. *, P < 0.05; **, P < 0.01 vs. their respective controls. AU, Arbitrary units.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, In Vitro, Incubation, Construct, Infection