Journal: bioRxiv

Article Title: Modeling motor neuron resilience in ALS using stem cells

doi: 10.1101/399659

Figure Lengend Snippet: (A) Time line depicting in vitro protocols for resistant oculomotor neurons (OMN) and vulnerable spinal motor neuron (SC MN) generation. ESC column reports the four cell lines used in this study. Patterning was performed for 5 days after ESC expansion and EB formation. mRNA-seq coupled with FACS was performed on EB dissociation day (day 9 of the protocol). Survival assay was performed after five days from EB dissociation. (B-C) SC MNs generated from E14.1 ESCs express Hb9, Islet1 and NF200, OMNs generated from E14.1 ESCs over-expressing the transcription factor Phox2a under the Nestin enhancer co-express Islet1 and NF200 in absence of Hb9. Scale bar in c 60 μm. (D) Percentage of Hb9+/Islet1+ cells in SC MN (64.1±5.2%, mean±SEM, n=4) and OMN (6.3±1.3%, mean±SEM) cultures respectively. (For quantification, experiments were run in quadruplicates including two technical replicates per experiment, with at least 120 Islet1+ cells counted per condition). (E) Microphotographs showing Islet1+/Tuj1+ cells in OMN cultures, (F) OMNs also express the specific marker Phox2a as indicated by asterisks. Scale bars in e and g 100 μm. (G) Quantification of Islet1+ over Tuj1+ cells demonstrates that half neuronal population appears to be Islet1+ (47.5±5.9%, mean±SEM, experiments performed in quadruplicates with two technical replicates each, total number of Tuj1+ cells counted: 1325). Quantification of Phox2A+ over Islet1+ cells (experiments performed in quadruplicates with two technical replicates each, total number Islet1+ cells counted: 746) indicates that 62±5.2% (mean±SEM) of the Islet1+ population is also Phox2a+. All quantifications were performed 5 days after EB dissociation (mean ± SEM). (H) PCA of OMN and SC MN samples based on all genes expressed confirmed cell differential identities. mRNA-seq analysis of OMNs and SC MNs isolated by FACS was performed after 5 days of patterning (day 9 of the protocol). (I) 1,017 differentially expressed genes were found between the two different cell types (adjusted P<0.05). Heatmap shows the top 500 most significant differentially expressed genes by adjusted p-value. (J) Heatmap of expected progenitors and motor neuron transcripts but also OMN and SC MN specific transcripts obtained in the two generated motor neuron populations. (K) Using the top 100 DEGs obtained from our RNAseq analysis we could separate datasets originating from in vivo microarray studies on early postnatal (J) and adult (L) rodent OMNs and SC MNs. (M) Venn diagram showing gene sets enriched either in brain stem cultures or OMN-specific as revealed by PAGODA analysis, (N) gene sets preferentially found in spinal cord cultures or MN-specific.

Article Snippet: The following primary antibodies were used: NF200 (1:1000, #AB5539 Millipore), Tuj1 (1:1000, MRB-435P Covance), Hb9 (1:10, #81.5C10 DSHB), Islet1 (1:500, #ab20670 abcam; 1:100, #40.2D6 DSHB), Phox2A (1:1000, gift of Prof JF Brunet), GFP (1:1000, #ab13970 abcam), phospho-AKT (Ser473) (1:50, #3787 Cell Signaling), activated beta-catenin (1:1000, #05-665 Millipore), alpha-catenin (1:1000, #AB153721 AbCam), ESYT1 (1:100, #HPA016858), BrdU (1:10, #G3G4 DSHB).

Techniques: In Vitro, Clonogenic Cell Survival Assay, Generated, Expressing, Marker, Isolation, In Vivo, Microarray

Journal: bioRxiv

Article Title: Modeling motor neuron resilience in ALS using stem cells

doi: 10.1101/399659

Figure Lengend Snippet: (A) Oculomotor neurons (OMNs) and spinal motor neurons (SC MNs) express similar mRNA levels of glutamate ionotropic receptor AMPA, NMDA and kainate type subunits. The heatmap shows log2 RPKM values of these subunits and no separate clustering of the two cell types is observed. (B-C) Immunohistochemistry performed on generated OMNs and SC MNs at D1 in vitro in control conditions; similar levels of glutamate ionotropic receptor kainate type subunit 5 (Grik5) are found in both cell types. Scale bar in d 60 μm. (D-E) Microphotographs presenting SC MN and OMN response to kainic acid induced toxicity (20 μM) for a week. Scale bars in f = 100 μm. (F) Curves represent percentages of MN survival over time in OMN and SC MN cultures. OMNs were visualized as NF200+Islet1+Hb9- clls, while SC MNs as NF200+Islet1+Hb9+ cells. OMNs show increased survival to KA toxicity at D7 (mean ± SEM, 2way ANOVA and Tukey’s multiple comparison test, F(9, 56)=2.333, *P=0.0261, SC MNs n=4; OMNs n=5) when compared to SC MNs (experiments were performed at least in quadruplicates, with technical replicates and with at least 130 motor neurons counted per condition in each experiment). Analysis of the length of neuronal processes in both oculomotor and spinal motor neuron cultures exposed to kainic acid for seven days. showed that oculomotor neurons were unaffected by kainic acid while spinal motor neurons displayed a shortening of neurites (G). (H-K) Sholl analysis was performed on OMN at D7 survival assay in control and KA20 conditions to further assess individual MN arborization complexity during toxicity. Scale bar = 100 μm. (I) Sholl mask was applied to individual OMNs after specifying the radius from the center of the soma of the neuron and created concentric circles every 25 μm of increasing radius. (J) Comparison of average number of neurite intersections of OMN in control and KA20 toxicity conditions with radial step size of 25 μm. OMNs did not show reduction in arborization (multiple t test, n = 10 per condition). (K) Schematic depicting identification of neurite segments by Sholl analysis. Color code is assigned depending on arbor localization from the soma in an inside-out manner following the given radius. Multiple intersections within the same segment display the same colour.

Article Snippet: The following primary antibodies were used: NF200 (1:1000, #AB5539 Millipore), Tuj1 (1:1000, MRB-435P Covance), Hb9 (1:10, #81.5C10 DSHB), Islet1 (1:500, #ab20670 abcam; 1:100, #40.2D6 DSHB), Phox2A (1:1000, gift of Prof JF Brunet), GFP (1:1000, #ab13970 abcam), phospho-AKT (Ser473) (1:50, #3787 Cell Signaling), activated beta-catenin (1:1000, #05-665 Millipore), alpha-catenin (1:1000, #AB153721 AbCam), ESYT1 (1:100, #HPA016858), BrdU (1:10, #G3G4 DSHB).

Techniques: Immunohistochemistry, Generated, In Vitro, Clonogenic Cell Survival Assay

Journal: Nature Communications

Article Title: PBRM1 loss defines a nonimmunogenic tumor phenotype associated with checkpoint inhibitor resistance in renal carcinoma

doi: 10.1038/s41467-020-15959-6

Figure Lengend Snippet: a Pbrm1 knockout validation in Renca cells at protein levels by western blot, and b at mRNA levels by real-time PCR. Renca cell were treated with or without 1 ng/ml IFNγ for 8 h. c IFNγ-induced JAK-STAT1 expression and phosphorylation in Renca cells. Control KO or Pbrm1 KO (clone #18) Renca cells were treated with 1 ng/ml IFNγ for 2 or 8 h. Cell lysates were analyzed by immunoblot using antibodies against PBRM1, STAT1, P-STA1 Y701, P-STAT1 S727, JAK2, P-JAK2 Y1007/1008, JAK1, P-JAK1 Y1034/1035, IRF1. β-actin was used an internal control. d IFNγ-induced gene expression in Renca cells. Control KO or Pbrm1 KO (clone #18) Renca cells were treated with 1 ng ml IFNγ for 8 h. mRNA expression of Stat1 , Cxcl9 , Irf1 , and Icam1 were detected by real-time PCR. Gapdh was used as internal control. e IFNγ-induced CXCL9 secretion. Renca cells were cultured in serum-free medium and treated with 1 ng/ml IFNγ for 4 or 10 h. The concentration of CXCL9 was analyzed using Quantikine® ELISA kit. f IFNγ-induced JAK-STAT1 expression and phosphorylation in 786-O cells. Control knockdown (Con KD) or PBRM1 knockdown ( PBRM1 KD) 786-O cells were treated with or without 10 ng/ml IFNγ for 2 h. Cell lysates were analyzed by immunoblot using antibodies against PBRM1, STAT1, P-STA1 Y701, JAK2, P-JAK2 Y1007/1008, and IRF1. β-actin was used an internal control. g IFNγ-induced gene expression in 786-O cells. 786-O cells were cultured in DMEM with 10% FBS, and treated with 10 ng/ml IFNγ for 8 h. mRNA expression of STAT1 , CXCL9 , and IRF1 were detected by real-time PCR. GAPDH was used as internal control. Unpaired t -test was performed with GraphPad Prism 7.03. * P < 0.05 and ** P < 0.001, compared with control knockout or knockdown cells. All data are representative of three independent experiments. Data in the bar graphs represent mean ± S.D., n = 3. Source data are provided as a Source Data file.

Article Snippet: PBRM1 antibody (A301-591A) was from Bethyl Laboratories.

Techniques: Knock-Out, Biomarker Discovery, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Phospho-proteomics, Control, Gene Expression, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Knockdown

Journal: Nature Communications

Article Title: PBRM1 loss defines a nonimmunogenic tumor phenotype associated with checkpoint inhibitor resistance in renal carcinoma

doi: 10.1038/s41467-020-15959-6

Figure Lengend Snippet: a BRG1 and STAT1 binding to Cxcl9 and Cxcl10 promoter in Renca cells. b BRG1 and SP1 binding to Ifngr2 promoter. Chromatin immunoprecipitation (ChIP) with BRG1, STAT1 or SP1 antibody as indicated in figures was performed using SimpleChIP® Plus Enzymatic Chromatin IP Kit. Isotype IgG was used as negative control. Immunoprecipitated DNA was amplified and quantified by real-time PCR. Protein relative occupancy on promoter was expressed as a percent of the total input chromatin. Con KO or Pbrm1 KO Renca cells were treated with 1 ng/ml IFNγ for 2 h for IFNγ-induced STAT1 binding to Cxcl9 or Cxcl10 promoter. c IFNγ receptor subunits, Ifngr1 and Ifngr2 , mRNA expression detected by real-time PCR. Gapdh was used as internal control. d IFNγ receptor subunits, IFNGR1 and IFNGR2, protein expression was detected by western blot. β-actin was used an internal control. e IFNGR2 membrane expression was detected by flow cytometry. Unpaired t -test was performed with GraphPad Prism 7.03. * P < 0.05 and ** P < 0.001, compared with control knockout cells. Data in the bar graphs represent mean ± S.D. Source data are provided as a Source Data file.

Article Snippet: PBRM1 antibody (A301-591A) was from Bethyl Laboratories.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Negative Control, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot, Membrane, Flow Cytometry, Knock-Out

Journal: Nature Communications

Article Title: PBRM1 loss defines a nonimmunogenic tumor phenotype associated with checkpoint inhibitor resistance in renal carcinoma

doi: 10.1038/s41467-020-15959-6

Figure Lengend Snippet: a The confidence interval plots represented the differences in these genes between the two groups ( Pbrm1 KO minus Pbrm1 WT). The x -axis is the difference in group means of Pbrm1 knockout group minus Pbrm1 wild-type group, so negative values correspond to genes downregulated in the Pbrm1 knockout group. The lines depicted 95% confidence intervals, and genes with a significant difference between the groups (adjusted P -value <0.05) were marked in orange. The P -values were obtained from a two-sample t-test on the log2-transformed values, and the resulting P -values were adjusted for multiple comparisons using the Benjamini–Hochberg method across the gene set. b mRNA expressions of Ifng , Cxcl9 , Cxcl10 , and Pdcd1 and c the mRNA expression of Pbrm1 , Ifngr1 , Ifngr2 , and Cd274 were detected by real-time PCR. Each dot represents the mean value of triplicated tumor samples. Unpaired t -test was performed with GraphPad Prism 7.03. d The coordinated differences of the genes as listed in ( a ) across the two groups assessed by gene set enrichment analysis (GSEA). e T cell infiltration and quantification. Murine Renca tumor microarrays, with triplicate formalin-fixed tissue cores for each case, were immunohistochemically stained with antibodies against CD3, CD8, CD4, PD-1, and P-STAT1 Y701. The percentages of positively stained cells were analyzed using inForm software. Unpaired t-test was performed with GraphPad Prism 7.03. Scale bar, 100 µm. f Multiplex Opal Immunofluorescence staining. The slides were stained with primary antibodies against CD8 and PD-1, corresponding HRP conjugated secondary antibodies, and subsequently TSA dyes to generate Opal signal (CD8, 520 nm; PD-1, 570 nm). PD-1 Opal signals are artificially colored as red. Scale bar, 50 µm. g Immunomodulatory gene expression signature score in pre-malignant murine kidneys following loss of Vhl alone ( Vhl −/− ), or Vhl in combination with Pbrm1 ( Vhl −/− Pbrm1 −/− ). Student t -test. h Gene expression-based inference of total T cell and CD8 T-cell infiltrates in pre-malignant murine kidneys following loss of Vhl alone, or Vhl in combination with Pbrm1 . Student t -test. Data in the graphs represent mean ± S.D. Source data are provided as a Source Data file.

Article Snippet: PBRM1 antibody (A301-591A) was from Bethyl Laboratories.

Techniques: Knock-Out, Transformation Assay, Expressing, Real-time Polymerase Chain Reaction, Staining, Software, Multiplex Assay, Immunofluorescence, Gene Expression

Journal: Nature Communications

Article Title: PBRM1 loss defines a nonimmunogenic tumor phenotype associated with checkpoint inhibitor resistance in renal carcinoma

doi: 10.1038/s41467-020-15959-6

Figure Lengend Snippet: a The confidence interval plots represented the differences in immunomodulatory genes between the two groups (mutant PBRM1 minus wild-type PBRM1 ) in TCGA KIRC dataset. MUT, mutant PBRM1 ; WT, wild-type PBRM1 . The lines depicted 95% confidence intervals, and genes with a significant difference between the groups (adjusted P -value <0.05) were marked in orange. The P -values were obtained from a two-sample t -test on the log2-transformed values, and the resulting P -values were adjusted for multiple comparisons using the Benjamini–Hochberg method across the gene set. b Comparison of gene expression in tumors with mutant PBRM1 (MUT) versus wild-type PBRM1 (WT) by GSEA using the immunomodulatory genes as listed in ( a ) and other predefined immune-related gene sets in the TCGA, IMmotion150 and ICGC patient cohorts. c Comparison of CD8 infiltrates as assessed by immunohistochemistry for CD8 in the IMmotion150 patient cohort. Rank-sum test. d CD8 immunostaining levels positively correlated with gene expression-based inference. Spearman correlation and associated P -value inset. e Gene expression-based inference of CD8 T cell infiltrates in patients stratified by PBRM1 mutation status. Rank-sum test. f Comparison of immune cell PD-L1 expression in patients from the IMmotion150 cohort stratified by PBRM1 mutation status. Cochran–Armitage test for trend. g Treatment naive RCC tumor microarray from 20 untreated RCC patients, including 15 samples with wild-type PBRM1 and 5 with PBRM1 mutations, were immunohistochemically stained with antibodies against CD3, CD45RO, CD8, and CD4. Each tumor was triplicated, and the n values indicate the number of the intact cores. The percentages of positively stained cells were analyzed using inForm software. Non-parametric Mann–Whitney test was performed with GraphPad Prism 7.03. Scale bar, 100 µm. h Multiplex Opal immunofluorescence staining. The slides were stained with primary antibodies against CD8 and PD-1, corresponding HRP conjugated secondary antibodies, and subsequently TSA dyes to generate Opal signal (CD8, 520 nm; PD-1, 620 nm). Scale bar, 50 µm. Data in the graphs represent mean ± S.D. Source data are provided as a Source Data file.

Article Snippet: PBRM1 antibody (A301-591A) was from Bethyl Laboratories.

Techniques: Mutagenesis, Transformation Assay, Comparison, Gene Expression, Immunohistochemistry, Immunostaining, Expressing, Microarray, Staining, Software, MANN-WHITNEY, Multiplex Assay, Immunofluorescence

Journal: Nature Communications

Article Title: PBRM1 loss defines a nonimmunogenic tumor phenotype associated with checkpoint inhibitor resistance in renal carcinoma

doi: 10.1038/s41467-020-15959-6

Figure Lengend Snippet: a Correlogram between the expression of immunomodulatory and angiogenic genes IMmotion 150 cohort (top line of each square), TCGA cohort (middle line of each square) and ICGC cohort (bottom line of each square). b Patients with mutant PBRM1 demonstrated increased CD31 immunostaining levels in the IMmotion150 cohort. Rank-sum test. c CD31 immunostaining levels positively correlated with angiogenesis expression score. Spearman correlation and associated P -value inset ( N = 119). d PBRM1 mutated tumors were associated with increased angiogenesis score in 3 indicated patient cohorts. Rank-sum test. e Pbrm1 knockout was associated with increased angiogenesis score in pre-malignant murine kidneys. Student t -test. f Pbrm1 knockout Renca tumors demonstrated increased CD31 immunostaining levels. Student t -test. Data in the bar graphs represent mean ± S.D. Source data are provided as a Source Data file.

Article Snippet: PBRM1 antibody (A301-591A) was from Bethyl Laboratories.

Techniques: Expressing, Mutagenesis, Immunostaining, Knock-Out

Journal: Nature Communications

Article Title: PBRM1 loss defines a nonimmunogenic tumor phenotype associated with checkpoint inhibitor resistance in renal carcinoma

doi: 10.1038/s41467-020-15959-6

Figure Lengend Snippet: a Early treatment with anti-PD-1 blockade. PD-1 antibodies were administrated at day 3, day 6, and day 9 after tumor inoculation. First dose was 400 µg/mouse, and the following two doses were 200 µg/mouse. b Delayed treatment with anti-PD-1 blockade. Anti-PD-1 antibody (200 µg/mouse) was administrated every third day once the tumors reached 100-200 mm 3 . Treatment schemas, In vivo tumor growth rates and survival rates of mice are shown. Two-way ANOVA and Log-rank (Mantel-Cox) analyses were performed with GraphPad Prism 7.03. Data in the graphs are means ± SEM. UnTX, untreated control. ns, P > 0.05, * P < 0.05, and ** P < 0.001. c Patient response in the IMmotion150 cohort following treatment with either atezolizumab (Atezo) or atezolizumab in combination with bevacizumab (Atezo + Bev). Mono, PBRM1 mutation only, Dual, PBRM1 mutation in combination with a BAP1 or SETD2 mutation; WT, wild type; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease. Cochran–Mantel–Haenszel test. d Comparison of patient response rate, defined as either a complete or partial response, in the IMmotion150 cohort. Cochran–Mantel–Haenszel test. e Overall survival of RCC patients treated with ICB from the MSKCC IMPACT cohort stratified by PBRM1 status. Log-rank test. f Overall survival of ccRCC patients from TCGA stratified by PBRM1 mutation status. Log-rank test. g Model for PBRM1 mediated IFNγ-STAT1 signaling and tumor immune microenvironment modulating. PBRM1 ensures IFNγ-induced STAT1 activity and autonomous expression of downstream genes involved in T-cell recruitment (e.g., CXCL9 ). Infiltrating, activated T cells in turn produce more IFNγ which stimulates tumor cells to secrete immunostimulatory chemokines and cytokines, and in parallel upregulate checkpoint pathways on T cells and tumors cells. Thus, the immunogenic TME of PBRM1 proficient tumors is primed to respond to ICB. On the other hand, PBRM1 loss reduces IFNγ-STAT1 signaling and downstream T cell attracting factors, which prevents T cell infiltration and IFNγ secretion. Such a non-immunogenic TME of PBRM1 mutated tumors blunts ICB response. Source data are provided as a Source Data file.

Article Snippet: PBRM1 antibody (A301-591A) was from Bethyl Laboratories.

Techniques: In Vivo, Control, Mutagenesis, Comparison, Activity Assay, Expressing

Journal: Nature medicine

Article Title: Enhancer mapping uncovers phenotypic heterogeneity and evolution in patients with luminal breast cancer

doi: 10.1038/s41591-018-0091-x

Figure Lengend Snippet: A) Global Kaplan-Meier analysis summarize univariate analysis for 22278 genes included in the Affymetrix microarray platform. Hazard Ratios are plotted in the X axis B) SLC9A3R1 RNA levels pre- and post- short-term aromatase inhibitor treatment in responder and non-responder patients 61 . Oestrogen-dependent expression of progesterone receptor mRNA is shown as comparison C) Silencing SLC9A3R1 leads to proliferation arrest in response to estrogen stimulation in MCF7 and estrogen independent growth in LTED cells. Proliferation assays were conducted in biological triplicate. Symbol and error bars indicate average and 95% confidence intervals. Asterisks represent significance at P<0.05, 0.01, 0.001 and 0.0001 after two-way ANOVA with Bonferroni’s correction D) RIs for the SLC9A3R1 enhancer within all the individual patients included in the current study. SLC9A3R1 enhancer location and its 3D interactions are shown in the top right inset E) SLC9A3R1 enhancer ranking analysis of available Epigenome Roadmap H3K27ac datasets. Tissues are displayed from the strongest to the weakest SLC9A3R1 enhancer activity (based on RI). Representative IHC analysis of normal tissues stained with a SLC9A3R1 antibody are shown (Scale bars, 50 μm). F-G) YY1 and SLC9A3R1 IHC analysis of BC patients profiled using H3K27ac ChIP-seq. Predicted activity (RI) of YY and SLC9A3R1 enhancers is shown on the X axis. The number of cells positively stained for YY1 and SLC9A3R1 protein is indicated on the Y axis. Representative images are shown in the inbox. We stained one slide for each patient. Linear regression R square, confidence intervals and representative staining are also shown.

Article Snippet: For SLC9A3R1 (HPA9672 and HPA27247, Atlas Antibodies Cat#HPA009672, RRID:AB_1857215 and Atlas Antibodies Cat#HPA027247, RRID:AB_10601162 respectively) the following conditions were used.

Techniques: Microarray, Expressing, Comparison, Activity Assay, Staining, ChIP-sequencing

Journal: Nature medicine

Article Title: Enhancer mapping uncovers phenotypic heterogeneity and evolution in patients with luminal breast cancer

doi: 10.1038/s41591-018-0091-x

Figure Lengend Snippet: A) Theoretical framework of the analysis. The relative size of phenotypic clones can be tracked using enhancer ranking (RIs). Phenotypic clones can be positively or negatively selected during BC progression in response to endocrine therapies. B) Expanding or contracting phenotypic clones were defined based on the RI-ratio in primary and metastatic samples (RI P /RI M ). Distribution of RI-ratio shows that YY1 enhancers RI does not change significantly during progression compared to other enhancers, while SLC9A3R1 RI ranks among the enhancers with stronger increase in activity during progression. Vertical bars represent 1σ (Standard Deviation) increments from the population median C) Scatterplot of YY1 and SLC9A3R1 enhancer ranking according to patient stage. Bars indicate mean and 95% confidence intervals. Asterisks represent significance at P<0.05 after students two-tail T-Test D) IHC staining for YY1 and SLC9A3R1 positive cells in an independent matched longitudinal cohort of 22 ERα breast cancer patients (Scale bars, 100 μm). All normal and primaries are treatment naïve. All metastatic have received endocrine therapies (Tamoxifen or Aromatase inhibitors). Statistical significance was calculated using a pair-wise, two-tail T-test. Representative images are also shown E) Enhancer and promoter stratification based on frequency of usage in primary and metastatic patients. Percentages were calculated for each regulatory region for each stage (primary and metastatic) and differential was then derived and plotted on the X-axis. All enhancers and promoters called in were used. PE and ME were called by taking the top 1/1000 in the distribution that also satisfied a Fisher-exact test p<0.05. F) Dot-plot represent RI indexes for all PE (324) and ME (301) are plotted. As a control, RI for common enhancers (CE=320) were also plotted. Bottom plot: permutation was used to assess changes in RI in 50 randomly selected sets of 320 CE. Bow and whiskers represent median and 1-99 percentile for P-Value distribution. A Wilcoxon matched-pairs signed rank test was used to test for statistical significance G) Kaplan-Meier analysis using 1427 ERα-positive patients and averaged RNA expression of genes associated with PE or ME regulatory regions. Confidence interval for PE (0.39-0.61). Confidence interval for ME (1.1-1.67). Comparison of survival curves was performed using a Log-rank (Mantel-Cox) test. Genes were assigned considering CTCF insulated perimeters. Multivariate correction for the comparisons is also shown H) Pathway analysis for genes associated with PE or ME regulatory regions. Pathways were identified using GREAT and are listed in order of significance (symbols indicate qValue).

Article Snippet: For SLC9A3R1 (HPA9672 and HPA27247, Atlas Antibodies Cat#HPA009672, RRID:AB_1857215 and Atlas Antibodies Cat#HPA027247, RRID:AB_10601162 respectively) the following conditions were used.

Techniques: Clone Assay, Activity Assay, Standard Deviation, Immunohistochemistry, Derivative Assay, Control, RNA Expression, Comparison

Journal: PLoS ONE

Article Title: Prognostic role of FUT8 expression in relation to p53 status in stage II and III colorectal cancer

doi: 10.1371/journal.pone.0200315

Figure Lengend Snippet: (A) FUT8 mRNA expression was significantly upregulated in primary tumors compared to normal colon mucosa. (B) In five independent datasets of colorectal cancer, higher levels of FUT8 mRNA expression were consistently observed in tumors with wild-type p53 than those of mutant p53.

Article Snippet: Primary rabbit polyclonal anti-FUT8 antibody (HPA043410, Prestige Antibodies Powered by Atlas Antibodies, Sigma-Aldrich, Co. LLC.

Techniques: Expressing, Mutagenesis

Journal: PLoS ONE

Article Title: Prognostic role of FUT8 expression in relation to p53 status in stage II and III colorectal cancer

doi: 10.1371/journal.pone.0200315

Figure Lengend Snippet: (A) FUT8 protein expression in colon carcinoma [T] and adjacent colon mucosa [N]. (B) FUT8 was not expressed by non-neoplastic colon mucosal cells. (C) FUT8 staining was typically found in cytoplasm of tumor cells. (D) Occasionally, concomitant cytoplasmic and membranous staining of FUT8 in tumor cells can be found. (E) p53-positive tumor showing strong nuclear staining in cancer cells. (F) p53-negative tumor showing no nuclear staining. Magnification: (A,E,F) x100; (B,C,D) x400.

Article Snippet: Primary rabbit polyclonal anti-FUT8 antibody (HPA043410, Prestige Antibodies Powered by Atlas Antibodies, Sigma-Aldrich, Co. LLC.

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Prognostic role of FUT8 expression in relation to p53 status in stage II and III colorectal cancer

doi: 10.1371/journal.pone.0200315

Figure Lengend Snippet: Clinicopathological characteristics of colorectal cancer patients according to FUT8 expression.

Article Snippet: Primary rabbit polyclonal anti-FUT8 antibody (HPA043410, Prestige Antibodies Powered by Atlas Antibodies, Sigma-Aldrich, Co. LLC.

Techniques: Expressing, Immunohistochemistry

Journal: PLoS ONE

Article Title: Prognostic role of FUT8 expression in relation to p53 status in stage II and III colorectal cancer

doi: 10.1371/journal.pone.0200315

Figure Lengend Snippet: (A,B) FUT8 protein or mRNA expression had no significant impact on survival in both cohorts. (C) In patients with p53-negative tumors, FUT8 protein expression was significantly associated with better survival in the IHC cohort. (D) In patients with wild-type p53 tumors, FUT8 mRNA expression tended to be associated with better survival in the microarray cohort. (E,F) In patients with p53-positive tumors or mutant p53 tumors, FUT8 protein or mRNA expression showed no association with survival.

Article Snippet: Primary rabbit polyclonal anti-FUT8 antibody (HPA043410, Prestige Antibodies Powered by Atlas Antibodies, Sigma-Aldrich, Co. LLC.

Techniques: Expressing, Microarray, Mutagenesis

Journal: PLoS ONE

Article Title: Prognostic role of FUT8 expression in relation to p53 status in stage II and III colorectal cancer

doi: 10.1371/journal.pone.0200315

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis for disease-free survival in patients with stage II and III colorectal cancer.

Article Snippet: Primary rabbit polyclonal anti-FUT8 antibody (HPA043410, Prestige Antibodies Powered by Atlas Antibodies, Sigma-Aldrich, Co. LLC.

Techniques: Immunohistochemistry, Microarray

Journal: PLoS ONE

Article Title: Prognostic role of FUT8 expression in relation to p53 status in stage II and III colorectal cancer

doi: 10.1371/journal.pone.0200315

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis for disease-free survival in patients with stage II and III colorectal cancer according to FUT8 expression and the p53 status.

Article Snippet: Primary rabbit polyclonal anti-FUT8 antibody (HPA043410, Prestige Antibodies Powered by Atlas Antibodies, Sigma-Aldrich, Co. LLC.

Techniques: Expressing, Immunohistochemistry, Microarray, Mutagenesis

Journal: Therapeutic Advances in Medical Oncology

Article Title: SOX11: friend or foe in tumor prevention and carcinogenesis?

doi: 10.1177/1758835919853449

Figure Lengend Snippet: The molecular structure of SOX11. The SOX11 protein is composed of 441 amino acids. SOX11 contains two functional domains, the N-terminal HMG domain and the conserved TAD. SOX11, sex-determining region Y-related high-mobility-group box transcription factor 11; HMG, high mobility group; TAD, C-terminal transactivation domain.

Article Snippet: MCL , 53 , IHC , Rabbit polyclonal (Atlas Antibodies AB) , SOX11-negative patients show worse prognosis and shorter OS. , Wang et al. .

Techniques: Functional Assay

Journal: Therapeutic Advances in Medical Oncology

Article Title: SOX11: friend or foe in tumor prevention and carcinogenesis?

doi: 10.1177/1758835919853449

Figure Lengend Snippet: Overview of SOX11 function reported in diverse types of cancer.

Article Snippet: MCL , 53 , IHC , Rabbit polyclonal (Atlas Antibodies AB) , SOX11-negative patients show worse prognosis and shorter OS. , Wang et al. .

Techniques: Expressing, Over Expression, Biomarker Discovery, Migration, Methylation, DNA Methylation Assay

Journal: Therapeutic Advances in Medical Oncology

Article Title: SOX11: friend or foe in tumor prevention and carcinogenesis?

doi: 10.1177/1758835919853449

Figure Lengend Snippet: Overall carcinogenic actions of SOX11 on the hallmarks of tumor biology. SOX11 exerts tumor-stimulative effects through increasing cell proliferation, repressing cell differentiation, inducing angiogenesis, and promoting metastasis. BCL6, B-cell lymphoma 6; BNIP3, B-cell lymphoma 2 (BCL2)/adenovirus E1B 19 kDa protein-interacting protein 3; CIC, cancer-initiating cell; PAX5, Paired box protein 5; PDGFA, platelet-derived growth factor A; SETMAR, SET domain and mariner transposase fusion gene; SOX11, Sex-determining region Y-related high-mobility-group box transcription factor 11; TANK, TRAF family member-associated NF-κB activator.

Article Snippet: MCL , 53 , IHC , Rabbit polyclonal (Atlas Antibodies AB) , SOX11-negative patients show worse prognosis and shorter OS. , Wang et al. .

Techniques: Cell Differentiation, Derivative Assay

Journal: Therapeutic Advances in Medical Oncology

Article Title: SOX11: friend or foe in tumor prevention and carcinogenesis?

doi: 10.1177/1758835919853449

Figure Lengend Snippet: Different prognostic significance of SOX11 in tumor cases.

Article Snippet: MCL , 53 , IHC , Rabbit polyclonal (Atlas Antibodies AB) , SOX11-negative patients show worse prognosis and shorter OS. , Wang et al. .

Techniques: Expressing, Microarray, Over Expression

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 1. The IJM region is necessary for collagen-induced DDR2 activation. (a) Overall topology and alignment of the transmembrane (TM) domain and the intracellular juxtamembrane region (IJM) of DDR1a and DDR2. The IJM was divided into three regions: JM1, JM2 and JM3. (b) Schematic diagram of various DDR2 constructs used in our study. (c) HEK293T cells transiently transfected with plasmids encoding the full-length DDR2 and F-DJM1-JM2 mutant were stimulated by Type I collagen for 60 min. Tyrosine phosphorylation of the F-DJM1-JM2 mutant was inhibited compared to that of the full-length DDR2. (d) HEK293T cells were transfected with plasmids encoding full-length DDR2, F- DJM1 and F-DJM2 and were stimulated by collagen. The F-DJM2 mutant showed a significant decrease in tyrosine phosphorylation. **p < 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Activation Assay, Construct, Transfection, Mutagenesis, Phospho-proteomics

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 2. DDR2 dimerizes via the JM2 of the IJM region. (a and b) HEK293T cells were transiently cotransfected with plasmids encoding TM- JM1-JM2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blot analysis showed that the cytoplasmic domains of DDR2 bind to each other via the intact TM-JM1-JM2 domain and form homodimers. Asterisks indicate the expected size of TM-JM1-JM2. (c) HEK293T cells were transfected with plasmids encoding F-DJM1 and F-DJM2 mutants. A crosslinking assay showed that dimers of F-DJM1 were not changed compared to full-length DDR2, whereas dimers were significantly decreased for F-DJM2.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Immunoprecipitation, Western Blot, Transfection

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 3. JM2 has a dominant-negative effect on DDR2 activation. (a and b) HEK293T cells were cotransfected with plasmids encoding full- length DDR2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blotting showed that the full-length DDR2 and TM-JM1-JM2 bind to each other to form heterodimers. The immunoprecipitates obtained with anti-IgG antibodies were used as a negative control. (c) H1299 cells transfected with plasmids encoding full-length DDR2 and TM-JM1-JM2 and HeLa cells were lysed, and the whole cell lysates (W) were separated into the plasma membrane (P) and cytosol (C) fractions. EGFR and a-tubulin were used as positive controls for the plasma mem- brane and cytosol fractions, respectively. Endogenous full-length DDR2 (HeLa cells), forced-expressed full-length DDR2 and TM-JM1-JM2 pro- teins were appropriately localized in the plasma membrane. (d) HEK293T cells were transfected with plasmids encoding a C-terminally myc- tagged full-length DDR2 and TM-JM1-JM2. Only under the permeabilized condition, full-length DDR2-myc and TM-JM1-JM2-myc were visual- ized, indicating that the C-termini of these proteins were located in the cytosol and not extracellular space. Full-length DDR2 was used as a positive control. Bar, 50 mm. (e) HEK293T cells were cotransfected with plasmids encoding full-length DDR2 (500 ng) and an increasing amount of TM-JM1-JM2 (100, 300 and 500 ng) as indicated and then stimulated with collagen. Tyrosine phosphorylation gradually decreased with an increasing amount of TM-JM1-JM2. (f) HeLa cells were transiently transfected with a plasmid encoding TM-JM1-JM2 and were then stimulated. Tyrosine phosphorylation was significantly decreased in endogenous DDR2. **p < 0.01, Student’s t-test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Dominant Negative Mutation, Activation Assay, Immunoprecipitation, Western Blot, Negative Control, Transfection, Clinical Proteomics, Membrane, Positive Control, Phospho-proteomics, Plasmid Preparation

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 4. JM2 regulates the collagen-binding affinity of DDR2. (a and b) HEK293T cells transiently transfected with various DDR2 constructs were harvested, and protein expression was verified by Western blot- ting (a). Collagen-binding affinities were reduced in F-DJM2, F-DJM1- JM2 and extra mutants but not the F-DJM1 mutant in a dose- dependent manner (b). Nontransfected (NC) and TM-JM1-JM2 samples were used as negative controls. **p< 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Binding Assay, Transfection, Construct, Expressing, Western Blot, Mutagenesis

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 5. Colony formation and proliferation of tumor cells are suppressed by overexpression of JM2. (a and b) Formalin-fixed tissue micro- array slides were used in immunohistochemistry experiments. DDR2 was overexpressed in bladder, testis, lung, kidney, prostate and stom- ach cancers. Bar, 50 mm. (c) Stable TM-JM1-JM2–expressing H1299 cells were stimulated by collagen and then harvested. Immunoprecipitation and Western blot analysis showed that tyrosine phosphorylation of DDR2 was decreased by TM-JM1-JM2 overexpres- sion (labeled JM1/2), but phosphorylation of DDR1 was unaffected. (d) A colony-forming assay of H1299 cells showed that the number and projected area of colonies were decreased by TM-JM1-JM2 overexpression. Bar, 100 mm. (e) Proliferation of H1299 cells was assessed by cell counting (left) and an MTT assay (right). Cell proliferation was inhibited by TM-JM1-JM2 overexpression. **p < 0.01, Student’s t-test; control, nontransfected cells; Mock, empty vector stably transfected cells; JM1/2, TM-JM1-JM2 stably transfected cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Over Expression, Microarray, Immunohistochemistry, Expressing, Immunoprecipitation, Western Blot, Phospho-proteomics, Labeling, Cell Counting, MTT Assay, Control, Plasmid Preparation, Stable Transfection, Transfection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ANLN-induced EZH2 upregulation promotes pancreatic cancer progression by mediating miR-218-5p/LASP1 signaling axis

doi: 10.1186/s13046-019-1340-7

Figure Lengend Snippet: ANLN was upregulated in pancreatic cancer. a , ANLN expression was significantly upregulated in 174 pancreatic cancer tissues compared with that in 62 normal tissues ( P < 0.001, http://medical-genome.kribb.re.kr/GENT/ ). b , The genetic alteration frequency of ANLN was evaluated in four independent cohorts (QCMG, Nature 2016; TCGA, PanCancer Atlas; TCGA, Provisional; UTSW, Nat Commun) from cBioportal for Cancer Genomic (www.cbioportal.org/). c , Genetic abnormalities of the copy number of the ANLN gene were plotted in two pancreatic cancer datasets (Pancreatic Adenocarcinoma-TCGA, PanCancer Atlas and Pancreatic Adenocarcinoma-TCGA, Provisional) from cBioportal for Cancer Genomic ( www.cbioportal.org/ ). d , The association between ANLN expression in pancreatic cancers and survival time was analyzed by Kaplan–Meier survival analysis in the Human Protein Atlas ( www.proteinatlas.org/ ). e , Representative photomicrographs from ANLN IHC staining of eighty pancreatic cancer tissues and ten cancer-adjacent normal tissues are shown in the upper panel (× 200 and × 400 magnification). Kaplan-Meier survival curve of the overall survival according to high and low ANLN expression in pancreatic cancer tissues is shown in the lower panel ( P < 0.001). f , The ANLN protein expression levels were increased in five pancreatic cancer cell lines (AsPC-1, PANC-1, BxPC-3, MIA PaCa-2 and SW1990) compared with those in the hTERT-HPNE cell line. **, P < 0.01 compared with the hTERT-HPNE cell line

Article Snippet: The sections were incubated with a mouse monoclonal antibody against human ANLN (1200; Abcam, Cambridge, United Kingdom) overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ANLN-induced EZH2 upregulation promotes pancreatic cancer progression by mediating miR-218-5p/LASP1 signaling axis

doi: 10.1186/s13046-019-1340-7

Figure Lengend Snippet: Cox proportional hazard models for prognostic factors

Article Snippet: The sections were incubated with a mouse monoclonal antibody against human ANLN (1200; Abcam, Cambridge, United Kingdom) overnight at 4 °C.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ANLN-induced EZH2 upregulation promotes pancreatic cancer progression by mediating miR-218-5p/LASP1 signaling axis

doi: 10.1186/s13046-019-1340-7

Figure Lengend Snippet: ANLN functioned as an oncogene in pancreatic cancer. a , The ANLN protein expression levels in BxPC-3 and SW1990 cells transfected with ANLN siRNA (ANLN RNAi) or the scramble control (NC) were analyzed by Western blot. b , Proliferation of BxPC-3 and SW1990 cells transfected with ANLN siRNA (ANLN RNAi) or the scramble control (NC) was determined by the CCK-8 assay. c , ANLN knockdown significantly inhibited the colony forming ability of BxPC-3 and SW1990 cells. d and e , The migration and invasion properties of the same cells described in b were determined by the transwell migration and invasion assay. f , Knockdown effect of ANLN silencing lentiviral vectors was confirmed by Western blot. g , ANLN knockdown in BxPC-3 cells repressed the tumor growth. n = 5 per group. ** P < 0.01

Article Snippet: The sections were incubated with a mouse monoclonal antibody against human ANLN (1200; Abcam, Cambridge, United Kingdom) overnight at 4 °C.

Techniques: Expressing, Transfection, Control, Western Blot, CCK-8 Assay, Knockdown, Migration, Invasion Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ANLN-induced EZH2 upregulation promotes pancreatic cancer progression by mediating miR-218-5p/LASP1 signaling axis

doi: 10.1186/s13046-019-1340-7

Figure Lengend Snippet: Global changes in gene expression in BxPC-3 cells transfected with ANLN siRNA. a , The heat map of the differentially expressed genes with a fold change of greater than 2.5 or less than − 2.5 in ANLN RNAi relative to NC. b , A volcano plot showing the differentially expressed genes with a fold change of greater than 2.5 or less than − 2.5 in ANLN RNAi relative to NC. c , The top twenty-five regulated Gene ontology (GO) biological process terms. d , The heat map of 54 cell-cell adhesion-related genes. e , The selected candidate genes from microarray experiments were confirmed by qRT-PCR after ANLN RNAi transfection in BxPC-3 cells. ** P < 0.01 compared with the NC group

Article Snippet: The sections were incubated with a mouse monoclonal antibody against human ANLN (1200; Abcam, Cambridge, United Kingdom) overnight at 4 °C.

Techniques: Gene Expression, Transfection, Microarray, Quantitative RT-PCR

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ANLN-induced EZH2 upregulation promotes pancreatic cancer progression by mediating miR-218-5p/LASP1 signaling axis

doi: 10.1186/s13046-019-1340-7

Figure Lengend Snippet: LASP1 re-expression partially reversed the effects of ANLN knockdown on proliferation, colony formation, migration and invasion in pancreatic cancer cells. a , LASP1 expression vectors rescued LASP1 expression in BxPC-3 and SW1990 cells transfection with ANLN RNAi. b , CCK-8 analysis indicated that LASP1 re-expression reversed the suppressive effects of ANLN knockdown on pancreatic cancer cell growth. c , The restoration of LASP1 expression reversed the suppressive effects of ANLN knockdown in colony formation. d and e , The restoration of LASP1 expression reversed the suppressive effects of ANLN knockdown in migration and invasion. ** P < 0.01

Article Snippet: The sections were incubated with a mouse monoclonal antibody against human ANLN (1200; Abcam, Cambridge, United Kingdom) overnight at 4 °C.

Techniques: Expressing, Knockdown, Migration, Transfection, CCK-8 Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ANLN-induced EZH2 upregulation promotes pancreatic cancer progression by mediating miR-218-5p/LASP1 signaling axis

doi: 10.1186/s13046-019-1340-7

Figure Lengend Snippet: MiR-218-5p was involved in the ANLN-induced LASP1 expression and pancreatic cancer progression. a , MiR-218-5p inhibitor (anti-miR-218) obviously reversed the ANLN knockdown-induced miR-218-5p expression. b , The LASP1 protein levels were partly restored in the cells cotransfected with ANLN RNAi and miR-218-5p inhibitor (anti-miR-218) compared with the protein levels in the cells cotransfected with ANLN RNAi and inhibitor control (anti-con). c , CCK-8 analysis revealed that miR-218-5p inhibitor (anti-miR-218) rescued the inhibition of cell proliferation in BxPC-3 and SW1990 cells transfected with ANLN RNAi. d , Partial restoration of the suppressed cell growth was observed by colony formation after miR-218-5p inhibition. e and f , miR-218-5p inhibition in BxPC-3 and SW1990 cells partly reversed the suppressive effects of ANLN knockdown in migration and invasion. ** P < 0.01

Article Snippet: The sections were incubated with a mouse monoclonal antibody against human ANLN (1200; Abcam, Cambridge, United Kingdom) overnight at 4 °C.

Techniques: Expressing, Knockdown, Control, CCK-8 Assay, Inhibition, Transfection, Migration

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ANLN-induced EZH2 upregulation promotes pancreatic cancer progression by mediating miR-218-5p/LASP1 signaling axis

doi: 10.1186/s13046-019-1340-7

Figure Lengend Snippet: EZH2 was responsible for ANLN-induced pancreatic cancer progression by mediating miR-218-5p/LASP1 signaling axis. a , EZH2 downregulation significantly inhibited the expression of LASP1 protein in BxPC-3 and SW1990 cells. b , MiR-218-5p inhibitor (anti-miR-218) obviously reversed the EZH2 knockdown-induced miR-218-5p expression. c , The LASP1 protein levels were partly restored in the cells cotransfected with EZH2 RNAi and anti-miR-218 compared with the protein levels in the cells cotransfected with EZH2 RNAi and inhibitor control (anti-con). b , EZH2 re-expression obviously reversed the ANLN knockdown-induced miR-218-5p expression. e , EZH2 re-expression obviously reversed the expression levels of LASP1 protein impaired by ANLN knockdown. f , CCK-8 analysis revealed that ectopic expression of EZH2 reversed the inhibition of cell proliferation in BxPC-3 and SW1990 cells transfected with ANLN RNAi. g , Partial restoration of the suppressed cell growth was observed by colony formation after LASP1 re-expression. h , Ectopic expression of EZH2 in BxPC-3 and SW1990 cells partly reversed the suppressive effects of ANLN knockdown in migration and invasion. ** P < 0.01

Article Snippet: The sections were incubated with a mouse monoclonal antibody against human ANLN (1200; Abcam, Cambridge, United Kingdom) overnight at 4 °C.

Techniques: Expressing, Knockdown, Control, CCK-8 Assay, Inhibition, Transfection, Migration

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ANLN-induced EZH2 upregulation promotes pancreatic cancer progression by mediating miR-218-5p/LASP1 signaling axis

doi: 10.1186/s13046-019-1340-7

Figure Lengend Snippet: ANLN knockdown repressed the levels of EZH2 and LASP1 expression in vivo. a , The levels of ANLN, EZH2 and LASP1 expression in tissues from xenograft tumor models established with the stable ANLN-silenced BxPC-3 cells or the stable scramble control BxPC-3 cells were determined by IHC. b , Western blot analysis in tumors as in ( a ). ** P < 0.01

Article Snippet: The sections were incubated with a mouse monoclonal antibody against human ANLN (1200; Abcam, Cambridge, United Kingdom) overnight at 4 °C.

Techniques: Knockdown, Expressing, In Vivo, Control, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ANLN-induced EZH2 upregulation promotes pancreatic cancer progression by mediating miR-218-5p/LASP1 signaling axis

doi: 10.1186/s13046-019-1340-7

Figure Lengend Snippet: Schematic diagram for the effect of ANLN on pancreatic cancer progression. ANLN upregulation promotes cell proliferation, colony formation, migration and invasion by inducing EZH2. ANLN-induced EZH2 expression leads to the downregulation of miR-218-5p, which inhibits LASP1 expression by directly targeting the 3’UTR of LASP1

Article Snippet: The sections were incubated with a mouse monoclonal antibody against human ANLN (1200; Abcam, Cambridge, United Kingdom) overnight at 4 °C.

Techniques: Migration, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ANLN-induced EZH2 upregulation promotes pancreatic cancer progression by mediating miR-218-5p/LASP1 signaling axis

doi: 10.1186/s13046-019-1340-7

Figure Lengend Snippet: Correlations between ANLN expression and clinicopathological characteristics of patients with pancreatic cancer

Article Snippet: The sections were incubated with a mouse monoclonal antibody against human ANLN (1200; Abcam, Cambridge, United Kingdom) overnight at 4 °C.

Techniques: Expressing

Journal: Microbiome

Article Title: Distinct B cell subsets in Peyer’s patches convey probiotic effects by Limosilactobacillus reuteri

doi: 10.1186/s40168-021-01128-4

Figure Lengend Snippet: Phenotypic and transcriptional distinction between B cell subsets in Peyer’s patches. a Flow cytometry of PPs live CD3 - CD19 + B220 + B lymphocytes (gated as shown in Fig. S1A) and separated into the small and large B cell subsets by forward scatter area and expressed as mean FSC-A (size), and the number of small-B and large-B cells per mg tissue ( n = 6 mice left panel; n = 24 mice right panel). b Imaging flow cytometry of the areas (μm 2 ) of small-B and large-B cell from three independent experiments. c , d Immunohistochemistry of PPs stained with anti-B220 (magenta), anti-CD138 (yellow) and Hoechst (blue) in c or anti-B220 (white), anti-GL7 (green), anti-Ki67 (cyan), anti-IgA (yellow), and anti-IgD (blue) in d . Scale Bars equal 100 μm or 10 μm in closed-up reviews. e Enrichment of gene ontology categories (Biological Process, BP) for genes differentially expressed in large B (upper panel) versus small B cells (lower panel) determined by microarray of lin - CD19 + B220 + cells sorted on FSC-A ( n = 4 samples per group), the number of genes in each functional category is shown. Data were adjusted by false discovery rate control (FDR). f, g Displays of heat maps of genes expression by q-RT-PCR in sorted small and large B cells. The expression was normalized to the mean value of large B and each column represents one sample. h , i Heat maps and histograms depicting expression of surface markers on small and large B cells ( n = 6 mice per group). The frequency of indicated markers (normalized to the mean value of large B cells). i MFI of B cells positive for GL7 and S1PR1, and percentage in each subset. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 using two-tailed Student’s t test

Article Snippet: Antibodies raised against the following mouse antigens were used: B220 (AF647, cloneRA3-6B2, BioLegend), CD3 (AF488, clone17A2, BioLegend), CD21/CD35 (AF594, clone7E9, BioLegend), CD138 (unconjugated, clone281-2, BioLegend), CD279/PD-1 (AF647, clone29F.1A12, BioLegend), GL7 (FITC, cloneGL7, BD Bioscience), IgA (FITC, cloneC10-3, BD Bioscience), IgD (Pacific blue, 11-26c2a, BioLegend), Ki67 (unconjugated, cloneMIB-1, Abcam), DEFa3 (unconjugated, RBM6, antibodies-online), pimonidazole (Pacific Blue, Hypoxyprobe), and Phalloidin (AF555).

Techniques: Flow Cytometry, Imaging, Immunohistochemistry, Staining, Microarray, Functional Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Microbiome

Article Title: Distinct B cell subsets in Peyer’s patches convey probiotic effects by Limosilactobacillus reuteri

doi: 10.1186/s40168-021-01128-4

Figure Lengend Snippet: L. reuteri expands the GC-like B cell population and reprograms its functional gene signature. a , c Immunohistochemistry of the GL7 + area stained with anti-B220 (magenta), anti-GL7 (green) and anti-Ki67 (cyan). Scale bars equal 100 μm. Representative images of PPs from the control or L. reuteri -treatment are shown in a and c , and the corresponding quantification shown in Fig. S3G, H. b , d Flow cytometry quantification of GL7 + B cells (in CD3 − CD19 + B220 + , n = 5–6 mice per group) and Ki67 + cells ( n = 5–6 mice per group). e In vivo proliferation assay was performed by flow cytometry following i.p . injection of EdU. A histogram depicting EdU expression in GL7 − , GL7 + or EdU + CD19 + B220 + B cells and the number of EdU + GL7 + B cells ( n = 6 mice per group). f Expression of Mki67 , Gsk-3α , and Hif-1α in large GC-like B cells from control or L. reuteri -treated mice (GOI, gene of interest; A.U., arbitrary unit). g q-RT-PCR analysis of gene expression levels ( n = 6 mice per group) and expression of TGFβR1 (microarray analysis). h Flow cytometry analyzing the numbers of TGFβ1 + B cells as well as the TGFβ1 levels (MFI, n = 6–18 mice per group). i The PPs tissue was analyzed for cytokine/chemokine production by Multi-Plex Mesoscale and ELISA, normalized to tissue protein content ( n = 6 mice per group). j Expression of α germline transcripts ( αGT ) in PPs ( n = 6 mice per group). k The numbers of B cells of different subsets in PPs in untreated mice and in response to L. reuteri R2LC and L. reuteri R2LC_ΔADO ( n = 6 mice per group). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01 using Student’s t test or ANOVA with Tukey’s post hoc test

Article Snippet: Antibodies raised against the following mouse antigens were used: B220 (AF647, cloneRA3-6B2, BioLegend), CD3 (AF488, clone17A2, BioLegend), CD21/CD35 (AF594, clone7E9, BioLegend), CD138 (unconjugated, clone281-2, BioLegend), CD279/PD-1 (AF647, clone29F.1A12, BioLegend), GL7 (FITC, cloneGL7, BD Bioscience), IgA (FITC, cloneC10-3, BD Bioscience), IgD (Pacific blue, 11-26c2a, BioLegend), Ki67 (unconjugated, cloneMIB-1, Abcam), DEFa3 (unconjugated, RBM6, antibodies-online), pimonidazole (Pacific Blue, Hypoxyprobe), and Phalloidin (AF555).

Techniques: Functional Assay, Immunohistochemistry, Staining, Flow Cytometry, In Vivo, Proliferation Assay, Injection, Expressing, Reverse Transcription Polymerase Chain Reaction, Microarray, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) Immunohistochemistry showing reduced methylated H3K79 levels (H3K79me2) that reflect loss of DOT1L activity in damaged areas from osteoarthritic patients (OA) as compared to their corresponding preserved areas and to cartilage from non-OA patients. Images are representative of images from four different patients. Scale bar, 400 μm. ( b ) Heat maps of differential mRNA expression determined by quantitative PCR in chondrocytes treated with DOT1L inhibitor EPZ-5676 (EPZ) or vehicle (V) from passage 0 (P0) until P2, and from preserved versus damaged areas in OA cartilage. The colour code represents the mean expression level of six and four independent patient samples respectively. ( c ) Immunoblot analysis showing decreased methylated H3K79 levels in mouse articular chondrocytes after intra-articular injection of EPZ into C57Bl/6 wild-type mouse knees. The image is representative of one experiment with protein extracts pooled from two or three mice per condition. Unprocessed original scans of blots are shown in . ( d , e ) C57/Bl6 wild-type mouse knees were injected with EPZ (5 mg kg –1 ) or vehicle and killed after 2 or 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( d ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( e ). One experiment was performed with n =10 and 5. Representative images from the 4 week evaluation are shown. * P <0.05 (two-tailed t -test). Error bars indicate mean±s.e.m.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Immunohistochemistry, Methylation, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) KEGG pathway enrichment analysis of microarray data obtained from human articular chondrocytes treated with EPZ-5676 or vehicle. Nominal P values by EASE modified Fisher Exact test using the DAVID analysis tool (see Methods section) are shown. n =5 independent patient-derived cell cultures. ( b ) Co-immunoprecipitation (Co-IP) using an anti-DOT1L antibody showing interaction between DOT1L and β -catenin in human articular chondrocytes, that is increased upon Wnt activation by LiCl and disrupted upon DOT1L inhibition. The image is representative of three experiments. ( c ) TOP/FOP reporter assay in human articular chondrocytes after Wnt stimulation by LiCl and DOT1L inhibition by EPZ. Activity is compared to untreated cells (dotted line). n =3 biologically independent experiments. *** P <0.001 by one-way ANOVA. ( d , e ) LEF1 , TCF1 and c-MYC expression measured by quantitative PCR in chondrocytes treated with EPZ-5676 and LiCl ( d ) or in LiCl-treated chondrocytes transfected with siRNA directed against DOT1L or scrambled siRNA (siDOT1L or siSCR, respectively) ( e ). Data are from one experiment with three technical replicates. ( f ) Immunohistochemistry demonstrating increased TCF1 levels in the articular cartilage of C57/Bl6 wild-type mice after injection of EPZ-5676. The images are representative of three different animals. Scale bar, 200 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Microarray, Modification, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Activation Assay, Inhibition, Reporter Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Immunohistochemistry, Injection

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: All experiments were performed in healthy human articular chondrocytes: treated as indicated with DOT1L inhibitor EPZ-5676, Wnt activator LiCl, SIRT1 antagonist EX527 or SIRT1 agonist SRT1720; or transfected with DOT1L or scrambled siRNA. All data are presented as mean±s.e.m. ( a ) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) analysis of DOT1L and methylated H3K79 and ( b ) acetylated H3K9 (H3K9Ac) and methylated H3K4 (H3K4me3) as markers of active transcription on the transcriptional start site (TSS) of Wnt target genes. Data are from two to five experiments. ( c ) Expression levels of TCF1 Wnt target gene measured by quantitative PCR in chondrocytes transfected with indicated specific or scrambled siRNA (siSCR). Data are from one experiment with technical triplicates. ( d ) TCF1 expression measured by quantitative PCR in the presence of SIRT1 agonist and antagonist. Data from two experiments each with technical triplicates. ( e ) Co-IP analysis using the indicated antibodies demonstrating the interaction of DOT1L and SIRT1. The image is a representative image of three biologically independent experiments. ( f ) SIRT1 activity relative to vehicle-treated cells (dotted line). Data are from three biologically independent experiments. * P <0.05, *** P <0.001 by one-way ANOVA. ( g ) ChIP-qPCR analysis of SIRT1, PPARGC1A, GCN5 and EP300 binding on the TCF1 promoter and ( h ) TCF1 expression after siRNA transfection with indicated specific or scrambled siRNA. Data from two biologically independent experiments.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Methylation, Expressing, Co-Immunoprecipitation Assay, Activity Assay, Binding Assay

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a – c ) Inactivation of SIRT1 protects against DOT1L inhibitor-induced osteoarthritis: ( a ) C57/Bl6 wild-type mouse knees were injected with DOT1L inhibitor EPZ-5676 (5 mg kg −1 ) and SIRT1 inhibitor EX527 (1.25 mg kg –1 ), or vehicle (V) and killed after 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( a ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( b ). One experiment was performed with n =3 (vehicle), 8 (EPZ) and 10 (EPZ+EX527). * P <0.05 by one-way ANOVA. Error bars indicate mean±s.e.m. ( c ) Immunohistochemistry of TCF1 in the indicated groups. TCF1 levels are increased after EPZ treatment and normalized by additional EX527 treatment. The images are representative of three different animals. Scale bar, 200 μm. ( d , e ) Loss of DOT1L function causes severe growth retardation as demonstrated by skeletal staining ( d ) and histology of the growth plate ( e ) of 4-week-old Dot1l fl/fl ;Col2-Cre −/− (Cre-neg) and Dot1l fl/fl ;Col2-Cre +/− (Dot1l Cart-KO ) mice. ( f , g ) Increased TCF1 levels in Dot1l Cart-KO mice as shown by immunohistochemistry in the indicated mice strains in the articular cartilage ( f ) and growth plate ( g ). The images are representative of three different animals. Scale bar, 200 and 100 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Injection, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: Upon Wnt signalling activation, DOT1L-containing complexes bind Wnt target gene chromatin. DOT1L interacts with SIRT1 and inhibits its function, preventing Wnt pathway hyper-activation. When Wnt signalling is activated in the absence of DOT1L function, high SIRT1 activity mediates the recruitment of transcriptional activators to LEF1 and TCF1 genes. High Wnt signalling leads to deleterious downstream effects and loss of cartilage homeostasis.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Activation Assay, Activity Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Immunohistochemistry staining with antibody against phosphorylated Smad2 (pSmad2) and TGFβ receptor II (TβRII) showed increased nuclear signal for pSmad2 in the invasive ECdnT organotypic cultures. Scale bar 50 micron. (b) Analysis of immunohistochemistry staining for TβRII and pSmad2 in 83 ESCC cases in a tissue microarray shows no significant correlation. Fisher’s exact test, two tailed p= 0.3182. (c) Five paired normal adjacent and ESCC tissues (GSE17531) were analyzed for INHBA mRNA expression, which identified upregulation of INHBA in four ESCC samples. (d) Waterfall plot of a publically available dataset (GSE23400) represented upregulation of INHBA in the ESCC (grey bars) samples vs. normal (black bars).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Immunohistochemistry, Staining, Microarray, Two Tailed Test, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures with either fetal esophageal fibroblasts (FEF) or cancer-associated fibroblasts (CAF) embedded in the underlying matrix. Immunofluorescence staining with antibody against αSMA (green) and podoplanin (red) showed similar expression pattern in the cultures. Scale bar is 50 micron. (b) Activin A concentration in conditioned media from organotypic cultures is higher in invasive cultures as measured using indirect ELISA. * p=0.003, ** p= 0.005, *** p=0.03 (c) Stimulation of epithelial cells with Act A in monolayer plastic culture demonstrated phosphorylation of Smad. Neutralizing antibody against Activin (nAb) prevented the induction of pSmad2 by Act A. Following stimulation with Act A or with conditioned media from organotypic culture increased expression of vimentin was detected after 48 hours by Western Blot. The increase was reversed in the presence of neutralizing antibody (nAb). (d) Inhibition with the Act A antagonist, Follistatin, or a pan-TGFβ inhibitor A83-01 suppressed MMP-9 secretion in E, EC and ECdnT cells as measured by gelatin zymography. Upper bands reflect pro-MMP, lower bands activated, cleaved MMP (arrow).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Immunofluorescence, Staining, Concentration Assay, Indirect ELISA, Phospho-proteomics, Western Blot, Inhibition, Zymography

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures in the presence of recombinant Activin A (Act A), its antagonist Follistatin or a neutralizing antibody against Activin A (nAb); H&E staining. Stimulation with Act A inhibited invasion of E and EC cells, but failed to suppress ECdnT cell invasion. Follistatin increased cell invasion in all cell types, while the neutralizing antibody prevented invasion of E and EC cells, without an effect on ECdnT cells. (b) Immunohistochemistry staining with ki67-antibody showed no differences in cell proliferation. Scale bars are 50 micron. (c) Indirect ELISA with antibody against Act A measured increased levels after addition of recombinant Act A in fibroblasts (FEF) and ECdnT. Untreated ECdnT cells (Control) secreted higher baseline levels of Act A than FEF, which were reduced by Follistatin. (d) TGFβ1 concentration was increased in response to stimulation with Act A and overall baseline secretion was higher in control ECdnT cells than fibroblasts as measured by indirect ELISA. Follistatin inhibited TGFβ1 secretion.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Recombinant, Staining, Immunohistochemistry, Indirect ELISA, Control, Concentration Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Separating the cellular matrix and epithelium of the organotypic cultures growing ECdnT cells through a collagen I layer, dashed lines, prevented cell invasion in the absence (control) and presence of Act A (+Act A). When the cellular matrix of the organotypic culture was treated with puromycin to kill the embedded fibroblasts before the ECdnT cells were seeded, epithelial formation occurred but invasion was inhibited with and without Act A stimulation. (b) Treatment of ECdnT organotypic cultures with a pan-MMP inhibitor, GM6001, suppressed cell invasion, which was not restored in the presence of Act A. Untreated (no tx) control ECdnT cells in organotypic culture invaded into the underlying matrix. TGFβ1 treatment inhibited epithelial cell invasion. Scale bars are 50 micron. (c) Immunohistochemistry showed nuclear localization of phosphorylated Smad (pSmad2, red) in control and Act A stimulated conditions. Collagen IV, red, was disrupted in invasive cultures after Act A treatment. Loss of the fibroblasts (FEF), labeled green with antibody against vimentin (no staining in the lower panels), had no effect on the nuclear localization of pSmad2. The collagen IV layer was not disrupted in non-invasive cultures in the absence of FEFs.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Control, Immunohistochemistry, Labeling, Staining

Journal: iScience

Article Title: Tmem30a protects against podocyte injury through suppression of pyroptosis

doi: 10.1016/j.isci.2024.109976

Figure Lengend Snippet:

Article Snippet: RNase-free water , TIANGEN , Cat# RT121-02.

Techniques: Recombinant, Saline, Lysis, Western Blot, Marker, Membrane, Immunohistochemistry, Protein Concentration, Staining, Modification, Microarray, Software

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 1. Loss of NF1 reduces RAC1-driven melanoblast migration. A. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 9 h and 12 h using either WT or NF1+/−melanoblasts (MB) in the presence of a RAC1 activator (CN04). B. RAC1 activity was measured by G-lisa in WT and NF1+/−melanoblasts (MB). C. Scratch-like migration assay after 3 h, 6 h, 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1). D. Expression status of NF1 and expression of phosphorylated and non-phosphorylated ERK and AKT in NF1+/−melanoblasts by western blot. α-actinin was used as a loading control. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody. E. Scratch-like migration assay representing the percentage of cell coverage after 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1) and in the presence or absence of a RAC1 activator (CN04). *: SCR vs. siNF1, #: -CN04 vs. +CN04. F. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody in the presence or absence of a RAC1 activator (CN04). **P < 0.01, *P < 0.05, ns: not significant (unpaired Student's t-test). All error bars represent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Activity Assay, Transfection, Expressing, Western Blot, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 2. Loss of NF1 increases melanoma migration and is associated with increased PREX1 expression. A. NF1 mRNA expression under NF1 silencing with two siRNAs (NF1.6 and NF1.11) in SK-mel-23, Mel501, and SK-mel-103 melanoma cell lines. B. PREX1 mRNA expression under NF1 silencing with two siRNAs in SK-mel-23, Mel501, and SK- mel-103 cell lines. C. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 12 h and 24 h under NF1 silencing in SK-mel-23, Mel501, and SK-mel- 103 cell lines. D. Scratch-like migration assay as in C, after additional transfection with siRNA control (scramble) or with PREX1 siRNA (siPREX1). E. Scratch-like migration assay as in C. in the absence (control) or presence (RAC1 inhibitor) of a RAC1 inhibitor. ***P < 0.001, **P < 0.01, *P < 0.05 (unpaired Student's t-test). All error bars rep- resent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Expressing, Transfection, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 4. PREX is upregulated in low NF1 expressing melanoma metastases. A. Representative microphotographs of Tissue Microarray (TMA) containing primary and metastatic melanoma samples analysed by immunohistochemistry using a specific antibody against NF1, RAC1 and PREX1. Bar, 100 μm. B. Scoring of the immunohistochemistry staining was performed according to our previously described protocol [24]. Duplicates of valid punch samples are represented for each condition. Significance was tested using two-tailed t-test with *P < 0.05 and ns: not significant.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Expressing, Microarray, Immunohistochemistry, Staining, Two Tailed Test