Journal: Cell Reports Methods

Article Title: Retrospective cell lineage reconstruction in humans by using short tandem repeats

doi: 10.1016/j.crmeth.2021.100054

Figure Lengend Snippet: Duplex MIPs-based cell lineage workflow (A) Design of duplex MIPs precursor: desired targets are selected from our cell lineage database and precursors are designed. (B) Duplex MIPs preparation: duplex MIPs precursors are synthesized on a microarray, pooled and amplified by PCR. The product is digested by MlyI to remove the universal adaptors (orange and green), purified and diluted to obtain active duplex MIPs. (C) Duplex MIPs and template DNA are mixed together to allow annealing of the targeting arms (blue and yellow) to the regions flanking the targeted sequence in the template DNA, the products are then circularized by gap filling, facilitated by DNA polymerase and ligase. Linear DNA, including excess MIPs and template DNA, is eliminated by exonucleases digestion. The Illumina sequencing library is generated individually for each sample in a PCR step, facilitating addition of adaptors and barcodes. Libraries are pooled and sequenced by using Illumina next-generation sequencing platform, followed by analysis of the raw reads to detect mutations. The latter are then used to infer the cell lineage tree.

Article Snippet: Sample specific barcoding PCR was performed using dual-index barcoding primers and NEBNext Ultra II Q5 DNA Polymerase.

Techniques: Synthesized, Microarray, Amplification, Purification, Sequencing, Generated, Next-Generation Sequencing

Journal: Cell Reports Methods

Article Title: Retrospective cell lineage reconstruction in humans by using short tandem repeats

doi: 10.1016/j.crmeth.2021.100054

Figure Lengend Snippet:

Article Snippet: Sample specific barcoding PCR was performed using dual-index barcoding primers and NEBNext Ultra II Q5 DNA Polymerase.

Techniques: Recombinant, Purification, Sequencing, Software

Journal: Cell Reports

Article Title: Aryl Hydrocarbon Receptor Contributes to the Transcriptional Program of IL-10-Producing Regulatory B Cells

doi: 10.1016/j.celrep.2019.10.018

Figure Lengend Snippet:

Article Snippet: Negative CD43- Isolation Kit , Miltenyi Biotec , Cat# 130-049-801.

Techniques: Western Blot, Recombinant, Methylation, Adjuvant, Enzyme-linked Immunosorbent Assay, Isolation, cDNA Synthesis, SYBR Green Assay, DNA Library Preparation, Purification, Bicinchoninic Acid Protein Assay, Microarray, Ex Vivo, Generated, Software, Red Blood Cell Lysis, Staining, Lysis

Journal:

Article Title: pp32 Reduction Induces Differentiation of TSU-Pr1 Cells

doi:

Figure Lengend Snippet: Expression of IL-6 in TSU-Pr1 cells. TSU-Pr1 cells stably transfected with pp32 anti-sense express higher levels of IL-6 message as compared to parental TSU-Pr1 cells and vector-only control by RT-PCR analysis, which validates the cDNA microarray analysis (see Figure 6).

Article Snippet: Microarray Analysis of TSU-Pr1 Cell Lines This procedure was performed at The Johns Hopkins University Oncology Microarray facility by using a human glass 12K cDNA chip.

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Control, Reverse Transcription Polymerase Chain Reaction, Microarray

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Ascites in peritoneal carcinomatosis (PC) is biologically active (A) Kaplan-Meier curve showing survival of PC patients who underwent cytoreductive surgery (n = 121), stratified by the presence or absence of ascites. (B and C) Proliferation curves of (B) Colo-205 and (C) SNU-C1 cells treated with varying concentrations of ascites, measured by CellTitre-Glo assay. Data are represented as cell viability at day 5 relative to day 0. Dotted line represents cell viability of Colo-205 treated with 10% fetal bovine serum (FBS) at day 5 relative to day 0. Graph shows mean ± SD. (D and E) Migration of (D) Colo-205 and (E) SNU-C1 cells pre-treated with serum-free media (SFM), 10% FBS medium or 5% cell-free ascites (CFA) medium, assessed by transwell migration assay. Significant difference detected via unpaired two-sided t test between migration of cells pre-treated with SFM and 10% FBS or 5% CFA was denoted by ∗. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Significant difference detected via unpaired two-sided t test between migration of cells pre-treated with 10% FBS and SFM or 5% CFA was denoted by # . # p < 0.05, ## p < 0.01, ### p < 0.001. Graph shows mean ± SD. (F) Enriched signaling pathways in colorectal PC cell lines upon treatment with CFA, identified by comparing gene expressions of cells treated with 5% versus 0.1% CFA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (G and H) Treatment of (G) Colo-205 and (H) SNU-C1 cells with 5% CFA activated STAT3 signaling pathway through phosphorylation at Tyr705.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Glo Assay, Migration, Transwell Migration Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Identification and validation of prognostic putative activators of STAT3 signaling in ascites (A) Workflow to identify clinically significant putative secreted STAT3 activators. (B) Kaplan-Meier survival curve illustrating poorer survival in patients expressing three biomarkers as compared to patients expressing 0–2 biomarkers. (C) Characterization of cytokine profiles in colorectal PC ascites and benign ascites. Top 25% most highly abundant cytokines in colorectal PC ascites (by mean abundance) are shown. Heatmap displays Z score of normalized mean pixel density of duplicate cytokine spots on the array. (D) Downregulated signaling pathways upon PAI-1 inhibition in Colo-205 cells exposed to CFA with high PAI-1 levels (PC085), CFA with low PAI-1 levels (PC249) and no PAI-1 (FBS control), identified using RNA microarray. Only signaling pathways with differential downregulation are presented. IL6-JAK-STAT3 signaling pathway was significantly downregulated in high PAI-1 CFA-treated cells upon PAI-1 inhibition. Normalized enrichment scores <0 indicate pathway suppression and scores >0 indicate pathway activation. (D) is representative of two independent biological experiments. ∗p < 0.05.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Expressing, Inhibition, Microarray, Activation Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Correlating PAI-1 level in ascites and intracellular STAT3 activation in cancer cells revealed distinct subgroups associated with differing susceptibility to PAI-1 inhibition (A) PAI-1 prevalence in colorectal PC ascites (n = 54). (B) Correlation between colorectal PC ascitic PAI-1 concentrations and ascites-treated Colo-205 cells p-STAT3(Y705) was determined by Pearson correlation coefficient test ( r = 0.4691, p = 0.0003). (C) PAI-1 prevalence in ascites of various histological PC subtypes (n = 156). † indicates benign ascites. (D) Correlation between various histological PC ascitic PAI-1 concentrations and ascites-treated Colo-205 cells p-STAT3(Y705) was determined by Pearson correlation coefficient test ( r = 0.6476, p < 0.0001). (A–D) PAI-1 concentrations are plotted on a log 2 scale to transform skewed data to normal distribution. p-STAT3(Y705) level was shown as optical density reading at 450 nm (OD450). (E) Untransformed values of PAI-1 and p-STAT3(Y705) levels from (D) were used for stratification strategy to identify patient subpopulations who might benefit from PAI-1 inhibition. Using 20 ng/mL PAI-1 level and 0.2 OD450 p-STAT3(Y705) level as cut-off values, three distinct subgroups of samples were observed: (i) high PAI-1 and high p-STAT3 levels, termed PAI-1 paracrine addicted (PPA) group (yellow region), (ii) low PAI-1 and high p-STAT3 levels, termed co-activators predominant (CAP) group (pink region) and (iii) low PAI-1 and low p-STAT3 levels, termed alternative pathways activation (APA) group (blue region). Each dot represents one patient ascites. Colors in each panel represent the various histological PC subtypes. All histological subtypes with less than five samples are grouped into other histological subtypes.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Activation Assay, Inhibition

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Cells dependent on PAI-1 to activate STAT3 are most susceptible to PAI-1 inhibition (A) Effect of TM5441 (PAI-1 inhibitor) on CFA-treated Colo-205 cells. Representative inhibitor dose-response curves of PPA group (yellow), CAP group (pink), APA group (blue) and FBS (control, black) demonstrated a left shift, indicating responsiveness to PAI-1 inhibition. (B) Differential sensitivity to TM5441 corresponding to the three subgroups, with PPA (n = 18) being the most sensitive to PAI-1 inhibition, followed by CAP (n = 59) and APA (n = 17). Graph shows mean ± SD. (C–E) Effect of (C) Napabucasin (STAT3 inhibitor), (D) BEZ235 (dual PI3K/mTOR inhibitor) and (E) Mitomycin C (conventional chemotherapeutic agent – DNA crosslinker) on the three subgroups of ascites-treated Colo-205 cells. Representative inhibitor dose-response curves of PPA group (yellow; n = 3), CAP group (pink; n = 3), APA group (blue; n = 1) and FBS (control, black). Targeting PAI-1, a dominant paracrine factor in ascites, was more effective than targeting downstream signaling pathway activated by ascites, proliferation pathway or DNA synthesis. (F) Evaluation of STAT3 suppression in Colo-205 cells treated with PPA CFA (PC085 and PC383), CAP CFA (PC249), APA CFA (PC010) and various concentrations of TM5441 by ELISA. STAT3 activation was shown as p-STAT3(Y705) and total STAT3 ratio at the indicated concentration relative to DMSO vehicle. Cells exposed to PPA CFA relied on PAI-1 to activate STAT3 as they required lower concentrations of TM5441 to suppress STAT3 activation. Graph shows mean ± SEM. Data in (A-E) are representative of at least three independent biological experiments and (F) is representative of two independent biological experiments. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Inhibition, DNA Synthesis, Enzyme-linked Immunosorbent Assay, Activation Assay, Concentration Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet:

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Bradford Protein Assay, Sequencing, Microarray, Cell Culture, Software

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: 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: International Journal of Biological Sciences

Article Title: DNMT3a promotes LUAD cell proliferation and metastasis by activating the HDAC7 signalling pathway

doi: 10.7150/ijbs.96509

Figure Lengend Snippet: High expression of HDAC7 predicts poor prognosis in LUAD. a Kaplan-Meier survival analysis of patients represented in the Kaplan-Meier Plotter database stratified by HDAC7 expression. b Representative images of HDAC7 IHC staining in LUAD and adjacent nontumour tissues. Scale bars, 200 μm and 20 μm (inset). c Statistical analysis of HDAC7 expression based on IHC staining in tumour tissue and adjacent nontumour tissue of 119 LUAD patients. Statistical analysis of HDAC7 expression based on IHC staining of samples from 119 LUAD patients stratified into subgroups of T classification, N classification and clinical stage. Kaplan-Meier survival analysis of 119 LUAD patients based on HDAC7 expression. d Comprehensive Kaplan-Meier survival analysis of 119 LUAD patients stratified by DNMT3a and HDAC7 expression based on tissue microarray IHC results. e Correlation analysis of DNMT3a and HDAC7 expression in LUAD tissues. f Representative images and statistical analysis of the colony formation assay. Colonies were visualized by crystal violet staining. Representative images and statistical analysis of the EdU incorporation assay. The results were calculated as the ratio of the number of EdU-positive cells (red fluorescence) to the total number of Hoechst 33342-positive cells (blue fluorescence). Scale bar, 100 μm (inset). g Representative wound healing assay images and results. The migration ability was quantified as the mean scratch area at each time point. The initial scratch area (0 h) was set as 100%. Scale bars, 100 μm (inset). h Representative transwell migration assay images and results. Scale bars, 200 μm (inset). * p < 0.05. ** p < 0.01.

Article Snippet: According to standard practice, immunohistochemical (IHC) staining of paraffin-embedded tissue microarray sections was performed using primary antibodies of anti-DNMT3a (1:400, 20954-1-AP, Proteintech) and anti-HDAC7 (1:100, #33418, Cell Signaling Technology (CST)).

Techniques: Expressing, Immunohistochemistry, Microarray, Colony Assay, Staining, Fluorescence, Wound Healing Assay, Migration, Transwell Migration Assay