Journal: British Journal of Cancer

Article Title: Regulation of E3 ubiquitin ligase-1 ( WWP1 ) by microRNA-452 inhibits cancer cell migration and invasion in prostate cancer

doi: 10.1038/bjc.2016.95

Figure Lengend Snippet: Effects of WWP1 knockdown on cell proliferation, migration, and invasion in PCa cells and expression of WWP1 in clinical PCa specimens. ( A ) Cell proliferation was determined by XTT assays. ( B ) Cell migration activity was determined using uncoated Transwell polycarbonate membrane filters. ( C ) Cell invasion activity was determined by Matrigel invasion assays. ( D ) Representative image of IHC of WWP1 in the tissue microarray. ( E ) WWP1 was upregulated in PCa compared with PIN and normal tissue in the tissue microarray. * P <0.0001. The bars indicate s.d.'s.

Article Snippet: The tissue microarray was obtained from Provitro (Berlin, Germany; Cat #401 2209, Lot #146.1 P020212, 26–46).

Techniques: Knockdown, Migration, Expressing, Activity Assay, Membrane, Microarray

Journal: Scientific Reports

Article Title: STAT1 is essential for the inhibition of hepatitis C virus replication by interferon-λ but not by interferon-α

doi: 10.1038/srep38336

Figure Lengend Snippet: ( A ) STAT2 knockout (KO) clones #1 and #2 were established from Huh-7.5 cells expressing a STAT2 sgRNA. DNA and amino acid sequences surrounding the sgRNA target sequences (blue) are shown. The protospacer adjacent motif (PAM) and the mutation in each allele are shown in green and red, respectively. ( B ) NT sgRNA-expressing cells and STAT2 KO cells (clones #1 and #2) were infected with HCVcc and treated with IFN-α (1,000 U/ml) for the indicated times. The expression levels of NS5A, PKR, and STAT2 were evaluated by immunoblotting (IB). ( C ) NT sgRNA-expressing cells and STAT2 KO cells were infected with HCVcc and treated with IFN-α (1,000 U/ml) for 96 h. Intracellular HCV RNA levels were quantified by real-time PCR and normalized to control values. Data represent the mean ± S.D. ( n = 3). * P < 0.01. NS, not significant.

Article Snippet: Anti-phospho-STAT2 (600-401-A93S) antibody was purchased from Rockland Immunochemicals.

Techniques: Knock-Out, Clone Assay, Expressing, Mutagenesis, Infection, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Scientific Reports

Article Title: STAT1 is essential for the inhibition of hepatitis C virus replication by interferon-λ but not by interferon-α

doi: 10.1038/srep38336

Figure Lengend Snippet: ( A , B ) STAT3 knockout (KO) clones #1, #2 were established from Huh-7.5 cells expressing STAT3 sgRNAs #1 and #2, respectively. STAT6 knockout (KO) clones #1 and #2 were established from Huh-7.5 cells expressing STAT6 sgRNAs #1 and #2, respectively. ( A ) Cells were infected with HCVcc and treated with IFN-α (1,000 U/ml) for 72 h. The expression levels of NS5A, PKR, IRF9, STAT1, STAT2, STAT3, and STAT6 were evaluated by immunoblotting (IB). ( B ) Cells were infected with HCVcc and treated with IFN-α (1,000 U/ml) for 96 h. Intracellular HCV RNA levels were quantified by real-time PCR and normalized to control values. Data represent the mean ± S.D. ( n = 3). * P < 0.01. NS, not significant.

Article Snippet: Anti-phospho-STAT2 (600-401-A93S) antibody was purchased from Rockland Immunochemicals.

Techniques: Knock-Out, Clone Assay, Expressing, Infection, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Scientific Reports

Article Title: STAT1 is essential for the inhibition of hepatitis C virus replication by interferon-λ but not by interferon-α

doi: 10.1038/srep38336

Figure Lengend Snippet: ( A ) NT sgRNA-expressing Huh-7.5 cells, STAT1 knockout (KO) cells (clones #1 and #2), and STAT2 KO cells (clone #1) were infected with HCVcc and treated with IFN-λ (1,000 U/ml) for the indicated times. The expression levels of NS5A, PKR, STAT1, and STAT2 were evaluated by immunoblotting (IB). ( B ) NT sgRNA-expressing cells, STAT1 KO cells (clones #1 and #2), and STAT2 KO cells (clone #1) were infected with HCVcc and treated with IFN-λ (1,000 U/ml) for 96 h. Intracellular HCV RNA levels were quantified by real-time PCR and normalized to control values. Data represent the mean ± S.D. ( n = 3). * P < 0.01. NS, not significant.

Article Snippet: Anti-phospho-STAT2 (600-401-A93S) antibody was purchased from Rockland Immunochemicals.

Techniques: Expressing, Knock-Out, Clone Assay, Infection, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Scientific Reports

Article Title: STAT1 is essential for the inhibition of hepatitis C virus replication by interferon-λ but not by interferon-α

doi: 10.1038/srep38336

Figure Lengend Snippet: ( A ) NT sgRNA-expressing Huh-7.5 cells and STAT1 knockout (KO) cells (clone #2) were infected with HCVcc and treated with IFN-α (1,000 U/ml) or IFN-λ (1,000 U/ml) for the indicated times. The expression levels of NS5A, PKR, IRF9, STAT1, and STAT2 were evaluated by immunoblotting (IB). ( B ) NT sgRNA-expressing cells, STAT1 KO cells (clones #1 and #2), and STAT2 KO cells (clone #1) were treated with IFN-α (1,000 U/ml) or IFN-λ (1,000 U/ml) for the indicated times. The levels of PKR and MX1 mRNAs were evaluated by real-time PCR and normalized to control values. Data represent the mean ± S.D. ( n = 3). ( C ) NT sgRNA-expressing cells and STAT1 KO cells (clone #1) were treated with IFN-α (1,000 U/ml) or IFN-λ (1,000 U/ml) for 24 h. Microarray analysis was performed. Fold changes relative to untreated NT sgRNA-expressing cells were calculated. Probe sets that showed >1.5-fold increase in response to both IFN-α and IFN-λ in NT sgRNA-expressing cells but showed little changes (within 1.5-fold) due to STAT1 KO were selected. Heat maps were generated using the microarray data. See for a full list of the selected probe sets and fold changes.

Article Snippet: Anti-phospho-STAT2 (600-401-A93S) antibody was purchased from Rockland Immunochemicals.

Techniques: Expressing, Knock-Out, Infection, Western Blot, Clone Assay, Real-time Polymerase Chain Reaction, Control, Microarray, Generated

Journal: Scientific Reports

Article Title: STAT1 is essential for the inhibition of hepatitis C virus replication by interferon-λ but not by interferon-α

doi: 10.1038/srep38336

Figure Lengend Snippet: ( A , B ) NT sgRNA-expressing Huh-7.5 cells, STAT1 knockout (KO) cells (clones #1 and #2), and STAT2 KO cells (clone #1) were treated with IFN-α (1,000 U/ml) ( A ) or IFN-λ (1,000 U/ml) ( B ) for the indicated times. Phosphorylation of STAT1 and STAT2 was evaluated by immunoblotting (IB).

Article Snippet: Anti-phospho-STAT2 (600-401-A93S) antibody was purchased from Rockland Immunochemicals.

Techniques: Expressing, Knock-Out, Clone Assay, Phospho-proteomics, Western Blot

Journal: Endocrinology

Article Title: Thyroid Hormone Receptor β Suppression of RUNX2 Is Mediated by Brahma-Related Gene 1–Dependent Chromatin Remodeling

doi: 10.1210/en.2018-00128

Figure Lengend Snippet: BRG1 expression is reduced in thyroid cancer cell lines and thyroid cancer tissue samples. (A) Representative immunoblot illustrates BRG1, Baf57, and Baf60 protein expression in thyroid cell lines derived from a spectrum of thyroid cancer subtypes. (B) The histogram illustrates quantitation of BRG1, Baf57, and Baf60 protein levels, standardized to β-actin, averaged from three independent experiments performed in triplicate. Error bars are SD; significance compared with nonmalignant cells (Nthy-ORI) is indicated. (C) The graph illustrates BRG1, Baf57, and Baf60 mRNA levels, standardized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), averaged from three independent experiments performed in triplicate. Error bars are SD; significance compared with nonmalignant cells (Nthy-ORI) is indicated. (D) Publically available microarray expression data (GSE76039, GSE3467), analyzed using GEOR2, reveals differential expression of BRG1 in thyroid cancer patient tumor samples. Error bars are SD; significance compared with normal thyroid tissue is indicated. *P < 0.05; **P < 0.01; ****P < 0.0001. ADU, arbitrary densitometric units; ATC, anaplastic thyroid cancer; PDTC, poorly differentiated thyroid carcinoma; PTC, papillary thyroid cancer.

Article Snippet: β -Actin , WB , Rockland Immunochemicals; 600-401-886 , AB_2223521 , 1:10,000.

Techniques: Expressing, Western Blot, Derivative Assay, Quantitation Assay, Microarray, Quantitative Proteomics

Journal: Cell Reports

Article Title: Polarity inversion reorganizes the stem cell compartment of the trophoblast lineage

doi: 10.1016/j.celrep.2023.112313

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-RFP , Rockland , Cat# 600-401-379, RRID: AB_2209751.

Techniques: Recombinant, Cell Culture, Synthesized, Microarray, Expressing, Software

Journal: Cell Reports Medicine

Article Title: Single-Cell Analyses Identify Dysfunctional CD16 + CD8 T Cells in Smokers

doi: 10.1016/j.xcrm.2020.100054

Figure Lengend Snippet: scRNA-Seq and Mass Cytometry Profiling of Human PBMCs from Smokers (n = 4) and Nonsmokers (n = 4) (A) Experimental design: cryopreserved PBMCs from smokers and nonsmokers were thawed for scRNA-seq and mass cytometry. (B) Uniform Manifold Approximation and Projection (UMAP) scRNA-seq plot: ∼45,000 PBMCs colored by major cell type. (C) Force directed layout (FDL) of mass cytometry: ∼1 million cells colored by major cell type. (D) Canonical gene expression markers for major cell types: CD8T cells ( CD3D , CD8A ), CD4T cells ( CD3D , CD4 ) natural killer T (NKT) cells ( CD3D , NCR3 ), natural killer (NK) cells ( NCR3 ), monocytes ( CD14 , FCGR3A [CD16]), dendritic cells (DCs; FCER1A ), and B cells ( MS4A1 ). (E) Cell surface protein expression for major cell types: CD8T cells (CD3, CD8a), CD4T cells (CD3, CD4), NKT cells (CD3, CD56), NK cells (CD56), monocytes (CD14, CD16), DCs (CD123) and B cells (CD19). (F and G) Major cell type frequency distributions by individual donor (nonsmokers, NS; smokers, SM) colored by cell type for scRNA-seq (F) and mass cytometry (G). (H) Major cell type frequencies showed strong correlation between scRNA-seq and mass cytometry (Pearson r = 0.99, r 2 = 0.98, p < 0.0001). Shapes represent matched individuals, NS (unfilled) and SM (filled), are colored by cell type.

Article Snippet: CD56 MicroBeads, human , Miltenyi Biotec , Cat# 130-050-401.

Techniques: Mass Cytometry, Gene Expression, Expressing

Journal: Cell Reports Medicine

Article Title: Single-Cell Analyses Identify Dysfunctional CD16 + CD8 T Cells in Smokers

doi: 10.1016/j.xcrm.2020.100054

Figure Lengend Snippet: Characterization of CD16 + CD8 T Cells (A–D) Mass cytometry confirms elevated proportion of CD16 + CD8 T cells in smokers. (A) FDL (n = 8) of CD8 T and NKT cells colored by cluster ID. (B and C) Cell surface marker intensity for NK marker CD56 (B) and CD16 (C) in nonsmokers (top, n = 4) and smokers (bottom, n = 4). (D) Frequency of CD16 + cells increases within CD8 T cells in smokers (filled, n = 4) compared to nonsmokers (unfilled, n = 4). Bar = median, ∗p < 0.05 by 1-tailed Mann-Whitney U test. (E and F) NK-associated transcriptome characteristics (n = 8) revealed in CD8T-8 cells by gene set enrichment analysis (GSEA). CD8T-8 cells were positively enriched (FWER < 0.05) for gene signatures of T cells induced to have NK-like phenotypes; split dot plot shows 25 genes within the induced T to NK gene set (E). CD8T-8 cells were negatively enriched (FWER < 0.05) for genes higher in naive CD8 T cells relative to NK cells; split dot plot shows 25 genes within the naive T versus NK gene set (F). Color intensity represents average per-cell expression (scale shows standard deviation), and circle size indicates the percentage of cells expressing each gene. (G and H) CD8T-8 cells show increased GZMB expression in smokers. (G) UMAP comparison of GZMB between nonsmokers’ (top, n = 4) and smokers’ (bottom, n = 4) cells. Cluster CD8T-8 is indicated by dotted circle. (H) Individual donor GZMB levels for CD8T-8 cells for nonsmokers (NS, n = 4) and smokers (SM, n = 4). (I and J) CD8T-8 cells have increased PRF1 expression in smokers. (I) UMAP comparison of PRF1 between nonsmokers’ (top, n = 4) and smokers’ (bottom, n = 4) cells. Cluster CD8T-8 is indicated by dotted circle. (J) Individual donor PRF1 levels for CD8T-8 cells for nonsmokers (NS, n = 4) and smokers (SM, n = 4).

Article Snippet: CD56 MicroBeads, human , Miltenyi Biotec , Cat# 130-050-401.

Techniques: Mass Cytometry, Marker, MANN-WHITNEY, Expressing, Standard Deviation, Comparison

Journal: Cell Reports Medicine

Article Title: Single-Cell Analyses Identify Dysfunctional CD16 + CD8 T Cells in Smokers

doi: 10.1016/j.xcrm.2020.100054

Figure Lengend Snippet:

Article Snippet: CD56 MicroBeads, human , Miltenyi Biotec , Cat# 130-050-401.

Techniques: Binding Assay, Recombinant, Modification, Saline, Staining, Multiplex Assay, Hybridization, DNA Methylation Assay, RNA Sequencing, Microarray, Software

Journal: Oncotarget

Article Title: Tumor suppressor FOXO3 regulates ribonucleotide reductase subunit RRM2B and impacts on survival of cancer patients

doi:

Figure Lengend Snippet: a) HeLa, H1299, and MDA-231 cells were treated with either control siRNA (C) or FOXO3 siRNA (F) for 72 hours, and harvested for Western blot analysis with antibodies anti-FOXO3, anti-RRM2B, anti-p53, and anti-β-ACTIN (as loading control). b) Cells were treated with control or FOXO3 siRNA for 72 hours. Samples from U2OS, MDA-231 and HeLa cells were analyzed by qPCR. H1299 cells were analyzed by PCR, and then relative expression was analyzed by image J software. c) H1299 cells were seeded onto the coverslips in culture dishes. Cells were treated with siRNA for 72 hours, fixed and then immunoflourescence assay was performed. DAPI served as nuclear marker. d) H1299 cells were transfected with indicated plasmids, and then harvested for Western blots and RNA expression analysis 48 hours later.

Article Snippet: Antibodies used were: RRM2B (produced by Rockland), anti-FOXO3a (Santa Cruz and Epitomics), γ-H2AX (Active Motif), p53 (Santa Cruz), GAPDH (Santa Cruz), VDAC1 (GeneTex), COX4 (GeneTex), and β-ACTIN (Sigma).

Techniques: Control, Western Blot, Expressing, Software, Marker, Transfection, RNA Expression

Journal: Oncotarget

Article Title: Tumor suppressor FOXO3 regulates ribonucleotide reductase subunit RRM2B and impacts on survival of cancer patients

doi:

Figure Lengend Snippet: a) The graph shows three putative FHREs on the RRM2B promoter, where FOXO3 could potentially bind to and activate RRM2B transcription. b) Cells were co-transfected with different FHRE of RRM2B-Luc and either control, WT, or FOXO3(A)3 plasmids as indicated, and then reporter assays were carried out. (means ± SEM, n = 2). c) Cells were co-transfected with WT or RRM2B-Luc (-964) mutants and either control or FOXO3(A)3 plasmids as indicated, and reporter assays were performed. (means ± SEM, n = 2). d) Cells were co-transfected with WT RRM2B-Luc and either control or FOXO3(A)3 plasmids as well as treated with control or FOXO3 siRNA and analyzed by reporter assay 48 hours later. (means ± SEM, n = 3).

Article Snippet: Antibodies used were: RRM2B (produced by Rockland), anti-FOXO3a (Santa Cruz and Epitomics), γ-H2AX (Active Motif), p53 (Santa Cruz), GAPDH (Santa Cruz), VDAC1 (GeneTex), COX4 (GeneTex), and β-ACTIN (Sigma).

Techniques: Transfection, Control, Reporter Assay

Journal: Oncotarget

Article Title: Tumor suppressor FOXO3 regulates ribonucleotide reductase subunit RRM2B and impacts on survival of cancer patients

doi:

Figure Lengend Snippet: a) Three pairs of H1299 cell lines expressing control vectors, RRM2BshRNA, RRM2B expressing vector, EGFP, and EGFP-FOXO3, were established and harvested for Western blots analysis with indicated antibodies. ( n > 3) bi)-biii) Stable H1299 cells mentioned above as three pairs were seeded onto 96 well plates, and the proliferation of the cells was analyzed by MTS assay 72h later. 4bi), n = 3; 4bii) and 4biii), n = 2 (means ± SEM). Cells were seeded onto the 96 well plates and the proliferation of cells was analyzed by MTS assay 72h later. (means ± SEM, n = 2)

Article Snippet: Antibodies used were: RRM2B (produced by Rockland), anti-FOXO3a (Santa Cruz and Epitomics), γ-H2AX (Active Motif), p53 (Santa Cruz), GAPDH (Santa Cruz), VDAC1 (GeneTex), COX4 (GeneTex), and β-ACTIN (Sigma).

Techniques: Expressing, Control, Plasmid Preparation, Western Blot, MTS Assay

Journal: Oncotarget

Article Title: Tumor suppressor FOXO3 regulates ribonucleotide reductase subunit RRM2B and impacts on survival of cancer patients

doi:

Figure Lengend Snippet: a) Representative photographs from 63 lung cancer microarray samples showing examples for RRM2B low, RRM2B high, FOXO3 low, and FOXO3 high. IHC staining was carried out with anti-RRM2B (Rockland), and FOXO3 (Epitomics) antibodies. b) The expression correlation between FOXO3 and RRM2B was shown here and analyzed by Cochran-Armitage Trend test. The P < 0.0001 c) Patient samples were separated into two groups, FOXO3 high/RRM2B high and FOXO3 low/RRM2B low. Calculation and comparison of survival functions was analyzed by the Kaplan-Meier method. The differences in the time to event distribution were examined by the log-rank test. P = 0.0454 d) The death rate and recurrence/metastasis rate were analyzed between the two groups indicated, and the differences were significant. P = 0.0202 for the death rate and P = 0.0031 for the recurrence/metastasis rate.

Article Snippet: Antibodies used were: RRM2B (produced by Rockland), anti-FOXO3a (Santa Cruz and Epitomics), γ-H2AX (Active Motif), p53 (Santa Cruz), GAPDH (Santa Cruz), VDAC1 (GeneTex), COX4 (GeneTex), and β-ACTIN (Sigma).

Techniques: Microarray, Immunohistochemistry, Expressing, Comparison

Journal: Scientific Reports

Article Title: Dipeptidyl peptidase-4 plays a pathogenic role in BSA-induced kidney injury in diabetic mice

doi: 10.1038/s41598-019-43730-5

Figure Lengend Snippet: Fibrogenic gene and EMT-related gene expression levels were induced in the BSA-injected diabetic mouse kidney; TENE treatment suppressed these gene expression levels. ( a ) Microarray analysis of the kidney samples. Heat map analysis of the gene expression. BSA injection (particularly in the diabetic mice) induced genes, such as DPP-4, TGF-β/smad3 signaling, CAV1, integrin β1 and the EMT program; TENE treatment suppressed these alterations ( n = 2 mice per group, and the average value is shown in the figure). Red indicates high and green indicates low relative expression levels. ( b – p ) qPCR analysis of the expression of the indicated genes in the kidney of mice in each group ( n = 7 mice per group). Gene expression was normalized to the control mice value. * P < 0.05, ** P < 0.01. Data are presented as mean ± s.e.m. ( q ) Representative western blot analysis. As a densitometric analysis, each protein level was normalized with actin. n = 7 per group were analyzed.

Article Snippet: A rabbit polyclonal anti-phospho smad3 (s423 and s425, 1:500) antibody was purchased from Rockland Immunochemicals (Gilbertsville, PA).

Techniques: Gene Expression, Injection, Microarray, Expressing, Control, Western Blot

Journal: Scientific Reports

Article Title: Dipeptidyl peptidase-4 plays a pathogenic role in BSA-induced kidney injury in diabetic mice

doi: 10.1038/s41598-019-43730-5

Figure Lengend Snippet: TENE treatment suppressed the crosstalk among DPP-4, integrin β1 and CAV1 via inhibition of TGF-β/smad3 signaling pathway in vitro . Duolink in situ analysis of (a-c) DPP-4/integrin β1, ( d – f ) DPP-4/CAV1 and ( g – i ) integrin β1/CAV1 in HK-2 cells with or without TGF-β1 (10 ng/ml) was performed by confocal microscopy (×1260). Scale bar: 50 μm in each panel. ( j ) Representative western blot analysis. As a densitometric analysis, each protein level was normalized with actin. n = 6 per group were analyzed. ( k – n ) Duolink in situ analysis of integrin β1/CAV1 in DPP-4 overexpressed HK-2 cells with or without TENE and SIS3. ( o ) Immunoprecipitation analysis revealed TGF-β treatment increased crosstalk among DPP-4, integrin β1 (ITGβ1) and CAV1. ( p ) Immunoprecipitation assay revealed TGF-β neutralization suppressed crosstalk among DPP-4, integrin β1 and CAV1 induced by DPP-4 overexpression.

Article Snippet: A rabbit polyclonal anti-phospho smad3 (s423 and s425, 1:500) antibody was purchased from Rockland Immunochemicals (Gilbertsville, PA).

Techniques: Inhibition, In Vitro, In Situ, Confocal Microscopy, Western Blot, Immunoprecipitation, Neutralization, Over Expression