Journal: Brain Pathology

Article Title: Galectin‐1 Is Implicated in the Protein Kinase C ε/Vimentin‐Controlled Trafficking of Integrin‐β1 in Glioblastoma Cells

doi: 10.1111/j.1750-3639.2008.00227.x

Figure Lengend Snippet: Transient transfection of galectin‐1 using targeted small interfering RNA (siRNA) decreases intracellular galectin‐1 protein. WB analysis of galectin‐1 expression levels at days 5, 7 and 9 post‐transfection in control (ctrl), scramble‐transfected (scr), and galectin‐1‐transfected (siGal1) U87 ( A ) and Hs683 ( C ) cells. Immunofluorescence (IF) analysis of galectin‐1 expression levels in U87 day 7 post‐transfection ( B ) and Hs683 day 5 post‐transfection ( D ) using scrambled or anti‐galectin‐1 siRNA. Top rows are the corresponding bright fields to the IF images.

Article Snippet: The Hs683 [American Type Culture Collection (ATCC) code HTB‐138], U87 (ATCC code HTB‐14), U373 (ATCC code HTB‐17) and T98G (ATCC code CRL‐1690) human GBM cell lines were obtained from the ATCC (Manassas, VA, USA) and maintained in our laboratory as detailed previously , , , .

Techniques: Transfection, Small Interfering RNA, Expressing, Control, Immunofluorescence

Journal: Brain Pathology

Article Title: Galectin‐1 Is Implicated in the Protein Kinase C ε/Vimentin‐Controlled Trafficking of Integrin‐β1 in Glioblastoma Cells

doi: 10.1111/j.1750-3639.2008.00227.x

Figure Lengend Snippet: Integrin‐α9 versus integrin‐β1 expression . A. Integrin‐α9, integrin‐β1 and actin expression across eight glioma cell lines by reverse transcription‐polymerase chain reaction (RT‐PCR) analysis. B. Quantitative RT‐PCR analysis of integrin‐α9 versus integrin‐β1 expression in glioma cell lines Hs683, U87 and U373. C. Hs683 expression levels of integrin‐α9 and integrin‐β1 under control (ctrl), scramble‐transfected (scr) or galectin‐1 siRNA‐transfected (siGal1) conditions obtained through microarray analysis using the Affymetrix Human Genome U133 set Plus 2.0.

Article Snippet: The Hs683 [American Type Culture Collection (ATCC) code HTB‐138], U87 (ATCC code HTB‐14), U373 (ATCC code HTB‐17) and T98G (ATCC code CRL‐1690) human GBM cell lines were obtained from the ATCC (Manassas, VA, USA) and maintained in our laboratory as detailed previously , , , .

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Control, Transfection, Microarray

Journal: Brain Pathology

Article Title: Galectin‐1 Is Implicated in the Protein Kinase C ε/Vimentin‐Controlled Trafficking of Integrin‐β1 in Glioblastoma Cells

doi: 10.1111/j.1750-3639.2008.00227.x

Figure Lengend Snippet: Galectin‐1‐targeted siRNA induces both a decrease of integrin‐β1 at the edges of the cell membrane and an increase in the intracellular accumulation of integrin‐β1 . A. U87 control wild‐type cells ( Aa,Ab ) and U87 cells containing stably transfected antisense galectin‐1 vectors ( Ac ) were co‐stained under non‐permeabilized conditions for integrin‐β1 [the red staining at the tip of actin stress fibers (in green fluorescence)], fibrillar actin (green) and globular actin (red staining inside the cells). Ab. Higher magnification of the control wild‐type ( Aa ) image. B. U87 control wild‐type cells ( Ba ) and U87 cells containing stably transfected antisense galectin‐1 vectors ( Bb ) were stained under permeabilized conditions for integrin‐β1 only (in green), without any staining to reveal actin ( Ba,Bb ). C. U87 and Hs683 cells were transiently transfected with either scrambled siRNA (scr) or siRNA targeted against galectin‐1 (siGal1). Cells were stained for integrin‐β1 (in red) under both permeablized and non‐permeabilized conditions on either day 5 post‐transfection (Hs683) or day 7 post‐transfection (U87).

Article Snippet: The Hs683 [American Type Culture Collection (ATCC) code HTB‐138], U87 (ATCC code HTB‐14), U373 (ATCC code HTB‐17) and T98G (ATCC code CRL‐1690) human GBM cell lines were obtained from the ATCC (Manassas, VA, USA) and maintained in our laboratory as detailed previously , , , .

Techniques: Membrane, Control, Stable Transfection, Transfection, Staining, Fluorescence

Journal: Brain Pathology

Article Title: Galectin‐1 Is Implicated in the Protein Kinase C ε/Vimentin‐Controlled Trafficking of Integrin‐β1 in Glioblastoma Cells

doi: 10.1111/j.1750-3639.2008.00227.x

Figure Lengend Snippet: Western blot (WB) and immunofluorescence (IF) analysis of vimentin and protein kinase C epsilon (PKCε) protein expression levels; galectin‐1‐targeted siRNA increases only vimentin protein levels in Hs683, induces increased perinulear amassment of both PKCε and vimentin, and diminishes the diffuse cytoplasmic staining of PKCε . WB analysis of PKCε and vimentin protein expression levels in U87 ( A ) or Hs683 ( B ) cells untreated (ctrl), scramble‐transfected (scr) or galectin‐1 siRNA‐transfected (siGal1) at days 5, 7 and 9 post‐transfection. IF analysis of PKCε localization in U87 ( C ) or Hs683 ( D ) scr or siGal1 at either day 5 (Hs683) or day 7 (U87) post‐transfection. IF analysis of vimentin localization in U87 ( E ) or Hs683 ( F ) cells scr or galectin‐1 siRNA‐transfected (siGal1) at either day 5 (Hs683) or day 7 (U87) post‐transfection. In all IF figures right columns represent the corresponding bright field images.

Article Snippet: The Hs683 [American Type Culture Collection (ATCC) code HTB‐138], U87 (ATCC code HTB‐14), U373 (ATCC code HTB‐17) and T98G (ATCC code CRL‐1690) human GBM cell lines were obtained from the ATCC (Manassas, VA, USA) and maintained in our laboratory as detailed previously , , , .

Techniques: Western Blot, Immunofluorescence, Expressing, Staining, Transfection

Journal: Cell Genomics

Article Title: The molecular consequences of androgen activity in the human breast

doi: 10.1016/j.xgen.2023.100272

Figure Lengend Snippet: AR activity drives fibroblast’s response to hormone-replacement therapy (A) UMAP depicting fibroblast subclusters (top) in snRNA-seq data (matrix 1 and 2, matrix-type fibroblasts; lipo-f, lipo-fibroblasts; vasc-f, vascular-like fibroblasts) and the distribution of patient samples in each gender identity (bottom). (B) Heatmap shows scaled average expression of snRNA-seq markers identified for each of the fibroblast subclusters. (C) Motif footprints for AR (AR-CisBP M03389_2.00) among trans-male and cis-female fibroblast cells. Top panel shows the transposase bias-corrected signal, and the bottom panel shows the transposase bias. (D) Right panel shows the enrichment of motifs among unique accessible chromatin peaks of fibroblast cells from cis-female and trans-male samples. Left panel shows the fraction of the peaks of the corresponding cells that overlap with the motif. (E) Average ratio of AR staining intensity in fibroblast nucleus compared with the cytoplasm in tissue regions of each sample type (p value, Wilcoxon; 1.2 × 10 −8 ). (F) Left panel boxplots show the fraction of cis-female (purple) and trans-male (orange) cells corresponding to five different classes of fibroblasts detected in tissue regions of CODEX microarray data (p values, Wilcoxon: fibr-main = 0.00011, fibr-epi = 0.0046). Right panel shows the scaled staining intensities of various markers that distinguish the five subtypes of fibroblasts. (G) Boxplots show per-region average distance for each of the five subtypes of fibroblasts to the most proximal epithelial cell. (H) Violin plots show the RNA expression of laminins LAMA2 (top) and LAMB1 (bottom) in fibroblast subclusters, split by sample type (adjusted p values, MAST: LAMA2 in lipo-f = 2.60 × 10 −63 , matrix 1 = 5.11 × 10 −175 , matrix 2 = 1.03 × 10 −69 , vasc-f = 1.99 × 10 −10 ; LAMB1 in lipo-f = 3.71 × 10 −35 , matrix 1 = 1.46 × 10 −55 , matrix 2 = 3.08 × 10 −87 , vasc-f = 2.58 × 10 −13 ). (I) AR binding sites (red markers) across genomic regions of LAMA2 and LAMB1. Gene bodies are shown (light green) with the promoter (arrow) and exon boundaries (dark green). Genomic window shows chromatin accessibility in cis-female (purple) and trans-male (orange) fibroblasts. (J) Boxplots show per-region average LAMA2 (left) and LAMB1 (right) staining intensities in the LAMB1 + fibroblast subtype on CODEX microarray data (p value, Wilcoxon: LAMA2 = 0.64, LAMB1 = 0.0079). (K) Boxplots show the average RNA expression of ITGB1 among luminal-HR + , luminal-HR – , and basal epithelial cells (adjusted p value, MAST in basal <2.22 × 10 −16 ).

Article Snippet: To study the staining patterns around epithelial cells in detail, we applied morphological operations to specifically isolate individual acini structures within the CODEX tissue microarray images ( C).

Techniques: Activity Assay, Expressing, Staining, Microarray, RNA Expression, Binding Assay

Journal: Cell Genomics

Article Title: The molecular consequences of androgen activity in the human breast

doi: 10.1016/j.xgen.2023.100272

Figure Lengend Snippet: Epithelial cells without hormone responsiveness lose contractile functions upon androgen therapy (A) Images from CODEX data showing mammary acini structures from cis-female (top) and trans-male (bottom) tissues marked by expression of ACTA2 (basal cells, purple), TP63 (basal cell nuclei, blue), and KRT8 (luminal cells, green). (B) Average area of acinar structures (left panel) and average area of acini border that was filled with ACTA2 signal (see Figure S7 C and ) among cis-female and trans-male tissues (p values, Wilcoxon: area = 0.026, ACTA2 coverage = 0.012). (C) UMAP of basal cell subclusters in snRNA-seq data (top) and the distribution of trans-male and cis-female cells across them (bottom). (D) RNA expression of ACTA2, OXTR (lactation markers), and TP63 in basal cells of trans-male and cis-female samples (adjusted p values, MAST: ACTA2 = 8.86 × 10 −296 , OXTR = 9.59 × 10 −262 , TP63 = 1.16 × 10 −96 ). (E) Module scores of enriched pathways overlaid on the basal cell UMAP (REAC, Reactome; KEGG, Kyoto Encyclopedia of Genes and Genomes). (F) Right panel shows the enrichment of motifs among unique accessible chromatin peaks from trans-male and cis-female basal cells. Left panel shows the fraction of peaks from the corresponding cells that overlap with the motif. (G) Kernel density estimation of module scores for selected altered structural pathways in luminal-HR – cells (p values, Wilcoxon: KEGG, adherens junction = 4.13 × 10 −285 ; KEGG, focal adhesion = 1.42 × 10 −255 ; KEGG, regulation of actin cytoskeleton <1.42 × 10 −255 ). (H) Average RNA expression (top) of integrin receptors from the “KEGG: regulation of actin cytoskeleton” pathway in luminal-HR – cells (adjusted p values, MAST: ITGA2 = 4.89 × 10 −201 , ITGB8 = 6.40 × 10 −267 ) and average expression of the ITGA2 and ITGB8 ligand FN1 in fibroblast subclusters and lymphatic endothelial cells (bottom) from trans-male and cis-female samples (adjusted p values, MAST: matrix 1 = 1.66 × 10 −54 , matrix 2 = non-significant [n.s.], lipo-f = 1.32 × 10 −16 , vasc-f = n.s., lymph. EC = 3.13 × 10 −99 ). (I) Fisher exact test odds ratio (x axis) and –log 10 p value (y axis) corresponding to enrichment of each motif among the chromatin accessibility peaks for the genes of the "WikiPathways: focal adhesion pathway.” Colors indicate log 2 fold change in gene expression of transcription factors corresponding to each motif. Gray motifs represent transcription factors without differential gene expression among luminal-HR – cells. Right panel shows the fraction of genes (left) and genes annotated within the focal adhesion pathway (right) that contain a chromatin peak with an ESRRG sequence motif (cisBP ESRRG_697). (J) Spatial distribution of epithelial, stromal, immune, and endothelial cells in an example breast tissue region from cis-female (top) and trans-male (bottom) samples. (K) Ratios of stromal to epithelial cells in the epithelial neighborhood (see Figure S8 C) among regions of cis-female and transgender male tissue in CODEX microarray data (p value, Wilcoxon: 0.0052).

Article Snippet: To study the staining patterns around epithelial cells in detail, we applied morphological operations to specifically isolate individual acini structures within the CODEX tissue microarray images ( C).

Techniques: Expressing, RNA Expression, Sequencing, Microarray

Journal: Cell Genomics

Article Title: The molecular consequences of androgen activity in the human breast

doi: 10.1016/j.xgen.2023.100272

Figure Lengend Snippet: Androgen therapy reduces epithelial vascularization through PPARG activity (A) Microscopic images show vascularization of two ductal structures in a cis-female (left) and a trans-male (right) breast tissue in CODEX microarray data. KRT8 (green) marks luminal cells, ACTA2 (purple) marks green cells, and CD31 (red) marks endothelial cells. Arrows point out (1) larger vessels with smooth muscle layer and (2) smaller vessels without smooth muscle layer. (B) UMAP shows vasculature subclusters detected in the snRNA-seq dataset. (left, blood endothelial cells; right, lymphatic endothelial cells; upper-mid, vascular accessory cells). (C) Boxplots show the proportions of vascular subclusters in each sample of the snRNA-seq data, split by gender ID (general linearized model [GLM] fitting a Poisson, p values: vein = 6.72 × 10 −45 , capillary = 5.31 × 10 −77 , artery = 3.58 × 10 −5 , lymph. EC = 1.33 × 10 −22 , and lymph. EC 2 = 0.0071). (D) UMAP (left) shows blood endothelial cells overlaid with scVelo stream plots. The scatterplot shows the ratio of spliced (horizontal axis) and unspliced RNA molecules (vertical axis) of PPARG among vein (blue), capillary (orange), and artery (green) blood endothelial cells. Dashed diagonal indicates the steady-state ratio. Top and bottom arcs indicate the estimated kinetic parameters of PPARG induction and repression, respectively. (E) PPARG GRN module score overlaid on UMAP plot among cis-female (left) and trans-male (right) blood endothelial cells. Barplot shows GRN importance scores of the top five genes coexpressed with PPARG . (F) Volcano plot shows the average log 2 fold change and –log 10 adjusted p value for differential expression of genes within the PPARG module among the trans-male and cis-female blood endothelial cells. Purple data points indicate genes with a chromatin accessibility peak overlapping the PPARG transcription factor sequence motif (CisBP PPARG_676) match. Barplots show the fraction of all genes (left) or genes within PPARG module (right) that contain a chromatin accessibility peak overlapping the PPARG transcription factor sequence motif (purple). (G) Boxplot shows average expression of PPARG in blood endothelial cells of cis-female (purple) and trans-male (orange) samples in snRNA-seq data. (H) Heatmap shows the log 2 fold change in expression of ligand (left)-receptor (right) pairs among cell types and vascular subclusters in the trans-male and cis-female samples. Colors indicate log 2 fold change in expression, and diameter of the circle shows the percent of cis-female cells expressing the gene.

Article Snippet: To study the staining patterns around epithelial cells in detail, we applied morphological operations to specifically isolate individual acini structures within the CODEX tissue microarray images ( C).

Techniques: Activity Assay, Microarray, Expressing, Sequencing

Journal: Cell Genomics

Article Title: The molecular consequences of androgen activity in the human breast

doi: 10.1016/j.xgen.2023.100272

Figure Lengend Snippet: Androgen therapy dominates helper T lymphocytes and reduces the presence of innate immunity (A) UMAP showing subclusters of all myeloid (left) and lymphoid (right) cells detected in the snRNA-seq data (CD8. CD8 + T cells; CD4, CD4 + T cells; T-effector, effector T cells; NK, natural killer cells; mono.DC, monocyte-derived dendritic cells; DC, dendritic cells). (B) Boxplots show the fraction of main immune cell subtypes within entire immune compartment in each sample (GLM p values, generalized linear model fitting a Poisson: CD4 = 0.00035, CD8 = 4.035 × 10 −13 , T effector = 0.045, NK = 0.00035, mono.DC = 0.017, macrophage = 0.52, monocyte = 0.055, DC = 0.0001). (C) Boxplot shows the proportion of macrophages within the periphery of epithelial cells in cis-female (purple) and trans-male (orange) tissue regions of the CODEX microarray data (p value, Wilcoxon: 0.003). (D) Kernel density estimates and boxplots show the module scores of immune-relevant Reactome pathways in macrophages of trans-male (orange) and cis-female (purple) samples (p values, Wilcoxon, class I major histocompatibility complex [MHC]-mediated antigen processing/presentation = 8.32 × 10 −17 , clathrin-mediated endocytosis = 3.64 × 10 −21 , toll-like receptor TLR1 TLR2 cascade = 3.89 × 10 −16 ). (E) Boxplots show the average RNA expression of PROS1 in basal cells of cis-female (purple) and trans-male (orange) samples (adjusted p value, MAST: 3.17 × 10 −192 ). (F) Volcano plot shows the average log 2 fold change and –log 10 adjusted p value assessing the differential expression of genes in trans-male macrophages compared with cis-female macrophages. Purple data points indicate scavenger receptors. (G) UMAP shows four immune cell staining sub-classes (macrophage, red; immune endo., green; immune main, blue; immune-epi., orange) according to the staining pattern in CODEX microarray data. Size of the data points indicates the distance to the most proximal epithelial cell. Boxplot (below) summarizes the average distance of each group of immune cells to their most proximal epithelial cell. (H) Microscopic images show staining of luminal (KRT8, green), basal (ACTA2, purple), and immune cells (CD45, red) within a trans-male (top) and a cis-female (bottom) breast tissue in CODEX microarray data. (I) Microscopic image shows IHC staining of luminal (KRT8, red), immune (CD45, green), and T-lymphocyte (CD3, purple) cells within a trans-male (top) and a cis-female (bottom) breast tissue. White cells are double-positive for CD45 and CD3. (J) Boxplot shows the ratio of immune cells (CD45 + ) expressing CD3 to those not expressing CD3 (T lymphocytes versus other immune cells) within the epithelial neighborhood of cis-female (purple) and trans-male (orange) breast tissues of IHC scan regions.

Article Snippet: To study the staining patterns around epithelial cells in detail, we applied morphological operations to specifically isolate individual acini structures within the CODEX tissue microarray images ( C).

Techniques: Derivative Assay, Microarray, RNA Expression, Expressing, Staining, Immunohistochemistry

Journal: Cell Genomics

Article Title: The molecular consequences of androgen activity in the human breast

doi: 10.1016/j.xgen.2023.100272

Figure Lengend Snippet: Testosterone induces PI3K pathway alterations with adipocytes showing distinct metabolic adaptations (A) Heatmap showing the log 2 fold change in RNA expression of PI3K activating receptors (taken from "KEGG: PI3K-Akt signaling pathway") among 10 breast cell types identified. The circle diameter indicates the fraction of cis-female cells of the cell type expressing the receptor. INS, circulating insulin secreted in the pancreas. (B) Heatmap showing log 2 fold changes in RNA expression of KEGG: PI3K-Akt signaling pathway downstream transcription factors within 10 breast cell types identified. (C) Pathway enrichment of tissue wide upregulated genes (>7 cell types) that have an NR4A1 motif in an enhancer (>4 cell types). Horizontal axis shows the odds ratio (one-sided Fisher’s exact test) comparing frequency of selected genes in the pathway versus background and vertical axis shows –log 10 p value of Fisher’s exact test (FDR < 0.05 = red, p value < 0.05 = yellow, n.s. = gray (PID, Pathway Interaction Database; WP, WikiPathways). (D) Module scores for the WikiPathways "WP: insulin signaling pathway" in all cell types, split by cis-female and trans-male origin (p value in adipocytes, Wilcoxon: 7.59 × 10 −4 ). (E) Representative images of computational segmentation of lipid vacuoles (left, see ), and resulting average area of adipocyte vacuoles per IHC scan region (p value Wilcoxon: 0.00059). (F) Sample averages of AZGP1 RNA expression in each cell type in trans-male and cis-female samples (adjusted p values, MAST: adipocyte = 5.95 × 10 −12 , basal = 2.42 × 10 −70 , blood EC = 8.67 × 10 −83 , fibroblast = n.s., luminal-HR − = 4.54 × 10 −302 , luminal-HR + = 0.00, lymph. EC = 2.83 × 10 −15 , lymphoid cells = 5.46 × 10 −12 , myeloid cells = 9.23 × 10 −8 ). (G) Microscopic CODEX image of a duct stained against AR (red), AZGP1 (green), and ACTA2 (purple) in a trans-male (top) and cis-female (bottom) breast tissue region of the tissue microarray. (H) GPAM co-expression module (GRNboost2, 95 th percentile, p value, Wilcoxon: <2.22 × 10 −16 ) score in cis-female and trans-male adipocytes. (I) TCF7L2 expression in trans-male and cis-female adipocytes (adjusted p value, MAST: 3.48 × 10 −105 ). (J) Differential expression of transcription factors in trans-male compared with cis-female adipocytes. Horizontal axis shows log 2 fold change in expression and the vertical axis shows –log 10 adjusted p value. Purple data points indicate transcription factors with accessible chromatin matching the AR sequence motif (CisBP AR_689). (K) Microscopic IHC image showing staining against nuclei (DAPI, blue), adipocytes (PLIN1, green), and TCF7L2 (purple). Boxplot shows the median staining intensity of TCF7L2 among IHC scan regions of cis-female and trans-male adipocytes (p value, Wilcoxon: 0.0069).

Article Snippet: To study the staining patterns around epithelial cells in detail, we applied morphological operations to specifically isolate individual acini structures within the CODEX tissue microarray images ( C).

Techniques: RNA Expression, Expressing, Staining, Microarray, Sequencing

Journal: Current Issues in Molecular Biology

Article Title: Development of a Novel Anti-CD44 Variant 4 Monoclonal Antibody C 44 Mab-108 for Immunohistochemistry

doi: 10.3390/cimb45030121

Figure Lengend Snippet: Immunohistochemical analysis using C 44 Mab-108 and C 44 Mab-46 against oral squamous cell carcinoma (OSCC) tissues. After antigen retrieval, the sections were incubated with 10 µg/mL of C 44 Mab-108 ( A , B , I , J ), 1 µg/mL of C 44 Mab-46 ( C , D , K , L ), and without the primary antibody (control) ( E , F , M , N ) followed by treatment with the Envision+ kit. The color was developed using 3,3′-diaminobenzidine tetrahydrochloride (DAB), and the sections were counterstained with hematoxylin. ( G , H , O , P ) Hematoxylin and eosin (HE) staining. ( Q , R ) Blocking of the C 44 Mab-108 reactivity to OSCC tissue by the CD44 peptide (aa 271–290) containing the C 44 Mab-108 epitope. After antigen retrieval, sections were incubated with C 44 Mab-108 (10 μg/mL) or C 44 Mab-108 (10 μg/mL) plus human CD44 peptide (aa 271–290, 10 μg/mL) followed by treatment with the Envision+ kit. The color was developed using DAB, and sections were counterstained with hematoxylin. Scale bar = 100 µm.

Article Snippet: The formalin-fixed paraffin-embedded (FFPE) OSCC tissue microarray (Product Code: OR601c, US Biomax Inc., Rockville, MD, USA) and the esophageal tissue microarray (Product Code: BC02011, US Biomax Inc.) were deparaffinized in xylene (Sigma-Aldrich Corp.) and rehydrated.

Techniques: Immunohistochemical staining, Incubation, Staining, Blocking Assay

Journal: Translational Cancer Research

Article Title: Functional analysis of long-chain non-coding RNA in oral squamous cell carcinoma

doi: 10.21037/tcr.2019.12.67

Figure Lengend Snippet: Clustered panels of differentially expressed lncRNAs (A) and mRNAs (B). Each column represents a sample; each row represents the degree of expression of a gene in different samples, the red refers to relatively high expression, and the green refers to the relatively low expression; the color in the upper left bar represents the fold change of gene expression; the top sample tree represents the clustering of similarity; the top color block represents the expected grouping of samples before the clustering analysis, and the samples with the same color are expected to be in the same group. LncRNA, long-chain non-coding RNA.

Article Snippet: The differentially expressed lncRNA and mRNAs were examined in 5 pairs of tissues with the 4×180 K lncRNA + mRNA Human Gene Expression Microarray V4.0 (CapitalBio Technology, Beijing, China).

Techniques: Expressing, Gene Expression, Blocking Assay

Journal: Translational Cancer Research

Article Title: Functional analysis of long-chain non-coding RNA in oral squamous cell carcinoma

doi: 10.21037/tcr.2019.12.67

Figure Lengend Snippet: Scatter plot of mRNA (A) and lncRNA (B). The number of genes with up-regulated and down-regulated expression was described in the upper left and lower right corners, respectively. The red line X2 is the threshold line of the up-regulated expression, the green line X(−2) is the threshold line of the down-regulated expression, and the middle gray line is the fitted line of the overall expression. The equations in the figure are the fitted line equations, and R represents the correlation coefficient. LncRNA, long-chain non-coding RNA.

Article Snippet: The differentially expressed lncRNA and mRNAs were examined in 5 pairs of tissues with the 4×180 K lncRNA + mRNA Human Gene Expression Microarray V4.0 (CapitalBio Technology, Beijing, China).

Techniques: Expressing

Journal: Translational Cancer Research

Article Title: Functional analysis of long-chain non-coding RNA in oral squamous cell carcinoma

doi: 10.21037/tcr.2019.12.67

Figure Lengend Snippet: Volcano plot of mRNA (A) and lncRNA (B). The horizontal ordinate is the log2 (P values) and the longitudinal ordinate is the log2 (fold change). The red refers to the up-regulated genes, the green refers to the down-regulated genes, and the black refers to genes without significant difference in the expression. Volcanoplot can intuitively reflect the number, significance, and reliability of differentially expressed genes. The closer to the right upper or left upper corner, the more significant the difference in the expression is between two groups. LncRNA, long-chain non-coding RNA.

Article Snippet: The differentially expressed lncRNA and mRNAs were examined in 5 pairs of tissues with the 4×180 K lncRNA + mRNA Human Gene Expression Microarray V4.0 (CapitalBio Technology, Beijing, China).

Techniques: Expressing

Journal: Translational Cancer Research

Article Title: Functional analysis of long-chain non-coding RNA in oral squamous cell carcinoma

doi: 10.21037/tcr.2019.12.67

Figure Lengend Snippet: Disease enrichment analysis of differentially expressed mRNA (top 30 terms). As shown in the figure, the diseases with the most enrichment of mRNAs included immune system diseases, primary immunodeficiency, muscular diseases, allergies and autoimmune diseases, skin and soft tissue diseases, skin diseases, musculoskeletal diseases, celiac diseases, cardiac diseases, inflammatory bowel disease, dilated cardiomyopathy, Graves’ disease, cardiovascular diseases, systemic sclerosis, systemic lupus erythematosus, vitiligo and rheumatoid arthritis.

Article Snippet: The differentially expressed lncRNA and mRNAs were examined in 5 pairs of tissues with the 4×180 K lncRNA + mRNA Human Gene Expression Microarray V4.0 (CapitalBio Technology, Beijing, China).

Techniques:

Journal: The EMBO Journal

Article Title: A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation

doi: 10.1038/emboj.2013.221

Figure Lengend Snippet: ATM-dependent regulation of lncRNA expression in response to DNA damage. (A) Experimental layout to identify ATM-dependent lncRNAs. Atm+/+ and Atm−/− mouse embryonic fibroblasts (MEFs) were treated with NCS (200 ng/ml) and harvested at indicated time points for microarray analyses. (B) The number of ATM-dependent lncRNAs upon DNA damage. (C) A representative group of ATM-dependent and DNA damage-induced lncRNAs. Green or red colour on the heat map indicates a decrease or an increase in the lncRNA level and colour intensities correspond to relative signal levels on a logarithmic scale. (D) Quantitative PCR validation of representative lncRNAs. Data represent the mean of three experimental replicates, with error bars depicting s.d.

Article Snippet: LncRNA in situ hybridization Breast cancer tissue microarray (no. {"type":"entrez-nucleotide","attrs":{"text":"BC081115","term_id":"51703523","term_text":"BC081115"}} BC081115 ) was purchased from Biomax, including 100 breast tumour samples and 10 control breast tissue samples.

Techniques: Expressing, Microarray, Real-time Polymerase Chain Reaction, Biomarker Discovery

Journal: The EMBO Journal

Article Title: A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation

doi: 10.1038/emboj.2013.221

Figure Lengend Snippet: LncRNA-JADE is induced after DNA damage. (A) Schematic illustration showing Jade1 and lncRNA-JADE genes in mouse and human. (B) Mouse and human lncRNA-JADE are induced in an ATM-dependent manner after DNA damage. (C) DNA damage positively regulates lncRNA-JADE promoter activity in an ATM-dependent manner. ATM-IN: ATM inhibitor. (D) Schematic illustration showing the NF-κB binding elements in the Jade1 promoter. DNA damage induces the activity of lncRNA-JADE promoter in an NF-κB-dependent manner. NF-κB-IN: NF-κB inhibitor. (E) Expression of LncRNA-JADE is regulated by ATM and NF-κB after DNA damage. IRF-1 (interferon response factor-1) is served as a positive control in the NF-κB signalling. Graphic data in this figure present the mean of three experimental replicates and error bars depict s.d.

Article Snippet: LncRNA in situ hybridization Breast cancer tissue microarray (no. {"type":"entrez-nucleotide","attrs":{"text":"BC081115","term_id":"51703523","term_text":"BC081115"}} BC081115 ) was purchased from Biomax, including 100 breast tumour samples and 10 control breast tissue samples.

Techniques: Activity Assay, Binding Assay, Expressing, Positive Control

Journal: The EMBO Journal

Article Title: A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation

doi: 10.1038/emboj.2013.221

Figure Lengend Snippet: LncRNA-JADE positively regulates histone H4 acetylation through Jade1. (A) H4 acetylation is induced after DNA damage. MCF7 cells were treated with NCS (500 ng/ml). H4Ac: total histone H4 acetylation; K5, K8, K12: Histone H4 acetylation at lysine 5, 8, or 12. (B) Jade1 knockdown abolishes the induction of H4 acetylation after DNA damage. (C) LncRNA-JADE positively regulates H4 acetylation and Jade1. Overexpression of lncRNA-JADE enhanced the induction of H4 acetylation and Jade1, and knockdown of lncRNA-JADE abolished the induction of H4 acetylation and Jade1. Semi-quantification of proteins is shown at the bottom.

Article Snippet: LncRNA in situ hybridization Breast cancer tissue microarray (no. {"type":"entrez-nucleotide","attrs":{"text":"BC081115","term_id":"51703523","term_text":"BC081115"}} BC081115 ) was purchased from Biomax, including 100 breast tumour samples and 10 control breast tissue samples.

Techniques: Knockdown, Over Expression

Journal: The EMBO Journal

Article Title: A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation

doi: 10.1038/emboj.2013.221

Figure Lengend Snippet: Brca1 binds lncRNA-JADE and mediates Jade1 induction in the DNA damage response via p300-containing transcription complex. (A) Schematic illustration showing the p300/CBP binding elements in the Jade1 promoter. (B) p300 physically interacts with the promoter region of Jade1 gene. Control or lncRNA-JADE knockdown MCF7 cells were treated with or without NCS (200 ng/ml) and cell lysates were immunoprecipitated with control IgG or p300 antibodies. The p300-binding activity of Jade1 promoter DNA was quantified by qPCR. (C) Brca1 interacts with p300 and this interaction is increased after DNA damage. (D) Jade1 promoter activity is induced in a Brca1-dependent manner after DNA damage. MCF-7 cells were infected with lentiviruses expressing control or Brca1 shRNA. The cells were transfected with pGL3-control vector (SV40 promoter) or Jade1 promoter-driven firefly luciferase expression vector and Renilla luciferase expression vector 2 days post infection. They were treated with NCS (500 ng/ml) 24 h after transfection and then harvested 16 h after treatment. Firefly luciferase activity was measured and normalized to the activity of Renilla luciferase. (E) LncRNA-JADE physically interacts with Brca1. 5′- and 3′-deletion mutants of lncRNA-JADE were generated as indicated. Two pairs of primers were used to detect the Brca1-binding sequences of lncRNA-JADE in RIP assays. Graphic data in this figure present the mean of three experimental replicates and error bars depict s.d.

Article Snippet: LncRNA in situ hybridization Breast cancer tissue microarray (no. {"type":"entrez-nucleotide","attrs":{"text":"BC081115","term_id":"51703523","term_text":"BC081115"}} BC081115 ) was purchased from Biomax, including 100 breast tumour samples and 10 control breast tissue samples.

Techniques: Binding Assay, Control, Knockdown, Immunoprecipitation, Activity Assay, Infection, Expressing, shRNA, Transfection, Plasmid Preparation, Luciferase, Generated

Journal: The EMBO Journal

Article Title: A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation

doi: 10.1038/emboj.2013.221

Figure Lengend Snippet: Biological functions of lncRNA-JADE in human MCF7 cells. (A) LncRNA-JADE positively regulates MCF7 cell proliferation. (B) Altering lncRNA-JADE expression affects DNA damage-induced cell-cycle arrest. Cell-cycle profiles were analysed by flow cytometry using propidium iodide-stained cells. (C) Knockdown of lncRNA-JADE increases cell apoptosis in the control and NCS-treated cells. The percentage of TUNEL-positive cells was summarized in the graph. (D) Knockdown of lncRNA-JADE increases the cell sensitivity to DNA damaging drugs NCS, Etopside, and Bleomycin. MCF7 cells were treated with DNA damaging agents as indicated and cultured for 48 h and cell viability was measured. Graphic data in this figure present the mean of three biological replicates and error bars depict s.d.

Article Snippet: LncRNA in situ hybridization Breast cancer tissue microarray (no. {"type":"entrez-nucleotide","attrs":{"text":"BC081115","term_id":"51703523","term_text":"BC081115"}} BC081115 ) was purchased from Biomax, including 100 breast tumour samples and 10 control breast tissue samples.

Techniques: Expressing, Flow Cytometry, Staining, Knockdown, Control, TUNEL Assay, Cell Culture

Journal: The EMBO Journal

Article Title: A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation

doi: 10.1038/emboj.2013.221

Figure Lengend Snippet: Knockdown of lncRNA-JADE inhibits mammary tumour growth in vivo. (A) Higher levels of lncRNA-JADE in human breast cancer tissues in comparison with normal breast tissues. In situ hybridization of lncRNA-JADE was performed on tissue microarray comprised of human normal breast and breast cancer tissues. (B) Correlation between lncRNA-JADE and Jade1 up-expression in breast cancer tissue cDNA array. The level of lncRNA-JADE and Jade1 was measured by RT–PCR. The lncRNA-JADE expression demonstrated a significant correlation with the Jade1 expression according to the Spearman correlation coefficient (r=0.6983 and P=0.0294). (C) Comparison of survival curves between patients with Jade1 overexpression and patients with normal Jade1 expression using TCGA data in breast invasive carcinomas. (D) Knockdown of lncRNA-JADE inhibits xenografted 4T1 tumour growth in vivo. One million luciferase expressing 4T1 cells stably expressing control or lncRNA-JADE shRNA were injected into the mammary fat pad of each Balb/cSCID mouse. Two weeks after injection, luciferase activity was measured and quantified by an IVIS device (left panel and upper right panel). Breast tumour size was measured in the mice for 24 days (bottom right panel). Graphic data present the mean of five mice and error bars depict s.d.

Article Snippet: LncRNA in situ hybridization Breast cancer tissue microarray (no. {"type":"entrez-nucleotide","attrs":{"text":"BC081115","term_id":"51703523","term_text":"BC081115"}} BC081115 ) was purchased from Biomax, including 100 breast tumour samples and 10 control breast tissue samples.

Techniques: Knockdown, In Vivo, Comparison, In Situ Hybridization, Microarray, Expressing, Reverse Transcription Polymerase Chain Reaction, Over Expression, Luciferase, Stable Transfection, Control, shRNA, Injection, Activity Assay

Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: Loss of the mucoid phenotype by a B. multivorans ATCC 17616 evolved colony is caused by reduction of bce gene expression. (A) Images of the mucoid colony morphologies of the ATCC 17616 ancestor and evolved 17616nmv colony grown in yeast extract-mannitol agar medium for 72 h. (B) Exopolysaccharide production in salts-mannitol medium by the ancestor and evolved colony estimated by recovery of the ethanol precipitate from culture supernatants. (C) Quantitative RT-PCR analysis of transcript levels of bceB , bceF , bceQ , and bceR genes in evolved 17616nmv compared to B. multivorans ATCC 17616, during growth in salts-mannitol medium up to 48 h at 37°C. Data are the average result from at least three independent growth cultures. Error bars indicate standard deviations. WT, wild type.

Article Snippet: Then, genes differentially expressed were mapped against ATCC 17616 coding sequence (CDS) to identify regions possibly acquired by horizontal gene transfer.

Techniques: Gene Expression, Quantitative RT-PCR

Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: Insertion of the IS 406 element in an intergenic region leads to increased expression of gene Bmul_0158 encoding a histone-like protein. (A) Using whole-genome sequencing of the 17616nmv strain, an insertion sequence of the IS 406 family was identified in the chromosome 1 intergenic region between genes Bmul_0157 and Bmul_0158. The nucleotide sequence of the intergenic region where the IS element was inserted is shown; the target sites of this IS element are shown in red, and partial sequences of the inverted repeats of the IS element are underlined. The two inverted orange triangles indicate the distance from IS 406 to the beginning of Bmul_0157 or Bmul_0158 coding sequences. nt, nucleotides. (B) Electrophoretic separation of the PCR products amplified from the genome of the ancestor ATCC 17616 (lane 1) and the evolved strain 17616nmv (lane 2) with expected sizes of 699 and 2,066 bp, respectively (red arrows in panel A indicate primer-binding sites). (C) qRT-PCR analysis of transcript levels of genes Bmul_0157 and Bmul_0158 and other hns -like genes in the evolved strain compared with the ancestor ATCC 17616 grown for 8 h in SM. (D) Amino acid sequence alignments of the C-terminal DNA-binding region of Mycobacterium tuberculosis Lsr2 ( ALB20845.1 ), Escherichia coli K-12 H-NS ( NP_415753.1 ), Salmonella enterica H-NS ( AUO51906.1 ), Acinetobacter baumannii ATCC 17978 H-NS ( QDQ68268.1 ), Burkholderia vietnamiensis Bv3F ( A4JS72.1 ), and B. multivorans ATCC 17616 Bmul_0158 ( ABX13853 ) are displayed. The conserved motif T/SXQ/RGRXPA implicated in DNA binding is highlighted in red. Asterisks indicate the amino acid residues that are identical in all proteins; one or two dots indicate semiconserved or conserved substitutions, respectively.

Article Snippet: Then, genes differentially expressed were mapped against ATCC 17616 coding sequence (CDS) to identify regions possibly acquired by horizontal gene transfer.

Techniques: Expressing, Sequencing, Amplification, Binding Assay, Quantitative RT-PCR

Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: Overexpression of the Bmul_0158 gene in wild-type Burkholderia strains reduces the mucoid phenotype of the colonies. (A) qRT-PCR analysis of transcript levels of genes Bmul_0158 and bceB in B. multivorans ATCC 17616 harboring the vector expressing Bmul_0158 from its own promoter (pLM20-4) compared with ATCC harboring the empty vector pBBR1MCS. (B) Mobilization of the vector (left panel) or pLM20-4 (right panel) to strains of B. multivorans , B. anthina , B. contaminans , and B. vietnamiensis followed by incubation in YEM agar medium supplemented with appropriate antibiotics for 3 days. Bar, 1 mm. (C) Quantification of the diameter of at least 20 randomly chosen colonies using the software Zen 3.1 from Zeiss. Colony diameter of strains overexpressing Bmul_0158 was significantly smaller than that for the ones carrying the empty vector. ***, P < 0.001 by Tukey’s honestly significant difference (HSD) multiple-comparison test.

Article Snippet: Then, genes differentially expressed were mapped against ATCC 17616 coding sequence (CDS) to identify regions possibly acquired by horizontal gene transfer.

Techniques: Over Expression, Quantitative RT-PCR, Plasmid Preparation, Expressing, Incubation, Software, Comparison

Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: The evolved variant has decreased surface hydrophobicity and forms smaller cellular aggregates. (A) Growth curves of B. multivorans ATCC 17616 and evolved 17616nmv in SM at 37°C as measured by optical density and counting of CFU. The evolved variant differed significantly from its ancestor regarding the optical density at 640 nm and CFU at the indicated time points. ***, P < 0.001 by Tukey’s HSD multiple-comparison test. (B to D) Light microscopy images of B. multivorans ATCC 17616 and evolved 17616nmv grown in SM at 37°C for 48 h (B), followed by dry-weight biomass determination (C) and by quantification of the percentage of aggregates and free cells (D). The evolved variant differed significantly from its ancestor regarding the percentage of cells in the form of aggregates. ***, P < 0.001 by Tukey’s HSD multiple-comparison test; ns, nonsignificant. (E) Relative surface hydrophobicity of B. multivorans ATCC 17616 and evolved 17616nmv. Bacterial suspensions cultured in SM were adjusted to an OD 640 of 0.6 (OD initial ). After the addition of n -hexadecane, the OD aq of the aqueous phase was measured. The hydrophobic activity (HP) was calculated from the formula % HP = [1 − (OD aq /OD initial ) × 100]. Values are the means of three independent experiments conducted in triplicates; error bars represent standard deviations. Cell surface hydrophobicity of the evolved variant was significantly lower than that of the ancestor. **, P < 0.01 by Tukey’s HSD multiple-comparison test.

Article Snippet: Then, genes differentially expressed were mapped against ATCC 17616 coding sequence (CDS) to identify regions possibly acquired by horizontal gene transfer.

Techniques: Variant Assay, Comparison, Light Microscopy, Cell Culture, Activity Assay, Cell Surface Hydrophobicity

Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: The evolved variant has increased motility but lower adhesion to a CF lung epithelial cell line. (A) Swimming motility assayed in 1% tryptone, 0.5% NaCl medium with 0.3% agar incubated at 37°C for 24 h and swarming motility assayed in Broomfield medium with 0.6% agar incubated at 37°C for 48 h were significantly higher for the strains overexpressing Bmul_0158 compared to the ancestors. ***, P < 0.001 by Tukey’s HSD multiple-comparison test. (B) Adhesion to CF lung epithelial cells by B. multivorans ATCC 17616 and evolved 17616nmv using an MOI of 10. The evolved variant differed significantly from its ancestor regarding the percentage of cells that adhere to the CF lung cell line. ***, P < 0.001 by Tukey’s HSD multiple-comparison test. (C) Susceptibility to the indicated antibiotics determined at 37°C after 24 h of incubation by measuring the diameter of cell growth inhibition. The evolved variant differed significantly from the ancestors only for resistance to kanamycin. ***, P < 0.001 by Tukey’s HSD multiple-comparison test.

Article Snippet: Then, genes differentially expressed were mapped against ATCC 17616 coding sequence (CDS) to identify regions possibly acquired by horizontal gene transfer.

Techniques: Variant Assay, Incubation, Comparison, Inhibition

Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: Functional distribution into COGs of genes that are differentially expressed shows enrichment of genes implicated in motility and intracellular trafficking, secretion, and vesicular transport. (A) qRT-PCR analysis of transcript levels of gene Bmul_0158 in B. multivorans ATCC 17616 grown in SM. Comparison for the different time points (t x ) was made against the expression level at 4 h (t 4 ). (B) qRT-PCR analysis performed in 17616nmv and B. multivorans ATCC 17616 and comparison with the microarray expression data. LB-FC, lower bound of fold change. (C) Clustering, based on biological function, of the differentially expressed genes with COG attributed. The percentage of genes differentially expressed within each category was calculated from the total number of B. multivorans genes attributed to each COG category. The cumulative distribution function (CDF) of the hypergeometric distribution was used to calculate enrichment of genes by COG. ***, P < 0.001; *, P < 0.05.

Article Snippet: Then, genes differentially expressed were mapped against ATCC 17616 coding sequence (CDS) to identify regions possibly acquired by horizontal gene transfer.

Techniques: Functional Assay, Quantitative RT-PCR, Comparison, Expressing, Microarray

Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: Mapping of the putative Bmul_0158 H-NS protein targets in the ATCC 17616 genome. Gene expression data were mapped onto circular representation of B. multivorans ATCC 17616 chromosomes using the software CGView. Downregulated genes are shown in red and upregulated genes in blue, located in the rings between FDAARGOS_623 and the GC content. To identify potentially horizontally acquired genomic regions, comparative BLASTP analyses between ATCC 17616 (outer rings) and B. multivorans AU1185 (light blue), FDAARGOS_623 (green), and BAA-247 (red) are included. Various downregulated genes or gene clusters not fully conserved between ATCC 17616 and other genomes were identified (blue boxes), being putative H-NS target sites. CDS, coding sequence.

Article Snippet: Then, genes differentially expressed were mapped against ATCC 17616 coding sequence (CDS) to identify regions possibly acquired by horizontal gene transfer.

Techniques: Gene Expression, Software, Sequencing

Journal: Applied and Environmental Microbiology

Article Title: A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans

doi: 10.1128/AEM.00369-21

Figure Lengend Snippet: The majority of the downregulated genes have upstream intergenic regions of low GC content. (A) Representation of the GC percentage of the upstream intergenic region of each differentially expressed gene obtained from the microarray data set. The average GC percentage of the B. multivorans ATCC 17616 genomic-wide intergenic regions was determined as being 63.4%. The cumulative distribution function of the hypergeometric distribution was used to calculate enrichment of genes above or below 63.4% GC content. (B) Schematic representation of the bce gene clusters directing the biosynthesis of cepacian, with indication of the GC percentage of three intergenic regions containing putative promoters for genes bceA and bceB to - K , bceO to - M , bceP to - R , and bceS - T as indicated by black boxes. The GC content plots were derived from the B. multivorans ATCC 17616 genome using the GC content calculator at Biologics International Corp.

Article Snippet: Then, genes differentially expressed were mapped against ATCC 17616 coding sequence (CDS) to identify regions possibly acquired by horizontal gene transfer.

Techniques: Microarray, Derivative Assay