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: Hormone-responsive epithelial cells of the trans-male breast are altered for genes that show sex bias in other tissues (A) UMAP of luminal-HR + snRNA-seq data showing detected subclusters with RNA-velocity streams overlayed (left) and gender identity (right). (B) RNA-velocity pseudotime ordering of trans-male and cis-female luminal-HR + cells. Time 0 (T0) in the center and respective endpoints of cis-female and trans-male lineages (T1) at the outer maxima. Annotation bars show gender identity and subcluster assignment of each cell. Rows are annotated with highly differentially expressed genes or subcluster markers. (C) Left panel overlays hormone receptor RNA expression on UMAP from (A). Right panel shows boxplots of hormone receptor staining intensities averaged across luminal-HR + cells in the CODEX data (p value, Wilcoxon: PGR = 0.00041). (D) Left panel shows per nucleus ChromVAR motif enrichment z scores for AR (Catalog of Inferred Sequence Binding Preferences [CisBP] M03389_2.00) in luminal-HR + snATAC-seq data (p value, Wilcoxon: <2.2 × 10 −16 ). Right panel shows average nuclear to cytoplasmic staining ratios for AR in luminal-HR + cells from each TMA region (p value, Wilcoxon: 0.00021). (E) RYR2 chromatin accessibility (top) for cis-female (purple) and trans-male (orange) luminal-HR + cells, with highlighted motif binding sites of AR, FOXA1, and CCCTC-binding factor (CTCF). The RYR2 gene body (light green) is shown with promoter (arrow) and exon boundaries (dark green). Also shown (center) is chromatin accessibility data for the genomic region in tissues with varying RYR2 expression and Hi-C data (bottom) comparing three-dimensional chromatin structure of the same region in PANC-1 (pancreas) and MCF-10A (breast) cell lines. (F) AR-motif binding sites (red markers) across open chromatin regions of the CUX2 locus in luminal-HR + cells from cis-females and trans-males. CUX2 gene body (light green), exon boundaries (dark green), and promoter (arrow) are shown below. (G) Average RNA (left, adjusted p value, model-based analysis of single-cell transcriptomics [MAST]: <2.2 × 10 −16 ) and per-region average staining intensity (right, p value, Wilcoxon: 0.027) of CUX2 in cis-female and trans-male tissues. (H) Effect sizes of CUX2 sex bias in GTEx tissues, as a function of median AR expression (vertical axis and dot size). Positive and negative values indicate female and male bias, respectively. (I) Chromatin accessibility (top) around the PGR locus in trans-male and cis-female luminal-HR + and luminal-HR – cells. The PGR gene body (light green) is shown with promoter (arrow) and exon boundaries (dark green). The one significantly altered chromatin peak is indicated by a gray shaded area (Wilcoxon false discovery rate [FDR] <0.05), magnified on the bottom left. Bottom right shows importance levels of transcription factors inferred through random forest analyses to co-bind with AR and determine the directionality of the transcriptional change, with the corresponding DNA binding sites at the PGR locus shown above the top panel. (J) Top left panel shows AR, JUN, and ESR1 chromatin footprints in cis-female and trans-male luminal-HR + cells. Bottom left panel shows average log 2 FC of chromatin accessibility in peaks containing no ESR1 motif, only ESR1 motifs, or both ESR1 and JUN motifs. Bottom right panel shows the fraction of chromatin peaks overlapping only ESR1 motifs or both ESR1 and JUN motifs among all, cis-female-specific, and trans-male-specific luminal-HR + peaks. Top right panel shows the fraction of peaks overlapping both ESR1 and JUN motifs that had in vitro ChIP-seq evidence for both JUN and ESR1 binding.

Article Snippet: Figure 4 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 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.

Techniques: RNA Expression, Staining, Sequencing, Binding Assay, Expressing, Hi-C, Single-cell Transcriptomics, In Vitro, ChIP-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: 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: Figure 4 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 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.

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: Figure 4 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 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.

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: Figure 4 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 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.

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

Journal: Scientific Data

Article Title: Genome-wide expression datasets of anti-VEGF and dexamethasone treatment of angiogenesis in the rat cornea

doi: 10.1038/sdata.2017.111

Figure Lengend Snippet: (1) sutures were placed intrastromally into the temporal cornea, and immediately followed by topical application of eye drops (IgG, anti- Vegf or dexamethasone). Eye drops were applied until the 48 h time point. At t=48 h, IVCM and slit lamp data was collected and used for phenotypic characterisation. (2) cornea tissue was harvested and used for RNA extraction, and RNA quality verified. (3) high quality RNA was used for target preparation for microarray hybridisation on to GeneChip Rat 230 2.0 microarray chips. The microarray chips were scanned and image files acquired. (4) CEL files were normalised using expression console software. The generated CHP together with the CEL files were submitted to Gene Expression Omnibus repository.

Article Snippet: Three groups of six rats each were treated with one of three topical treatments: IgG (Cat. No. 108-C, R&D Systems) at 20 μgml −1 , anti- Vegf (Cat. No AF 564, a neutralizing rat-specific goat polyclonal pan-VEGFA antibody, R&D Systems, Minneaplois MN, USA) at 20 μgml −1 or dexamethasone (Opnol, Clean Chemical, Sweden AB, Borlänge, Sweden) at 1 mgml −1 .

Techniques: Eye Drops, RNA Extraction, Microarray, Hybridization, Expressing, Software, Generated, Gene Expression

Journal: Scientific Data

Article Title: Genome-wide expression datasets of anti-VEGF and dexamethasone treatment of angiogenesis in the rat cornea

doi: 10.1038/sdata.2017.111

Figure Lengend Snippet: Dataset and sample description across treatment groups.

Article Snippet: Three groups of six rats each were treated with one of three topical treatments: IgG (Cat. No. 108-C, R&D Systems) at 20 μgml −1 , anti- Vegf (Cat. No AF 564, a neutralizing rat-specific goat polyclonal pan-VEGFA antibody, R&D Systems, Minneaplois MN, USA) at 20 μgml −1 or dexamethasone (Opnol, Clean Chemical, Sweden AB, Borlänge, Sweden) at 1 mgml −1 .

Techniques: Microarray, Control

Journal: Scientific Data

Article Title: Genome-wide expression datasets of anti-VEGF and dexamethasone treatment of angiogenesis in the rat cornea

doi: 10.1038/sdata.2017.111

Figure Lengend Snippet: ( a – d ) are signal intensity values correlated between control, IgG, anti- Vegf and dexamethasone treated samples respectively. ( e ) is an example of pathway enrichment analysis and ( f ) is a display of the genes involved in a selected pathway (PI3K-Akt signalling pathway).

Article Snippet: Three groups of six rats each were treated with one of three topical treatments: IgG (Cat. No. 108-C, R&D Systems) at 20 μgml −1 , anti- Vegf (Cat. No AF 564, a neutralizing rat-specific goat polyclonal pan-VEGFA antibody, R&D Systems, Minneaplois MN, USA) at 20 μgml −1 or dexamethasone (Opnol, Clean Chemical, Sweden AB, Borlänge, Sweden) at 1 mgml −1 .

Techniques: Control

Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: The differential expression and pan-cancer analysis of FGL1 in multiple databases, confirmed by tissue microarray immunohistochemical staining. (A,B) The mRNA expression of 19 ligands (A) and the corresponding 17 receptors (B) were explored in LUAD; the differential expression of FGL1 is significant and selected as the target gene. (C) The expression of FGL1 in various lung cancer subtypes, the number in the brackets referred to the number of articles corresponding to the tumor. (D) The pan-cancer analysis of FGL1 were made by the Oncomine, TIMER, and UALCAN databases, the number in the brackets referred to the number of articles corresponding to the tumor. (E) 3 GSE data sets (GSE 10072, GSE 33532, and GSE 32863) and Oncomine meta-analysis were used to explore the expression of FGL1 in LUAD. (F) 70 LUAD, 70 LUSC tissues, and 140 paired normal tissues were stained as a tissue microarray by immunohistochemical staining, and we confirmed the expression level of FGL1. **, P<0.01; ***, P<0.001; ****, P<0.0001. FGL1, fibrinogen-like protein 1; LUAD, lung adenocarcinoma.

Article Snippet: The microarray was put into an autoclaved citric acid buffer (pH 6.0) to boil for 15 minutes; 3% hydrogen peroxide was used to block the activity of peroxidase for a 20-minute incubation, and an anti- FGL1 polyclonal antibody (1:100 dilution; Proteintech, Wuhan, China) was used to incubate the microarray overnight at 4 °C.

Techniques: Quantitative Proteomics, Microarray, Immunohistochemical staining, Staining, Expressing

Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: The interactive network of FGL1. (A) The gene-gene interactive network of FGL1 was constructed by the GeneMANIA database. (B) The protein-protein interactive network was constructed by the STRING database. FGL1, fibrinogen-like protein 1.

Article Snippet: The microarray was put into an autoclaved citric acid buffer (pH 6.0) to boil for 15 minutes; 3% hydrogen peroxide was used to block the activity of peroxidase for a 20-minute incubation, and an anti- FGL1 polyclonal antibody (1:100 dilution; Proteintech, Wuhan, China) was used to incubate the microarray overnight at 4 °C.

Techniques: Construct

Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: The genetic alteration of FGL1 was presented by the cBioPortal database. +, the article included provides the corresponding data; −, the article included not provides the corresponding data; 5%*, the alteration frequency of deep deletion is 5%, significant in all alterations. FGL1, fibrinogen-like protein 1.

Article Snippet: The microarray was put into an autoclaved citric acid buffer (pH 6.0) to boil for 15 minutes; 3% hydrogen peroxide was used to block the activity of peroxidase for a 20-minute incubation, and an anti- FGL1 polyclonal antibody (1:100 dilution; Proteintech, Wuhan, China) was used to incubate the microarray overnight at 4 °C.

Techniques:

Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: FGL1-related immune infiltration and the correlation analysis with PD-L1. (A) Immune infiltration related to FGL1 was analyzed by the TIMER database. (B) The correlation analysis of FGL1 and PD-L1 (CD274) (GEPIA). FGL1, fibrinogen-like protein 1; PD-L1, programmed death-ligand 1; GEPIA, Gene Expression Profiling Interactive Analysis.

Article Snippet: The microarray was put into an autoclaved citric acid buffer (pH 6.0) to boil for 15 minutes; 3% hydrogen peroxide was used to block the activity of peroxidase for a 20-minute incubation, and an anti- FGL1 polyclonal antibody (1:100 dilution; Proteintech, Wuhan, China) was used to incubate the microarray overnight at 4 °C.

Techniques: Gene Expression

Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: The survival analysis of FGL1 in LUAD. (A,B) The survival analysis between FGL1 expression and DFS (A), OS (B) in the GEPIA database. (C) The expression plot and survival analysis of GSE 31210, analyzed by PrognoScan database. (D) The survival analysis between FGL1 deep deletion mutation and OS. (E) The expression of FGL1 in various clinical stages. FGL1, fibrinogen-like protein 1; LUAD, lung adenocarcinoma; DFS, disease-free survival; OS, overall survival.

Article Snippet: The microarray was put into an autoclaved citric acid buffer (pH 6.0) to boil for 15 minutes; 3% hydrogen peroxide was used to block the activity of peroxidase for a 20-minute incubation, and an anti- FGL1 polyclonal antibody (1:100 dilution; Proteintech, Wuhan, China) was used to incubate the microarray overnight at 4 °C.

Techniques: Expressing, Mutagenesis

Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: The knockdown of FGL1 and RNA-seq results. (A) The knockdown of FGL1 confirmed by western blot. (B) The knockdown of FGL1 confirmed by immunofluorescent staining. (C) Volcano map of differential gene changes in PC9 cells after FGL1 knockdown. (D) Volcano map of differential gene changes in Jurkat T cells after FGL1 knockdown. (E) The enrichment of differential genes in PC9 cells, analyzed by GSEA. **, P<0.01; ***, P<0.001. NC, negative control; KD, knockdown; LUAD, lung adenocarcinoma; FGL1, fibrinogen-like protein 1.

Article Snippet: The microarray was put into an autoclaved citric acid buffer (pH 6.0) to boil for 15 minutes; 3% hydrogen peroxide was used to block the activity of peroxidase for a 20-minute incubation, and an anti- FGL1 polyclonal antibody (1:100 dilution; Proteintech, Wuhan, China) was used to incubate the microarray overnight at 4 °C.

Techniques: Knockdown, RNA Sequencing, Western Blot, Staining, Negative Control

Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: The experiments related to cell proliferation. (A,B) Effects of FGL1 knockdown on the cycle of PC9 cells and Jurkat T cells. (C) Real-time cell analyzer presents the real-time condition of cell proliferation. (D) Colony formation confirms the cell proliferation of PC9 cells and HCC827 cells, the magnifications in the figure are 40× and 200×, respectively, both of them were stained with crystal violet. **, P<0.01; ***, P<0.001; ****, P<0.0001. NC, negative control; KD, knockdown; LUAD, lung adenocarcinoma; FGL1, fibrinogen-like protein 1.

Article Snippet: The microarray was put into an autoclaved citric acid buffer (pH 6.0) to boil for 15 minutes; 3% hydrogen peroxide was used to block the activity of peroxidase for a 20-minute incubation, and an anti- FGL1 polyclonal antibody (1:100 dilution; Proteintech, Wuhan, China) was used to incubate the microarray overnight at 4 °C.

Techniques: Knockdown, Staining, Negative Control

Journal: Scientific Reports

Article Title: Parathyroid Hormone-Like Hormone is a Poor Prognosis Marker of Head and Neck Cancer and Promotes Cell Growth via RUNX2 Regulation

doi: 10.1038/srep41131

Figure Lengend Snippet: ( A ) The mRNA expression correlation between RUNX2 and PTHLH in Taiwanese HNSCC microarray database (GSE37991) ( B ) The mRNA expression correlation between RUNX2 and PTHLH in HNSCC from the Oncomine™ database. The Pearson’s Correlation test was used to evaluate the statistical significance of correlation between RUNX2 and PTHLH expression. ( C and D ) PTHLH mRNA ( C ), cellular protein ( D ) expression after the enforced expression of ectopic RUNX2 in Ca9-22 cells. The RUNX2 band was cropped from at 55 kDa region. ( E ) RUNX2 ChIP-qPCR results on −800 bp and −500 bp RUNX2 binding regions of PTHLH promoter in Ca9-22 cells after enforced expression of ectopic RUNX2. ( F ) RUNX2 expression among HOK and HNSCC cell lines was analyzed by Western blotting . ( G ) RUNX2 mRNA levels in in 45 paired adjacent normal tissues and tumor tissues from HNSCC patients. RUNX2 protein levels in 40 paired adjacent normal tissues and tumor tissues from HNSCC patients. ( H ) Representative images from IHC staining of RUNX2 from paired HNSCC tissues. ( I ) The quantification of IHC results of RUNX2 IHC. ( J and K ) The RUNX2 mRNA ( J ) and protein ( K , ) expression after exposure to different concentration calcium cation (1.8, 2.4, and 3.0 mM). In ( C , E , G , I and J ) data from three independent experiments were presented as mean ± SEM. The statistical significance was analyzed by Student t-test. (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Slides were stained with polyclonal rabbit PTHLH antibody (1:200; GeneTex, Taiwan), monoclonal mouse RUNX2 antibody (1:20; Santa Cruz Biotechnology, CA), and monoclonal mouse Ki-67 antibody (1:100; Dako, Denmark).

Techniques: Expressing, Microarray, ChIP-qPCR, Binding Assay, Western Blot, Immunohistochemistry, Concentration Assay

Journal: Scientific Reports

Article Title: Parathyroid Hormone-Like Hormone is a Poor Prognosis Marker of Head and Neck Cancer and Promotes Cell Growth via RUNX2 Regulation

doi: 10.1038/srep41131

Figure Lengend Snippet: ( A and B ) The in vitro proliferation ( A ) and in vivo tumor growth ( B ) abilities in of Ca9-22 cells without (EV) or with RUNX2 overexpression. ( C and D ) The KI-67 staining result ( C ) and tumor image and weight ( D ) of figure ( B ). ( E and F ) The in vitro proliferation ( E ) and in vivo tumor growth ( F ) abilities of Cal-27 and SAS cells stably infected with non-silencing (NS) or 2 independent RUNX2 shRNA clones. ( G ) Tumor weight result of ( F ). In ( A , B , D , E,F and G ) data from three independent experiments were presented as mean ± SEM. The statistical significance was analyzed by Student t-test or ANOVA test. ***p < 0.001.

Article Snippet: Slides were stained with polyclonal rabbit PTHLH antibody (1:200; GeneTex, Taiwan), monoclonal mouse RUNX2 antibody (1:20; Santa Cruz Biotechnology, CA), and monoclonal mouse Ki-67 antibody (1:100; Dako, Denmark).

Techniques: In Vitro, In Vivo, Over Expression, Staining, Stable Transfection, Infection, shRNA, Clone Assay

Journal: Scientific Reports

Article Title: Parathyroid Hormone-Like Hormone is a Poor Prognosis Marker of Head and Neck Cancer and Promotes Cell Growth via RUNX2 Regulation

doi: 10.1038/srep41131

Figure Lengend Snippet: ( A and B ) In vitro proliferation assay for the RUNX2-PTHLH axis. The restoration of PTHLH was performed in RUNX2-silencing Cal-27 ( A ) and SAS ( B ) cells. ( C and D ) In vivo tumor growth assay of Cal-27 ( C ) and SAS ( D ) from ( A and B , n = 5). ( E and F ) The tumor images and tumor weights from ( C and D ) respectively. The statistical significance was analyzed by Student t-test or ANOVA test. ***p < 0.001.

Article Snippet: Slides were stained with polyclonal rabbit PTHLH antibody (1:200; GeneTex, Taiwan), monoclonal mouse RUNX2 antibody (1:20; Santa Cruz Biotechnology, CA), and monoclonal mouse Ki-67 antibody (1:100; Dako, Denmark).

Techniques: In Vitro, Proliferation Assay, In Vivo, Growth Assay

Journal: Scientific Reports

Article Title: Parathyroid Hormone-Like Hormone is a Poor Prognosis Marker of Head and Neck Cancer and Promotes Cell Growth via RUNX2 Regulation

doi: 10.1038/srep41131

Figure Lengend Snippet: ( A ) The most statistically significant pathways associated with PTHLH overexpression in Ca9-22 cells. Orange is activation pathways and blue is suppression. ( B ) mRNA expression after the enforced expression of ectopic PTHLH in Ca9-22 cells. ( C and D ) Clinical RNA expression profile of PTHLH stimulation cyclins and cell cycle regulation genes in Taiwanese HNSCC cohort ( C , GSE37991) and TCGA cohort ( D ). ( E ) Hypothetical model of RUNX2-PTHLH positive loop in HNSCC tumor growth. In ( B ) data from three independent experiments were presented as mean ± SEM. The statistical significance was analyzed by Student t-test. (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Slides were stained with polyclonal rabbit PTHLH antibody (1:200; GeneTex, Taiwan), monoclonal mouse RUNX2 antibody (1:20; Santa Cruz Biotechnology, CA), and monoclonal mouse Ki-67 antibody (1:100; Dako, Denmark).

Techniques: Over Expression, Activation Assay, Expressing, RNA Expression

Journal: Science immunology

Article Title: Core-binding factor β and Runx transcription factors promote adaptive natural killer cell responses

doi: 10.1126/sciimmunol.aan3796

Figure Lengend Snippet: (A) Graph shows mRNA expression of highest induced lymphocyte-specific transcription factors in splenic Ly49H+ NK cells sorted from uninfected and MCMV-infected animals on day 2 PI, as assessed by microarray [data provided by the Immunological Genome Consortium (41)]. Data are shown as fold change in microarray signal intensity for the infected versus uninfected samples (n = 3 biological replicates per group and representative of three separate experiments). Solid black bars denote significant up-regulation or down-regulation as compared with uninfected controls (P < 0.05, two-tailed unpaired Student’s t test). (B) Normalized counts of Runx1, Runx2, Runx3, and Cbfb in splenic Ly49H+ NK cells sorted from MCMV-infected mice on day 2 PI and uninfected mice (top) or in unstimulated (US) or IL-12 plus IL-18-treated (12 + 18; 16-hour stimulation) splenic NK cells (bottom), as assessed by RNA-seq (n = 2 to 3 biological replicates per group). (C) RNA-seq was performed on purified Ly49H+ WT NK cells and Stat4−/− NK cells from uninfected and MCMV-infected mixed BMC mice (day 2 PI). Normalized counts of Runx family members are shown (n = 2 to 3 biological replicates per group). (D) RNA-seq was performed on purified Ly49H+ and Ly49H− WT NK cells from uninfected and MCMV-infected mice (day 2 PI). Normalized counts of Runx1 and Runx3 are shown (n = 2 to 3 biological replicates per group). Data are presented as means ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: ChIP was performed as previously described ( 10 , 16 ), using 10 μg of rabbit polyclonal anti-STAT4 antibody (Santa Cruz Biotechnology, sc-468, clone C-20) or 1 μg of rabbit polyclonal anti-trimethyl-histone H3 (Lys4) antibody (H3K4me3; Millipore, 07473) followed by Illumina next-generation sequencing.

Techniques: Expressing, Infection, Microarray, Two Tailed Test, RNA Sequencing, Purification

Journal: Science immunology

Article Title: Core-binding factor β and Runx transcription factors promote adaptive natural killer cell responses

doi: 10.1126/sciimmunol.aan3796

Figure Lengend Snippet: Splenic NK cells (TCRβ−CD19−CD3ε−Ly6G−TER119−TCRγδ−NK1.1+) were sorted from WT mice and stimulated with IL-12 and IL-18 or media alone as a control (unstimulated). STAT4 ChIP was performed, followed by high-throughput DNA sequencing. (A) Proportions of STAT4 genome-wide occupancy at promoter (2 kb upstream and 0.5 kb downstream from TSS), intronic, exonic, or distal intergenic regions in cytokine-stimulated NK cells are shown. (B) RNA-seq was performed on splenic Ly49H+ WT NK cells and Stat4−/− NK cells sorted from mixed chimeras 2 days after MCMV infection. Venn diagram of overlap between differentially expressed genes (top; Padj < 0.05) identified through RNA-seq and reproducible STAT4-bound regions identified through ChIP-seq (bottom; IDR < 0.05). RNA-seq data were performed on n = 3 per condition. (C) Bar graphs depict the top 20 genes with greatest fold enrichment of STAT4 binding over input calculated by MACS2 in transcription factors that show differential expression in RNA-seq data. (D) Representative gene tracks for indicated core-binding factors from STAT4 ChIP-seq. ChIP-seq data are representative of three independent experiments with n = 15 to 20 pooled mice per group per experiment.

Article Snippet: ChIP was performed as previously described ( 10 , 16 ), using 10 μg of rabbit polyclonal anti-STAT4 antibody (Santa Cruz Biotechnology, sc-468, clone C-20) or 1 μg of rabbit polyclonal anti-trimethyl-histone H3 (Lys4) antibody (H3K4me3; Millipore, 07473) followed by Illumina next-generation sequencing.

Techniques: Control, High Throughput Screening Assay, DNA Sequencing, Genome Wide, RNA Sequencing, Infection, ChIP-sequencing, Binding Assay, Quantitative Proteomics

Journal: Science immunology

Article Title: Core-binding factor β and Runx transcription factors promote adaptive natural killer cell responses

doi: 10.1126/sciimmunol.aan3796

Figure Lengend Snippet: Splenic NK cells (TCRβ−CD19−CD3ε−Ly6G−TER119−TCRγδ−NK1.1+) were isolated from WT and Stat4−/− mice and stimulated with IL-12 and IL-18 or media alone as a control (unstimulated; unstim). H3K4me3 ChIP was performed, followed by high-throughput DNA sequencing. (A) Global proportions of H3K4me3 permissive marks at promoter, intronic, exonic, or distal inter-genic regions in cytokine-stimulated NK cells are shown. (B) Bar plots depict number of peaks that change on the basis of fold change (FC) of stimulated versus unstimulated NK cells. FC was calculated by taking the difference between log2-transformed normalized counts for each condition. Only peaks that showed a log2 FC greater than a magnitude of 1 were counted. (C) Meta-peak of all H3K4me3 promoter regions. Overlap of midpoints of ChIP fragments (defined as regions between properly paired sequence reads) for each TSS region was counted for each base pair ± 1 kb from the transcriptional start site. Line plot depicts average signal for all regions for each base pair. (D) Heat map of all H3K4me3 binding regions, with each row representing a single-peak region, row-clustered by normalized peak counts. Signal is displayed as normalized read counts over 5 kb centered at the peak region and is binned at 100-bp windows. (E) H3K4me3 signals from Cbfb, Runx1, Runx2, and Runx3 loci plotted as normalized fragment counts binned at 200 bp across a 10-kb window centered on the transcriptional start site. (F) Zoomed-in histograms of STAT4 ChIP and H3K4me3 ChIP reads mapped to Runx1 and Runx3 loci. Dashed box within boxed tracks indicate STAT4 ChIP called peak region. Data are representative of two independent experiments with n = 15 to 20 pooled mice per group per experiment.

Article Snippet: ChIP was performed as previously described ( 10 , 16 ), using 10 μg of rabbit polyclonal anti-STAT4 antibody (Santa Cruz Biotechnology, sc-468, clone C-20) or 1 μg of rabbit polyclonal anti-trimethyl-histone H3 (Lys4) antibody (H3K4me3; Millipore, 07473) followed by Illumina next-generation sequencing.

Techniques: Isolation, Control, High Throughput Screening Assay, DNA Sequencing, Transformation Assay, Sequencing, Binding Assay

Journal: Journal of Applied Physiology

Article Title: Impact of brief exercise on peripheral blood NK cell gene and microRNA expression in young adults

doi: 10.1152/japplphysiol.01341.2012

Figure Lengend Snippet: Comparison of microarray and RT-PCR gene expression data

Article Snippet: We used Affymetrix U133+2.0 arrays for gene expression and Agilent Human miRNA V2 Microarray for miRNAs.

Techniques: Microarray, Expressing

Journal: Cell reports

Article Title: Mineralocorticoid receptors dampen glucocorticoid receptor sensitivity to stress via regulation of FKBP5

doi: 10.1016/j.celrep.2021.109185

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sections were incubated with primary antibodies (goat anti-FKBP5 (F-14, Santa Cruz, 1:500), rabbit anti-GR (M-20, Santa Cruz, 1:1000) and mouse anti-MR (MABS496, clone 6G1, Millipore-Sigma, 1:100)) overnight at 4°C and labeled with AlexaFluor-conjugated secondary antibodies (1:1000)).

Techniques: Recombinant, SYBR Green Assay, Protease Inhibitor, RNAscope, Sequencing, Microarray, RNA Sequencing, Software

Journal: Nature Communications

Article Title: Neuromorphic electro-stimulation based on atomically thin semiconductor for damage-free inflammation inhibition

doi: 10.1038/s41467-024-45590-8

Figure Lengend Snippet: a The anatomy of sympathetic trunk and ES strategy model. In the top panel, the red line is abdominal aorta and the yellow line is SChG. b The temporal variation of IL-6 in tissue around the injury tendon after surgery. ELISA test for IL-6 between sham and non-ES groups at 0, 1, 2, 3 h after surgery. It shows that IL-6 increased significantly at 2 h after surgery, which provided the timepoint of ES. n = 3 mice; two-sided Student’s unpaired t test; for 0 h, ns, p = 0.0515; for 1 h, ns, p = 0.3224; for 2 h, * p < 0.05; for 3 h, **** p < 0.0001. c ELISA for IL-6 in the non-ES, t-ES and 2D ES groups. The level of IL-6 was decreased by 73.5% in the 2D ES group compared with the non-ES group. The 2D ES group with average currents of ~0.175 mA resulted in a further 70.6% drop in IL-6 level compared to the t-ES group with fixed currents at 0.175 mA, which suggests that the direct neuromorphic bionic spikes lowered the threshold for ES efficacy. And the level of IL-6 had no significant statistical difference between t-ES of 0.3 mA and 2D ES of 0.175 mA, which means that 2D ES achieved the same effect of t-ES but with a 41.7% lower stimulation current. n = 6 mice except for 0.175 mA t-ES and 2D ES groups which of n was 5 mice due to animal death; One-way ANOVA; F 5,27 = 25.89, p < 0.0001; post hoc Tukey test: * p < 0.05; ** p < 0.01; **** p < 0.0001; ns, p = 0.1125 (non-ES vs. 0.1 mA t-ES), p = 0.973 (non-ES vs. 0.175 mA t-ES), p = 0.7459 (2D ES). d The HE staining of lumbar SChG in the t-ES and 2D ES groups. 2D ES and 0.1 mA of t-ES causes little damage to sympathetic neurons, while 0.3 mA of t-ES shows crevices between neurons and connective tissues. Scale bar: 25 μm. n = 3 mice. e The TEM of sympathetic chain. The swelling rough endoplasmic reticulum (yellow arrow) and low electron density of mitochondrion (red arrow and colored shading) indicated that the 2D ES and 0.1 mA current has minimal damage to neurons compared to the 0.3 mA current. Maximum diameter of mitochondria after 0.3 mA stimulation was clearly larger than 2D ES and 0.1 mA, and the expansion of the mitochondria confirmed that a higher current caused damage to sympathetic neurons. n = 12; one-way ANOVA; F 2,33 = 45.99, p < 0.0001; post hoc Tukey test: **** p < 0.0001; ns, p = 0.7548. Scale bar: 0.5 μm. f Immunostaining of DAPI, IL-6 and CD68. IL-6 and CD68 were co-expressed and the level of IL-6 declined after 2D ES. Scale bar: 100 μm. g ELISA for NE. NE was significantly increased after 2D ES. n = 6 mice; one-way ANOVA; F 2,15 = 8.189, p = 0.0039; post hoc Tukey test: * p < 0.05, ** p < 0.01, ns, p = 0.8159. NE: noradrenaline, ES: electro-stimulation, sham: sham surgery and flexible electrode implantation without ES group, t-ES: tendon surgery, flexible electrode implantation and traditional commercial ES based on the Intan RHS2000 commercial stimulator group, 2D ES: tendon surgery, flexible electrode implantation and neuromorphic ES based on 2D FGM IDC group, TEM: transmission electron microscope, HE: hematoxylin-eosin staining, ns: no significance. Source data are provided as a file.

Article Snippet: After three rinses with PBS, the samples were reacted with fluorescent dye-conjugated secondary antibodies in immunostaining secondary antibody dilution buffer (no. E674005, BBI) at RT for 1.5 h. The samples were then well rinsed three times and carefully mounted with DAPI Fluoromount-G (no. 0100-20, Southern Biotech).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Immunostaining, Transmission Assay, Microscopy

Journal: Nature Communications

Article Title: Neuromorphic electro-stimulation based on atomically thin semiconductor for damage-free inflammation inhibition

doi: 10.1038/s41467-024-45590-8

Figure Lengend Snippet: a Heatmaps of RNA-sequencing results. R and W represent the 2D ES and non-ES groups, respectively. Adrb2 was significantly enriched compared with the other adrenergic receptor genes. b Volcano plots of candidate DEGs in the microarray datasets based on the screening criteria. Adrb2 showed no change between the non-ES and 2D ES groups. c Schematic of the intersectional genetic strategy used to generate Lyz2-Cre::Adrb2(f/f) mice. The statistical test was Wald test. d The percentage of ADRB2 + CD68 + in the CD68 + cells. These results verified the ablation of ADRB2 on monocytes/macrophages. n = 6 mice; two-sided Student’s unpaired t test; **** p < 0.0001. e ELISA for IL-6 in the non-ES and 2D ES groups of Lyz2-Cre::Adrb2(f/f) and Adrb2(f/f) mice. The decrease of IL-6 was prevented after 2D ES in Lyz2-Cre::Adrb2(f/f) mice. n = 6 mice; two-way ANOVA; F 1,20 = 63.53, p < 0.0001; p ost hoc Tukey test: *** p < 0.001; **** p < 0.0001; ns, p = 0.2134. f Immunostaining of DAPI, IL-6 and CD68. Scale bar: 100 μm. g The percentage of IL-6 + CD68 + cells among CD68 + cells. The results were in accordance with the ELISA data. n = 6 mice; two-way ANOVA; F 1,20 = 58.85, p < 0.0001; post hoc Tukey test: *** p < 0.001; **** p < 0.0001; ns, p = 0.9986. ES: electro-stimulation, non-ES: tendon surgery and flexible electrode implantation without ES group, 2D ES: tendon surgery, flexible electrode implantation and neuromorphic ES based on 2D FGM IDC group, ns: no significance. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Source data are provided as a file.

Article Snippet: After three rinses with PBS, the samples were reacted with fluorescent dye-conjugated secondary antibodies in immunostaining secondary antibody dilution buffer (no. E674005, BBI) at RT for 1.5 h. The samples were then well rinsed three times and carefully mounted with DAPI Fluoromount-G (no. 0100-20, Southern Biotech).

Techniques: RNA Sequencing, Microarray, Enzyme-linked Immunosorbent Assay, Immunostaining