Journal: The Journal of Biological Chemistry

Article Title: Laccaic Acid A Is a Direct, DNA-competitive Inhibitor of DNA Methyltransferase 1 *

doi: 10.1074/jbc.M113.480517

Figure Lengend Snippet: qPCR validation of LCA microarray analysis and changes in gene expression caused by treatment with control compound, anthraquinone-2-carboxylic acid

Article Snippet: Microarray Analysis Total RNA was isolated from 2 × 10 5 cells and used for microarray analysis (University of Iowa DNA Core Facility) in hybridization to Human Gene ST1.0 Array GeneChips (Affymetrix).

Techniques: Biomarker Discovery, Microarray, Gene Expression, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting CRABP-II overcomes pancreatic cancer drug resistance by reversing lipid raft cholesterol accumulation and AKT survival signaling

doi: 10.1186/s13046-022-02261-0

Figure Lengend Snippet: CRABP-II regulates cholesterol metabolic genes expression through cooperation with HuR. ( A ) Molecular and cellular function analysis by IPA software (Qiagen) based on gene expression microarray profiling. The altered lipid synthesis and accumulation functions upon CRABP-II knockout were listed. ( B ) Heat map of altered cholesterol metabolic genes. ( C, D, E ) Cholesterol metabolic genes expression assessed by Q-PCR. ( F ) Correlation between cholesterol metabolic genes and CRABP-II expression in human pancreatic cancer specimens by Pearson’s product-moment correlation coefficient analysis (PPMCC). Data shown here are combination of Pei Pancreas and Badea Pancrease datasets ( n = 75) from Oncomine. ( G ) Interaction between CRABP-II and HuR identified by co-immuprecipitation (co-IP). GR4000 cell lysis was incubated with anti-CRABP-II rabbit polyclonal antibody and the pull down proteins were separated and blotted with anti-HuR mouse monoclonal antibody. ( H ) Half-life of SREBP-1c mRNA assessed by actinomycin D treatment following with Q-PCR. ( I ) RNA-immunoprecipitation (RIP). The down pulled SREBP-1c mRNA from flagged-CRABP-II transfected CIIKO cells and empty vector transfected cells were assessed by Q-PCR. The actin mRNA was used as control. The experiment was repeated three times and the error bars present standard deviation (SD). **, p < 0.01

Article Snippet: Antibodies used in this study include: CRABP-II mouse mAbs (Millipore, MAB5488), CRABP-II rabbit polyclonal antibody (Proteintech, 10,225–1-AP), HuR (3A2, Santa Cruz, sc-5261), Flotilin-2 (Santa Cruz, sc-28320), GAPDH (Santa Cruz, sc-365062), and Actin (Santa Cruz, sc-1615), anti-Flag M2 mAb (Sigma, F9291), anti-Flag agarose beads (Clontech, #635,686), Ki67 (SP6, ThermoFisher, RM-9106-S0), ADRP (Novus, NB110-40,877), Caspas3 (Cell Signaling, #9662), PARP (Cell Signaling, #9542), AKT (Cell Signaling, #4691), mTOR (Cell Signaling, #2983), S6 (Cell Signaling, #2217), pAKT (S473, Cell Signaling, #9018), pmTOR (Cell Signaling, #5536), pS6 (Cell Signaling, #4858), and pGSK3β (Cell Signaling, #5558).

Techniques: Expressing, Cell Function Assay, Software, Gene Expression, Microarray, Knock-Out, Co-Immunoprecipitation Assay, Lysis, Incubation, RNA Immunoprecipitation, Transfection, Plasmid Preparation, Control, Standard Deviation

Journal: Cell reports

Article Title: Overlapping Activities of Two Neuronal Splicing Factors Switch the GABA Effect from Excitatory to Inhibitory by Regulating REST

doi: 10.1016/j.celrep.2019.03.072

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-calbindin D-28K monoclonal antibody (clone CB-955) , Acris Antibodies , Cat# AM08219SU-N; RRID: AB_1954252.

Techniques: Expressing, Plasmid Preparation, Recombinant, SYBR Green Assay, Reporter Assay, Isolation, Staining, Microarray, Clone Assay, Software

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 2. Upregulation of CXCL10 and MHC class II in human neutrophils in urine during BCG infusion therapy. (a,b) Comprehensive analysis of mRNA expression in urine-derived neutrophils compared to peripheral blood neutrophils. Blood and urine were collected from three patients after one week from the 6th BCG infusion. Comprehensive analysis of mRNA in neutrophils was performed using a DNA tip microarray. (a) Cluster analysis after adjustment and standardiza- tion. The mRNA expression in neutrophils obtained from urine samples (vertical axis) or periph- eral blood (horizontal axis) was analyzed. White lines indicate the thresholds for genes that are upregulated or downregulated > 2-fold between urine- and blood-derived neutrophils. A relatively higher expression in urine-derived neutrophils is indicated using arrows, including expression for CXCR3 ligands (CXCL9 and CXCL10) and MHC class II (HLA-DRB1, HLA-DPA1, and HLA-DQA1). (b) Volcano plot depicting the differentially expressed genes between peripheral blood-derived and urine-derived neutrophils after the 6th BCG infusion. The horizontal axis denotes the fold change in mRNA expression in neutrophils from the urine and blood, while the vertical axis represents the –log10 (p-value) for a t-test of differences in neutrophils from the blood and urine. These data represent the top 6000 genes of the –log10 (p-value). The gene expressions of CXCR3 ligands (CXCL9, CXCL10, and CXCL11) and MHC class II (HLA-DQA2, HLA-DPA1, and HLA-DQA1) were also detected as characteristic features of urine-derived neutrophils (arrows). (c,d) Representative data of intracellular- stained neutrophilic cells obtained via flow cytometric analysis. The CD33+CD15+ neutrophilic cells in the blood (c) or urine (d) samples were obtained from the same patient who was treated with 4th BCG infusions and are presented as CXCL10 MFI (upper panels) and HLA-DR MFI (lower panels). Gray-closed histograms indicate each background staining, and light blue line histograms denote the staining of CXCL10 or HLA-DR. (e–g) Comparison of intracellular expression of (e,f) CXCL10 and (g) HLA-DR in neutrophilic cells from the blood (open circle) and urine (closed circle) samples. These samples were collected after one week from the 2nd to the 6th BCG infusions (after each infusion). (e) ∆CXCL10 MFI was calculated as follows: ∆CXCL10 MFI = (MFI of PE-conjugated anti-CXCL10 mAb staining) −(MFI of PE-conjugated control IgG staining). (f) The neutrophilic cells

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Expressing, Derivative Assay, Microarray, Staining, Comparison, Control

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 3. Effect of BCG on CXCL10 and MHC-II expression in human or mice neutrophilic cells in vitro. Human (a,d) or mouse (b,e) peripheral blood was diluted ten-fold in 10% FCS RPMI1640, or mouse bone marrow cells (4 × 106/mL; c,f) were incubated with or without 4 µg/mL of BCG for 20 h. Following incubation, the expression levels of CXCL10 (a–c) and MHC class II (d–f) in human (CD33+CD15+) or mouse neutrophils (CD45+Ly6G+) were analyzed, as described in Figure S2. Statistical significance was calculated with the paired t-test, * p < 0.05 (n = 3). Abbreviations: BCG, Bacillus Calmette–Guérin; CXCL10, chemokine (C-X-C motif) ligand 10; HLA-DR, human major histocompatibility complex class II cell surface receptor; MFI, mean fluorescence intensity; and I-A/I-E, mouse major histocompatibility complex class II cell surface receptor.

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Expressing, In Vitro, Incubation, Immunopeptidomics, Cell Surface Receptor Assay

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 4. Upregulation of CXCL10 and MHC class II in monocytes and neutrophils in peritoneal effusion cells after BCG injections. Mice were injected with B16F10 cells (5 × 104 cells/100 µL/head), and the PECs were collected after two weeks. The PECs induced after one injection of BCG (40 µg/head) after 16 h and the PECs induced after five repeated injections of BCG (40 µg/head) after 16 h from the final injection are presented as “1-shot” and “5-shots”, respectively. These PECs were intracellularly stained with each antibody, and the relative expression (MFI) of CXCL10 and I-A/I-E was analyzed in CD45+Ly6C+ cells and CD45+Ly6G+ cells, respectively. (a–i) Representative flow cytometric analysis of mouse monocytes (Ly6C+ cells) and neutrophils (Ly6G+ cells) via flow cytometry. The (a–c) panels present flow cytometric analysis of the PECs induced 2 weeks after B16F10 cell injection (presented as “Tumor”). The (d–f) panels show representative flow cytometric analysis of the PECs induced 16 h after the administration of BCG (presented as “1-shot). The (g–i) panels indicate representative flow cytometric analyses of the PECs induced via five repeated BCG injections at one-week intervals. The PECs were collected 16 h after the final BCG admin- istration (presented as “5-shots”). The left panels (a,d,g) show CD45+ leukocytes presented with the gates of Ly6C+ cells (monocytic cells) and Ly6G+ cells (neutrophilic cells). (j–l) The number and proportion of myeloid cells (Ly6C+ and Ly6G+ cells) of the PECs. The peritoneal effusion cells obtained after injection of B16F10 cells are presented as “tumor” (open circles). The cells induced 16 h after a single administration of BCG are presented in the group “1-shot” (closed circles). The cells induced via five repeated injections of BCG are presented in the group “5-shots” (closed triangles). The (j) number of the cells in peritoneal fluid were counted using a hemocytometer, and the proportions of (k) Ly6C+ cells and (l) Ly6G+ cells in CD45+ leukocytes were analyzed via flow cytometry. (m–p) The intracellular expression levels of CXCL10 and MHC-II (I-A/I-E) in mouse monocytes (Ly6C+ cells) and neutrophils (Ly6G+ cells) after BCG injection. These PECs were intracellularly stained with each anti- body, and the relative expression (MFI) of (m,n) CXCL10 and (o,p) I-A/I-E was analyzed in (m,o) CD45+Ly6C+ cells and (n,p) CD45+Ly6G+ cells, respectively. Statistical analyses were per- formed using the Kruskal–Wallis test with the Dunn’s post-hoc test. Each bar is presented as the mean of data. * p < 0.05; ** p < 0.01; and ns, not significant. Abbreviations: PECs, peritoneal exudate cells; CXCL10, C-X-C motif chemokine ligand 10; BCG, Bacillus Calmette–Guérin; and MFI, mean fluorescence intensity.

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Injection, Staining, Expressing, Cytometry

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 7. CXCL10 and MHC class II expression in neutrophils induced via BCG was inhibited via partial neutrophil depletion using anti-Ly6G mAbs. BCG (40 µg/100 µL/head) was injected into the peritoneal cavity, following which the antibodies (100 µg/50 µL/head; control mAb, open circle; or anti-Ly6G mAb, closed circle) were injected into the

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Expressing, Injection, Control

Journal: Frontiers in Immunology

Article Title: RNA-Seq Analysis of IL-1B and IL-36 Responses in Epidermal Keratinocytes Identifies a Shared MyD88-Dependent Gene Signature

doi: 10.3389/fimmu.2018.00080

Figure Lengend Snippet: Type I and II interferon genes have time-dependent IL-1B and IL-36 responses. (A) Interferon gamma receptor 1 ( IFNGR1 ). (B) Interferon gamma receptor 2 ( IFNGR2 ). (C) Interferon alpha and beta receptor subunit 2 ( IFNAR2 ). In panels (A–C) , average FPKM (±1 SE) is shown for each group, and asterisks denote significant differences relative to the control (CTL) treatment at the corresponding time point (paired two-sample t -test; n = 2 or 3 per treatment). (D) Genes with interferon response factor 1 (IRF1) binding sites (5 kb upstream region). The middle 50% of FC estimates is shown for genes with 2+ IRF1 binding sites compared with genes with fewer IRF1 sites (magenta font, horizontal axis: P < 0.05, Wilcoxon rank sum test). The IRF1 position weight matrix is shown (right) along with the IRF1 tetrameric structure (bottom right; NCBI structure database). (E) IFN-induced gene signature scores. IFN-induced genes were identified from microarray studies of IFN-treated keratinocytes (left margin), and the average FC for these genes was calculated in IL-1B/IL-36 experiments (bottom margin). Left margin labels indicate the cytokine concentration (in ml), treatment duration, and GEO series accession number. All cytokine experiments were replicated with at least two samples per treatment. (F) Top 30 IFN-g-induced genes (identified from GSE36287). (G) Top 35 INFa-induced genes (identified from GSE36287). (H) Self-organizing maps (SOMs). The SOM layout was determined only from IFN-g-induced genes (i.e., 2,500 genes most strongly induced by IFN-g, GSE36287). Colors reflect average FC estimates for IFN-g-induced genes assigned to each SOM region (columns 1 and 2 on left). The final column (yellow–blue) displays the mean FC difference for each cytokine with respect to each SOM region (8 h mean FC–24 h mean FC; log 2 scale).

Article Snippet: MYD88-KO KCs including WT KCs were grown in 12-well plates, and cells were treated with recombinant IL-1 beta (10 μg/ml; R&D Systems # 201-LB-025), IL-36 gamma (10 μg/ml; R&D Systems # 6835-IL-010), IFN-gamma (50 μg/ml; R&D Systems # 285-IF-100), IL-17A (20 μg/ml; R&D Systems # 317-ILB-050), and/or TNF-alpha (10 μg/ml; R&D Systems # 210-TA-005) for 8 or 24 h. RNAs were isolated from cell cultures using Qiagen RNeasy plus kit (Cat # 74136).

Techniques: Control, Binding Assay, Microarray, Concentration Assay

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: HDAC1, HDAC2, and HDAC3 were upregulated in breast cancer tissues and correlated with worse prognosis in breast cancer patients. ( a ) Representative immunohistochemical (IHC) staining photos of HDAC1, HDAC2, and HDAC3 in breast specimens. HDAC1, HDAC2, and HDAC3 levels were obviously elevated in the tumor tissues compared to the non‐neoplastic adjacent tissues of patients with invasive ductal carcinoma (IDC). Yellow boxes indicated mammary ducts. Scale bars, 100 μm. ( b ) Representative IHC photos of three different kinds of HDAC3 subcellular localization. Scale bars, 100 μm. ( c – f ) Overall survival (OS) curves of 139 IDC patients with different HDAC1 ( c ) ( ) HDAC1 Low ( ) HDAC1 High ( ) HDAC1 low‐censored ( ) HDAC1 high‐censored, HDAC2 ( d ) ( ) HDAC2 Low ( ) HDAC2 High ( ) HDAC2 low‐censored ( ) HDAC2 high‐censored, cytoplasmic HDAC3 ( e ) ( ) HDAC3 C‐low ( ) HDAC3 C‐high ( ) HDAC3 C‐low‐censored ( ) HDAC3 C‐high‐censored, and nuclear HDAC3 ( f ) levels ( ) HDAC3 N‐low ( ) HDAC3 N‐high ( ) HDAC3 N‐low‐censored ( ) HDAC3 N‐high‐censored. According to another set of criteria in which cases with either high nuclear or cytoplasmic expression were classified into the C‐high/N‐high group and other cases were classified into the Others group, the overall survival curve of the 139 IDC patients was reproduced as Figure 1g ( ) HDAC3 Others ( ) HDAC3 C‐high/N‐high ( ) HDAC3 Others‐censored ( ) HDAC3 C‐high/N‐high‐censored. P values of the Kaplan‐Meier plots in (c‐g) were calculated by log‐rank test in IBM SPSS Statistics 19 software. ( h – j ) Kaplan‐Meier survival curves with log‐rank analysis were used to assess the correlation between HDAC1 ( h ) ( ) HDAC1 Low (≤ 75th percentile) ( ) HDAC1 High (> 75th percentile), HDAC2 ( i ) ( ) HDAC2 Low (≤ 75th percentile) ( ) HDAC2 High (> 75th percentile), and HDAC3 ( j ) ( ) HDAC3 Low (≤ 80th percentile) ( ) HDAC3 High (> 80th percentile) expression and overall survival of 4903 breast cancer patients in the bc‐GenExMiner platform (website: http://bcgenex.centregauducheau.fr ; all DNA microarray data, node mixed, ER mixed; optimized split for HDAC1 and 2, an 80th percentile customized cutoff for HDAC3).

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Software, Microarray

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: HDACs expression exhibited different roles in overall survival of IDC patients ( n = 16l)

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Expressing

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: Relationship between clinicopathological characteristics and HDACs expression in IDC patients ( n = 139)

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Expressing, Over Expression

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: HDACs expression exhibited different roles in the onset of brain metastasis of IDC patients ( n = 161)

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Expressing

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: The roles of HDACs expression and other clinicopathological characteristics played in the prognosis of breast cancer patients after brain metastasis ( n = 63)

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Expressing

Journal: Nature communications

Article Title: Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

doi: 10.1038/s41467-020-15425-3

Figure Lengend Snippet: Fig. 1 Pro-BMP9 and pro-BMP10 are equivalent ALK1-ligands. a Dose-dependent signalling assays in PAECs. Serum-starved PAECs were treated with different ligands at 2.48 pM (white bars), 8.27 pM (light grey bars) and 27.3 pM (dark grey bars) (using monomer molecular weight, equivalent to 0.03, 0.1 and 0.33 ng ml−1 BMP9 GF-domain concentration) for 1 h. Changes in the ID1 gene expression were monitored using RT-qPCR. Data were presented as fold change relative to untreated cells, and means ± SEM of three independent experiments are shown. Source data are provided as a Source Data file. b–d Volcano plots comparing changes in global gene expression in PAECs after pro-BMP9 or pro-BMP10 treatment. Serum-starved PAECs were treated with 25 pM of pro-BMP9 or pro-BMP10 (purity can be found on SDS-PAGE with silver staining in Supplementary Fig. 8a, lanes 1 and 4) for 1.5 h before RNA was extracted for microarray analysis. Four different primary PAEC lines were used. Red dots above the dashed line represent the changes in target genes with adjusted p values of less than 0.05. Several representative target genes are highlighted in c and d. Full list of genes can be found in Supplementary Data 1 and 2. e Affinity measurements of BMP9 and BMP10 for ALK1 using Biacore. A CM5 Biacore chip was immobilised with ALK1 dimer (ALK1-Fc) or monomer (in-house purified ALK1 ECD, purity can be seen in Supplementary Fig. 8a, lane 7). The sensorgrams of BMP9, pro-BMP9, BMP10 and pro- BMP10 binding raw data (in black lines) were overlaid with a global fit to a 1:1 model with mass transport limitations (red lines). f A summary of kinetic parameters for ligand-receptor interactions derived from the Biacore measurements in e.

Article Snippet: Human GF-domains of BMP2, BMP9 and BMP10, CV2, ALK1-Fc and ALK2-Fc were all purchased from R&D Systems.

Techniques: Molecular Weight, Concentration Assay, Gene Expression, Quantitative RT-PCR, SDS Page, Silver Staining, Microarray, Binding Assay, Derivative Assay

Journal: Nature communications

Article Title: Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

doi: 10.1038/s41467-020-15425-3

Figure Lengend Snippet: Fig. 2 Crystal structure of human BMP10:ALK1 complex at 2.3 Å. a Crystal structure of BMP10 (cyan) in complex with ALK1 (magenta), overlaid with the structure of BMP9:ALK1:ActRIIb complex (PDB:4FAO, in grey and semi-transparent). Mol A and Mol B are the two BMP monomers whose interfaces with ALK1 (Mol C) were analysed in b. b Comparison of the buried interface upon complex formation between BMP10:ALK1 and BMP9:ALK1 (from 4FAO). Total buried surface area and the contributing residues were calculated using PDBePISA server.

Article Snippet: Human GF-domains of BMP2, BMP9 and BMP10, CV2, ALK1-Fc and ALK2-Fc were all purchased from R&D Systems.

Techniques: Comparison

Journal: Nature communications

Article Title: Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

doi: 10.1038/s41467-020-15425-3

Figure Lengend Snippet: Fig. 3 Specificity determinants in the BMP9 and BMP10 subfamily. a Sequence alignment of representative ALK-binding BMPs. GF-domain sequences of ALK1-binding BMP9 and BMP10, ALK6-binding BMP14, ALK3-binding BMP2 and BMP4, as well as ALK2-binding BMP6 and BMP7 are aligned. Lines over and below the sequences highlight the residues at the type I and type II receptor-binding surface based on BMP10:ALK1 and BMP9:ALK1:ActRIIb structures, respectively. Asterisk (*) marks the residues that are conserved among at least 6 out of 7 aligned BMPs. Residues preferentially conserved between BMP9 and BMP10 are highlighted, in cyan for those at the type I site (conserved region 1), in blue for those at the type II site (conserved region 2) and in yellow for those outside receptor binding surface (conserved region 3). BMP10 D338 and P366 are also highlighted in cyan because they make conserved interactions with ALK1 in the crystal structure (Fig. 4). b Residues from conserved regions 1–3 plotted on BMP10 structure and labelled with full length proBMP10 residue numbers. Fifteen residues from conserved regions 1–3 are shown in spheres, coloured accordingly. The first Gly from conserved region 3 is not modelled in the crystal structure, and hence not plotted. c An overlay of BMP10 (grey) onto the structures of BMP9 (gold, from 4FAO)9, BMP2 (green, from 2GOO)31 and BMP7 (cyan, from 1M4U)61 is shown from the side view (left) and the top view (right). The red arrows indicate the unique insertion in BMP9 and BMP10.

Article Snippet: Human GF-domains of BMP2, BMP9 and BMP10, CV2, ALK1-Fc and ALK2-Fc were all purchased from R&D Systems.

Techniques: Sequencing, Binding Assay, Residue

Journal: Nature communications

Article Title: Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

doi: 10.1038/s41467-020-15425-3

Figure Lengend Snippet: Fig. 4 Conserved region 1 and ALK1-specificity determinants. a ALK1-binding residues are mapped onto the BMP10 surface (grey), with those conserved across all BMPs in Fig. 3a coloured in red, those from the BMP9 and BMP10 conserved region 1 in cyan, and other variable residues in yellow. b ALK1 (magenta cartoon) binding to BMP10 (surface), with residues interacting with BMP10 shown in sticks. c–f Detailed interactions between BMP10 and ALK1. g Sequence alignment of four BMP-binding type I receptors, ALK1, ALK2, ALK3 and ALK6, with the four specificity-determining residues in ALK1 highlighted in yellow. Loop 4 and loop 5 are the two loops surrounding the 310 helix (Supplementary Fig. 4). h Overlaid structures of BMP type I receptors. The structure of ALK1 in the BMP10:ALK1 complex (magenta) is overlaid onto ALK1 in BMP9:ALK1 complex (PBD:4FAO, orange), ALK3 (PDB:2GOO, light green) and ALK6 (PDB:3EVS, light grey). ALK1 residues highlighted in g are shown in sticks.

Article Snippet: Human GF-domains of BMP2, BMP9 and BMP10, CV2, ALK1-Fc and ALK2-Fc were all purchased from R&D Systems.

Techniques: Binding Assay, Sequencing

Journal: Nature communications

Article Title: Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

doi: 10.1038/s41467-020-15425-3

Figure Lengend Snippet: Fig. 5 Conserved region 2 and type II site analysis. a ActRIIb-binding residues (based on PDB:4FAO, ActRIIb in semi-transparent cartoon) are mapped onto BMP9 surface (grey), with those conserved across all BMPs in Fig. 3a coloured in red, those from conserved region 2 in blue, and other variable residues in yellow. b Type II binding surface of BMP9 (left) and BMP10 (right), showing as electrostatic surface (generated in PyMOL, red representing negatively charged and blue positively charged surface). ActRIIb is shown in orange, semi-transparent cartoon. c Residues from BMP9 conserved region 2 make three backbone β-sheet and one sidechain H-bond interactions with ENG (PDB:5HZW, ENG in green, BMP9 in cyan). BMP10 is overlaid onto BMP9 and shown in grey, with four conserved region 2 residues shown in blue spheres. Sidechains of other residues are omitted for clarity. d Sequence alignment of human BMP10 prodomain (hBMP10_pro) with mouse BMP9 prodomain (mBMP9_pro) and human BMP9 prodomain (hBMP9_pro). Residues at the BMP9-binding surface are highlighted in yellow and those that make direct interactions with BMP9 GF-domain are marked with *. Residues that make main chain interactions are also marked with ^. Only the prodomain regions that interact with BMP9 GF-domain are shown, and full-length alignment of hBMP9_pro and hBMP10_pro can be found in Supplementary Fig. 5. e Residues in conserved region 2 of BMP9 make four backbone H-bond β-sheet interactions with prodomain (PDB:4YCG; prodomain in orange, BMP9 in cyan. BMP10 is overlaid on BMP9 and shown in grey. Four conserved BMP10 residues are in blue spheres).

Article Snippet: Human GF-domains of BMP2, BMP9 and BMP10, CV2, ALK1-Fc and ALK2-Fc were all purchased from R&D Systems.

Techniques: Binding Assay, Generated, Sequencing

Journal: Nature communications

Article Title: Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

doi: 10.1038/s41467-020-15425-3

Figure Lengend Snippet: Fig. 7 CV2 does not inhibit BMP9 signalling. a Structural analysis. BMP10 (grey, with conserved region 2 residues in blue spheres) was overlaid onto the BMP2:CV2 structure (PDB:3BK3, CV2 in magenta and BMP2 in green). Four mainchain H-bonds that stabilise the BMP2:CV2 β-sheet interaction are shown. BMP9 has the same conformation as BMP10 in this region. b, c CV2 does not inhibit BMP9 signalling in PAECs. Serum-starved PAECs were treated with BMP9 or pro-BMP9 (at 1 ng ml−1 GF-domain concentration) without or with CV2 at 10-fold, 20-fold, 50-fold or 250-fold molar excess for 15 min to assess Smad1/5 phosphorylation using immunoblots (b) or for 1 h to assess ID1 gene expression using qPCR (c). One representative of three independent experiments is shown in b. Band intensity was quantified using Image J (version 1.51s). d CV2 inhibits BMP4 signalling in PASMCs. Serum-starved PASMCs were treated with BMP4 (25 ng ml−1) without or with CV2 at indicated molar excess for 15 min. Immunoblots and quantification were carried out as above. N = 3 independent experiments and one representative blot is shown. e CV2 inhibits BMP2 but not BMP9 signalling in C2C12 cells. Serum-starved C2C12 cells were treated with BMP2 (130 ng ml−1) or BMP9 (25 ng ml−1) without or with CV2 at the indicated molar excess for 68 h. ALP activity in the cell lysate were analysed (see Methods section). N = 7 independent experiments. For all panels, means ± SEM are shown. d, e One-way ANOVA for each BMP treatment group, Dunnett’s post hoc analysis against BMP alone-treated controls. Source data are provided as a Source Data file.

Article Snippet: Human GF-domains of BMP2, BMP9 and BMP10, CV2, ALK1-Fc and ALK2-Fc were all purchased from R&D Systems.

Techniques: Concentration Assay, Phospho-proteomics, Western Blot, Gene Expression, Activity Assay

Journal: Nature communications

Article Title: Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

doi: 10.1038/s41467-020-15425-3

Figure Lengend Snippet: Fig. 8 Modifying BMP9 signalling specificity by mutagenesis. A panel of BMP9 mutants were generated, and tested in vitro and in vivo as described in the Methods. a Mutant proteins were subject to endothelial cell signalling assays (at 0.3 ng ml−1 GF-domain concentration) using induction of ID1 gene expression in hPAECs as a readout, and osteogenic signalling assays (at 10 ng ml−1 GF-domain concentration) using ALP induction in C2C12 cells as a readout. Data were normalised to WT BMP9 and shown as fold change relative to WT upon mutation. Each treatment condition was repeated in 3–7 independent experiments alongside untreated and WT controls. The exact N number for each condition is given under the column. Means ± SEM are shown. b Recombinant WT pro-BMP9, pro-BMP9 D366E, pro-BMP10, as well as BMP2 GF-domain were subject to in vivo heterotopic bone-forming assays in the presence and absence of cardiotoxin. Each data point represents the HO result from an independent injection in one leg. N number for each treatment condition is given under each column. Data are presented as % ossification volume relative to the average of BMP2-treated controls. Means ± SEM are shown. c Representative CT images (left) and histological staining (right) of in vivo formed heterotopic bones after stimulation of indicated BMP molecules in the presence and absence of cardiotoxin. B: osteoid matrix; M: muscle cells. Scale bar = 500 µm.

Article Snippet: Human GF-domains of BMP2, BMP9 and BMP10, CV2, ALK1-Fc and ALK2-Fc were all purchased from R&D Systems.

Techniques: Mutagenesis, Generated, In Vitro, In Vivo, Concentration Assay, Gene Expression, Recombinant, Injection, Staining

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 1. TGFβ pathway signature increases in TNBC cells upon anchorage-independent culture and androgen receptor and SMAD3 are predicted up- stream regulators. (A) Metacore pathway analysis of BT549 microarray data showing SMAD3 connecting with other genes altered under anchorage- independent culture for 24 hours. Original data set from Barton et al (2015) (GEO record GSE95472). (B) GSEA pathway enrichment analysis showing changes in the TGFβ pathway in BT549 cells cultured in attached or under anchorage-independent culture for 24 hours. (C) Heatmap of significantly altered genes associated with the TGFβ pathway in BT549 cells grown in attached versus suspended culture conditions for 24 hours (n = 4). Gene list from KEGG database on TGFβ Signaling Pathway. Red asterisk: genes associated with the canonical TGFβ signaling pathway. (D) qRT-PCR for ca- nonical TGFβ signaling pathway gene expression in attached versus under anchorage-independent culture conditions at 48 hours in SUM159PT and BT549 cell lines (n = 6). Mean ± SD; *P < .05; **P < .01; ***P < .001. ****P < .0001.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Microarray, Cell Culture, Quantitative RT-PCR, Gene Expression

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 2. Anchorage-independent conditions increase TGFβ1 expression and secretion and nuclear pSmad3 localization and activity. (A) RPPA data showing changes in protein in BT549 cells grown in attached versus under anchorage-independent culture conditions for 24, 48, and 72 hours with biological triplicates at each condition and timepoint shown. Red, high relative expression levels; black, intermediate relative expression levels; green, low relative expression levels. Full panel of RPPA data in (30). (B) ELISA measuring changes in TGFβ1 in conditioned medium from three TNBC lines cultured in attached versus suspended conditions for 24 and 48 hours normalized to total live cell count as determined by trypan blue. (C) IHC and quantification of percentage of pixels corresponding to nuclear DAB staining for pSmad3 in MDA-MB-453, SUM159PT, and BT549 cells in at- tached versus suspended culture conditions for 24 hours. (D) 3TP-lux TGFβ reporter-linked luciferase activity normalized to renilla in SUM159PT cells grown in attached versus suspended culture. Mean ± SD; *P < .05; **P < .01; ***P < .001. ****P < .0001.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Cell Counting, Staining, Luciferase

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 3. Exogenous TGFβ increases AR protein levels and TGFβ inhibition decreases AR and cell viability in anchorage-independent conditions. (A) Western blot for pSmad2, Smad2, Smad3, ID1, AR, GR, and GAPDH in the SUM159PT cell line treated with recombinant TGFβ1 (10 ng/mL) over a time course of 24 hours. (B) qRT-PCR for AR in SUM159 and BT549 cells grown in attached conditions for 48 hours ± LY2109761 (10 µM). (C) Western blot for AR was performed on MDA-MB-453, SUM159PT, and BT549 cell lines grown in attached or suspended conditions for 48 hours ± LY2109761 (10 µM). (D) Western Blot to analyze the apoptosis marker cleaved-PARP in SUM159PT and BT549 cells in attached or suspended conditions for 48 hours ± LY2109761 or LY2157299 (10 µM). Densitometry values calculated as (cleaved-PARP/PARP)/GAPDH. (E) Trypan blue assay for percentage of dead cells in SUM159PT and BT549 cells in attached or suspended conditions for 48 hours ± LY2109761 or LY2157299 (10 µM). Western blot quantifica- tions performed by ratio of protein of interest to loading control and normalized to control. Mean ± SD; *P < .05; **P < .01; ***P < .001. ****P < .0001.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Inhibition, Western Blot, Recombinant, Quantitative RT-PCR, Marker, Control

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 4. Clinical data suggests a high AR and TGFB3 is associated with worse outcome. (A) Correlation analysis on the co-expression of AR and the TGFβ ligand TGFB3 in TNBC patients in the TCGA-BRCA (n = 123) and SCAN-B (n = 143) cohorts. (B) GSEA analysis on TGFB3 relating to the androgen response gene set from the HCI-009 PDX. (C) KMplotter was used to stratify basal BC patients stratified into ARhigh (top) or ARlow (bottom) populations. TGFB3 expression was then inter- rogated based on overall survival (OS, n = 241) and distant metastasis free survival (DMFS, n = 232)

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Expressing

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 5. AR ChIP-seq peaks are identified near SMAD3 and TGFB1. (A) Heatmaps show that differential AR genome binding in attached and sus- pended conditions with vehicle, DHT, and DHT+Enza treatments. (B) Heatmaps display the signal at differential DHT-induced AR binding sites that show a 2-fold or greater enrichment in the suspended condition. (C) Gene ontology biological processes found nearby AR bound sites that are more than 2-fold enriched in suspended cells using GREAT (63). (D) Genes involved in focal adhesion assembly that were identified as being 2-fold en- riched in under anchorage-independent culture by AR ChIP-seq. (E) AR ChIP-seq peaks identified near SMAD3 are shown. The scale bar at the top is labeled for each loci to indicate the relative distance from the canonical SMAD3 TSS along with the respective gene annotations. For each region, the AR ChIP-seq signal for each treatment condition in attached (black) and suspended (red) are shown. The peaks at each loci are scaled to the same height for accurate comparison but are not directly comparable between loci due to differences in ChIP-seq signal intensity. The chromosomal coord- inates of the area covered by each peak are shown below each region. (F) Browser tracks show AR binding near the TGFβ1 promoter. AR ChIP-seq signal within a 2000 bp window covering 500 bp upstream of the TGFβ1 TSS and 1500 bp into the 5′ UTR and first exon is shown. ChIP-seq signal is scaled to the same height for comparison and represents each treatment condition in attached (black) and suspended (red), with the chromosomal coordinates included at the bottom for reference.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: ChIP-sequencing, Binding Assay, Labeling, Comparison

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 6. SUM159PT cells are more sensitive to a combined treatment with Enza and TGFβ inhibitor than either drug alone, particularly after anchorage-independent culture. (A) SUM159PT cells were grown in triplicate in 6-well plates in attached conditions for 72 hours with either vehicle, LY1 (10 μM), Enza (40 μM), or both, followed by crystal violet assay. (B) SUM159PT cells were grown in triplicate in 6-well plates in attached conditions for 72 hours with the same treatments, followed by washout of drug and an additional 72 hours in culture, then crystal violet assay was performed. (C) SUM159PT cells were grown in under anchorage-independent culture on poly-hema coated plates for 48 hours with the treatments mentioned above, followed by replating of cells in attached conditions for an additional 48 hours. Crystal violet assay was performed after. Mean ± SD; *P < .05; **P < .01; ***P < .001. ****P < .0001.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Crystal Violet Assay

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 7. Proposed model of a positive feedback loop whereby TGFβ signaling increases AR expression, and AR increases core TGFβ pathway components.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Expressing