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: 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: Cell

Article Title: A Milieu Molecule for TGF-β Required for Microglia Function in the Nervous System

doi: 10.1016/j.cell.2018.05.027

Figure Lengend Snippet: (A) Immunostaining of 8 μm brain sections for LacZ and Iba1. (B and C) Immunostaining of 40 μm brain sections for Iba1 and CD68 (B) and 3D reconstruction (C). (D) Quantification of reactive microglia cells in somatomotor cortex region M1, mean ± SEM for n=3 mice; ***P=0.008, two-tailed t-test). (E-G) Immunophenotype of CD45low Mac1high CD39high microglia from day 21 mice and quantification of expression. (E) Representative gating comparing gates for WT (red) and KO (violet) microglia. (F-G). Quantitation with representative histograms for CD68 (F) and mean fluorescence intensity (G, MFI, mean ± SEM, n = 3 mice). *: p=0.012 to 0.02; ***: p=0.006; ****: p=0.0006. (H) Staining of F4/80+ Mac1+ PEC with TGF-β prodomain (TW7–16B4) or control antibodies with or without permeabilization. One representative of 4 mice. (I-K) High MW proTGF-β1 complexes in WT and not Lrrc33−/− cells or transfected (Tra.) or non-transfected (Non-tra.) cells. (I) Anti-proTGF-β1 WB of PEC (106/lane) or 33-G-X5 - proTGF-β1 L1.2 transfectant (105/lane) lysates. (J,K) Lysates from WT and Lrrc33−/− PEC and spleen cells, LRRC33 proTGF-β1 co-transfectants or untransfected cells, with or without IP with prodomain antibody TW7–16B4 or mouse IgG coupled to Sepharose, were subjected to non-reducing or reducing SDS 7.5% PAGE and WB with anti-denatured mouse LRRC33 (Noubade et al., 2014) or anti-proTGF-β1 Ratio of transfectant:native cell equivalents was 1:10. (L) TGF-β activation. PEC from adult WT or Lrrc33−/− mice or 1:1 cocultures of WT astrocytes with WT or Lrrc33−/− microglia were assayed for TGF-β production; N=3 mice, mean ± SEM; **P<0.01, *P<0.05 (unpaired Student’s t-test). (M) pSMAD. PEC and microglia from WT or Lrrc33−/− mice were assayed for fluorescence intensity with SMAD2 and phospho-SMAD2/3 antibodies by In-cell-western. Mean ± SEM; N=3 mice (3 replicates each). ****P<0.0001, unpaired Student’s t-test. (N) Integrin dependence of TGF-β activation. WT astrocyte and microglia co-cultures were assayed for TGF-β production using reporter cells in the presence of indicated inhibitors. Mean ± SEM, N=3 mice (3 replicates each); **P<0.01, *P<0.05 (unpaired Student’s t-test).

Article Snippet: Human LRRC33 and mouse proTGF-β1 cDNAs were from Origene (Rockville, MD) and mouse LRRC33 cDNA from GE Dharmacon (Lafayette, CO).

Techniques: Immunostaining, Two Tailed Test, Expressing, Quantitation Assay, Fluorescence, Staining, Transfection, Activation Assay, In-Cell ELISA

Journal: Cell

Article Title: A Milieu Molecule for TGF-β Required for Microglia Function in the Nervous System

doi: 10.1016/j.cell.2018.05.027

Figure Lengend Snippet: (A) Sequence alignment. Red asterisks (*) mark cysteines that disulfide link to proTGF-β1 (Wang et al., 2012). X1 and X5 mark GARP/LRRC33 chimaera exchange positions. (B) Phylogram of closest LRR-superfamily relatives of LRRC33. Trees were calculated with the NJ method on ectodomains aligned with MAFFT (G-INS-i, gap insertion and extension penalties of 3 and 1, respectively). (C) LRRC33 mRNA expression in murine hematopoietic cells from the ImmGen microarray database. (D) LRRC33 and TGF-β1 mRNA expression in human cancer cell lines in the Cancer Cell Line Encyclopedia; red dots: haematopoietic cell lines. (E) LRRC33 and TGF-β1 mRNA levels positively correlate in normal human tissue, datasets from BioGPS. (F, G) X-gal staining showing LacZ expression in 4-month-old WT, Lrrc33+/− and Garp+/− heterozygous mice. (H) Mouse brain RNAseq data (Zhang, 2014); relative gene expression is shown among 8 cell types isolated from the CNS with FPKM (Fragments Per Kilobase of transcript per Million mapped reads) value shown for the highest expressing cell type.

Article Snippet: Human LRRC33 and mouse proTGF-β1 cDNAs were from Origene (Rockville, MD) and mouse LRRC33 cDNA from GE Dharmacon (Lafayette, CO).

Techniques: Sequencing, Expressing, Microarray, Staining, Isolation

Journal: Cell

Article Title: A Milieu Molecule for TGF-β Required for Microglia Function in the Nervous System

doi: 10.1016/j.cell.2018.05.027

Figure Lengend Snippet: (A and B) Lysates of 293T cells transfected with indicated constructs (A) or culture supernatants (B) were immunoprecipitated (IP) and subjected to reducing SDS 10% PAGE and blotted (WB) as indicated. (C) Disulfide linkage. 293T cells transfected with indicated constructs were subjected to IP, 7.5% non-reducing or 10% reducing SDS-PAGE, and WB as indicated. (D) LRRC33 outcompetes LTBP for proTGF-β1 293T transfectant lysates were IP, subjected to non-reducing SDS 7.5% PAGE, and WB as indicated. (E) LRRC33-proTGF-β1 complex in THP-1 cells. THP-1 cells were treated with or without PMA (80 nM, 24 h) and cell lysates were IP with 1/8.8 to LRRC33 or mouse IgG control, reducing and non-reducing SDS 7.5% PAGE, and WB as indicated. (F) Flow cytometry. THP-1 cells treated with or without PMA were stained with anti-LRRC33 (1/8.8), anti-prodomain (TW4–2F8), anti-integrin αV (17E6) or anti-integrin β6 (7.1G10) and subjected to FACS. Numbers in histograms show specific mean fluorescence intensity. (G) Blockade of active TGF-β1 release. THP-1 cells treated with or without PMA were incubated with antibody 1/8.8 to LRRC33, 17E6 to αV integrin, or MAB240 to TGF-β1 and cocultured with TMLC to measure TGF-β activation. Data represent mean ± SEM of quadruplicate samples.

Article Snippet: Human LRRC33 and mouse proTGF-β1 cDNAs were from Origene (Rockville, MD) and mouse LRRC33 cDNA from GE Dharmacon (Lafayette, CO).

Techniques: Transfection, Construct, Immunoprecipitation, SDS Page, Flow Cytometry, Staining, Fluorescence, Incubation, Activation Assay

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: 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: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: Primers used.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques:

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: Overview of regulated genes and their known relevant function.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques: Microarray, Expressing, Migration

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: Regulation of NGC expression by QKI. (a) Heat map of expression of NGCs in monocytes and macrophages determined by RNA-seq from QKI patient and sibling in log2 scale (Lighter blue indicates higher expression; n = 1). (b) Fold change of NGC expression comparing QKI patient’s monocyte and macrophage to her sibling’s. (c) In macrophages treated with GapmeR antisense oligonucleotides, there is a significant positive correlation between QKI and SEMA7A expression measured by quantitative PCR analysis. Gene expression is expressed as copies per GAPDH. QKI: quaking.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques: Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction, Gene Expression

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: Visualisation of genomic annotations in 3′UTR region of SEMA7A gene. The region in 3′UTR of SEMA7A in the human genome (hg19 Chromosome 15 q24.1, bp 74,701,500–74,703,000) is annotated with aspects of miRNA binding sites, QKI targeting sites (experimental evidence based), QRE ( in silico alignment of ‘ACUAA motif’) and SEMA7A exon. The microRNA binding information was extracted from ‘miRbase’ and was filtered by expression in monocytes/macrophages and miRNA interaction score. Interactions between RNA binding proteins (RBPs) to genomic DNA were obtained from technique duplicates of eCLIP-seq in the myelogenous K562 cells.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques: Binding Assay, In Silico, Expressing, RNA Binding Assay

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: RNA-immunoprecipitation showing quaking protein binding to the 3′UTR of SEMA7A. (a) RNA-immunoprecipitation in Hek293T cells overexpression QKI5 using an IgG control or QKI5 Ab. QKI5 and GAPDH mRNA abundance in immune-precipitated fraction was determined by qPCR. Results are presented relative to IgG immunoprecipitation. Data are the mean ± SEM; n = 5; * P < 0.05. (b) RNA-immunoprecipitation in Hek293T cells overexpression QKI5 and 3′UTR of SEMA7A using an IgG control or QKI5 Ab. SEMA7A 3’UTR and GAPDH mRNA abundance in immune-precipitated fraction was determined by qPCR. Results are presented relative to IgG immunoprecipitation. Data are the mean ± SEM; n = 3; * P < 0.05.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques: RNA Immunoprecipitation, Protein Binding, Over Expression, Control, Immunoprecipitation

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: Role for SEMA7A in monocyte differentiation into pro-inflammatory macrophages. (a) Bright field microscope images from THP-I monocytes and macrophages. (b) Representative immunoblot analysis of SEMA7A or ACTB (loading control) in protein lysates of THP-I monocytes and macrophages. (c) Bright field microscope images from THP-I macrophages transduced with anti-SEMA7A shRNA (sh-SEMA7A) or scrambled shRNA (sh-Ctrl). Elongated phenotype indicated by #. (d) Ratio of TNF-α and IL-10 mRNA expression of sh-SEMA7A or sh-Ctrl THP-I macrophages. Data are the mean ± SEM; n = 4; * P < 0.05.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques: Microscopy, Western Blot, Control, Transduction, shRNA, Expressing