Journal: Journal of experimental & clinical cancer research : CR

Article Title: Upregulation of ERK-EGR1-heparanase axis by HDAC inhibitors provides targets for rational therapeutic intervention in synovial sarcoma.

doi: 10.1186/s13046-021-02150-y

Figure Lengend Snippet: Fig. 4 HDACi upregulate heparanase mRNA and protein. a Analysis of HPSE expression by qRT-PCR in SS cells treated with SAHA or FK228 for the indicated times. Data are reported as relative quantification with respect to untreated cells as calibration sample. Relative mRNA values are the mean ± SE from three independent experiments. b Western blot detection of heparanase polypeptides in CME-1 cells exposed to SAHA or FK228 for the indicated times. Image on the right is from a cropped blot (dashed line) from which lanes not of interest have been removed. c N-glycosylation inhibition does not modify electrophoretic mobility of heparanase polypeptides. CME-1 cells were treated with tunicamycin (2 μg/ ml for 24 h) and processed for Western blotting. As controls, the shift of PDGFRα bands shows the glycosylation inhibition and BIP upregulation indicates endoplasmic reticulum stress. In (b) and (c) anti-actin, −vinculin and -GAPDH blots are shown as loading control

Article Snippet: For treatment with human active recombinant heparanase (R&D systems, Minneapolis, MN) (Supplementary Table S1), cells plated in complete medium for 24 h were incubated in serum-free medium with or without 5 μg/ml recombinant enzyme for 24 h and 48 h.

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Western Blot, Glycoproteomics, Inhibition, Control

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Upregulation of ERK-EGR1-heparanase axis by HDAC inhibitors provides targets for rational therapeutic intervention in synovial sarcoma.

doi: 10.1186/s13046-021-02150-y

Figure Lengend Snippet: Fig. 9 Schematic representation of the proposed HDACi activated auto-sustaining pro-survival loop and its blockade by co-treatment with ERK pathway and heparanase inhibitors in SS cells. This figure was prepared using tools from Servier Medical Art (http://www.servier.fr/servier-medic al-art)

Article Snippet: For treatment with human active recombinant heparanase (R&D systems, Minneapolis, MN) (Supplementary Table S1), cells plated in complete medium for 24 h were incubated in serum-free medium with or without 5 μg/ml recombinant enzyme for 24 h and 48 h.

Techniques:

Journal: Science Advances

Article Title: Cryo-EM structure of the Hedgehog release protein Dispatched

doi: 10.1126/sciadv.aay7928

Figure Lengend Snippet: ( A ) Biochemical activity of HhN C85II ligand, assessed by luciferase luminescence (left) and MST (right). The data correspond to values of luciferase activity normalized to the values measured for the “Control” sample (medium without the ligand); n = 4 [for recombinant human sonic hedgehog (rhShhN), n = 2]. The affinity of HhN C85II for Dispatched–yellow fluorescent protein (YFP), determined by MST, was 6.2 ± 2.5 μM ( n = 3). a.u., arbitrary units; Norm., normalized; a.f.u., arbitrary fluorescence units. ( B and C ) Comparison of the density maps of Dispatched (B) and Dispatched-HhN C85II complex (C); the asterisk (B) indicates the cavity between the ECD1 and ECD2 regions, occupied by HhN C85II density in the map shown in (C). ( D ) Overview of the Dispatched-HhN C85II complex model; color coding for Dispatched is the same as in ; the Hh ligand is colored red. ( E ) View of the complex from the luminal/extracellular side of the membrane shows the orientation of the HhN C85II ligand. The Cα atoms of N-terminal residue Y100 and the C-terminal residue D248 in the model are shown as red spheres. Arrows indicate possible proximity of the cholesterol-modified C-terminal region of HhN to the SSD region of Dispatched. ( F and G ) Structural alignment of the ligand-free and ligand-bound Dispatched; the complex aligned to apo-Dispatched using the TM domain (F) or the ECD2 domain (G). The numbers correspond to RMSD between the atoms of the individual domains. The red arrows indicate the whole domain structural rearrangements that accompany HhN ligand binding.

Article Snippet: For experiments involving purified ligands, purified HhN C85II or recombinant human sonic hedgehog, rhShhN (R&D Systems, no. 8908-SH/CF), was sterile-filtered and added to the medium during transfection to a final concentration of 200 nM.

Techniques: Activity Assay, Luciferase, Control, Recombinant, Fluorescence, Comparison, Membrane, Residue, Modification, Ligand Binding Assay

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 6. Anti-MICA/B 7C6 mAb boosts anti-tumor cytotoxic function of peripheral NK cells from iCCA patients against the HuCCT-1 cell line. A): peripheral NK cell degranulation, evaluated as frequency of CD107a+ NK cells, in iCCA patients (n = 13) and HC (n = 16) in the presence of 7C6 mAb or IgG1-Fc. Parametric paired and unpaired t tests were used to compare data. B): dot plots showing the frequency of CD3-CD56+ CD107a+ NK cells in a HC and a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc. C): the proportion of circulating IFNγ+ NK cells in patients (n = 10) and HC (n = 11) in the presence of 7C6 mAb compared with IgG1-Fc. To compare paired data, the parametric t test and the non-parametric Wilcoxon t test were used. To compare unpaired data, the parametric t test and the non-parametric Mann-Whitney U test were used. D): representative dot plots showing the frequency of CD3-CD56+ IFNγ+ NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: MANN-WHITNEY

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 7. Anti-MICA/B 7C6 mAb boosts anti-tumor cytotoxic function of peripheral NK cells from iCCA patients against patient-derived iCCA cell lines. A): peripheral NK cell degranulation, evaluated as CD107a+NK frequency, in iCCA patients (n = 12) and HC (n = 8) in the presence of 7C6 mAb or IgG1-Fc using patient- derived primary tumor cell lines as targets. Parametric paired and unpaired t tests were used to compare data. B): dot plots showing the frequency of CD3-CD56 + CD107a+ NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc. C): proportion of circulating IFNγ+NK cells in patients (n = 10) and in HC (n = 8) in the presence of 7C6 mAb compared with IgG1-Fc. To compare paired data, we used the parametric t test and the non-parametric Wilcoxon t test. To compare unpaired data, the parametric t test and the non-parametric Mann-Whitney U test were used. D): representative dot plots showing the frequency of CD3-CD56+ IFNγ +NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: Derivative Assay, MANN-WHITNEY

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 8. 7C6 mAb enhances the anti-tumor effect of liver- and tumor-infiltrating NK cells in iCCA patients. A): Frequency of degranulating CD107a+NK cells in LIL (n = 13) and TIL (n = 10) of iCCA patients in the presence of anti-MICA/B 7C6 mAb or IgG1-Fc using autologous tumor-derived cell lines as targets. Parametric paired and unpaired t tests were used to compare data. B): representative dot plots showing the frequency of CD3-CD56+ CD107a+ LIL- and TIL-NK cells in the presence of 7C6 mAb or IgG1-Fc. C): proportion of IFNγ+ NK cells in LIL (n = 10) and TIL (n = 8) of iCCA patients in the presence of 7C6 mAb compared with IgG1-Fc using autologous tumor-derived cell lines as targets. The parametric t test and non-parametric Wilcoxon t test were used to compare paired data. The parametric t test was used to compare unpaired data.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: Derivative Assay

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 9. Cytotoxicity assay of HC PBMC, patient PBMC, LIL and TIL cells. A, B): Frequency of CFSE+LIVE/DEAD (LD)+ HuCCT-1 cell line targets when HC PBMC (n = 5), patient PBMC (n = 10), LIL (n = 8) and TIL (n = 5) were used as effector cells in the presence of 7C6 mAb and isotype control (IgG1). The parametric paired t tests were used to compare data. C, D): Frequency of CFSE+LD+ patient-derived cell line targets when HC PBMC (n = 5), patient PBMC (n = 8), LIL (n = 8) and TIL (n = 4) were used as effectors in the presence of 7C6 and isotype control. The parametric paired t test was used to compare data in panel C. The non-parametric Wilcoxon t test was used to compare paired data in panel D. Target cell death was determined as frequency of CFSE+LD+ cells.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: Cytotoxicity Assay, Control, Derivative Assay

Journal: Developmental cell

Article Title: Phosphorylation of Ci/Gli by Fused family kinases promotes Hedgehog signaling

doi: 10.1016/j.devcel.2019.06.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse Anti-Flag antibody Millipore Sigma Clone ID: M2; Cat#1804; RRID: AB_439685 Mouse Anti-HA (F-7) antibody Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 Mouse Anti-Myc antibody Santa Cruz Biotechnology Clone ID: 9E10; Cat#sc-40; RRID: AB_627268 Rabbit Anti-Myc antibody Abcam Cat#ab9106; RRID: AB_307014 Rat Anti-Ci antibody DSHB Cat#2A1; RRID: AB_2109711 Mouse Anti-Fu antibody DSHB Cat#22F10; RRID: AB_528258 Goat Anti-PKC ζ antibody Santa Cruz Biotechnology Clone ID: c-20; Cat#sc-216; RRID: AB_2300359 Mouse Anti-α-tubulin antibody Millipore Sigma Clone ID: DM1A; Cat#T9026; RRID: AB_477593 Mouse Anti-GFP antibody Takara Biol Cat#632380; RRID: AB_10013427 Mouse Anti-acetylated Tubulin antibody Millipore Sigma Clone ID:6-11b1; Cat#T7451; RRID: AB_609894 Goat Anti-Gli2 antibody R&D Systems Cat#AF3635; RRID: AB_2111902 Goat Anti-Gli3 antibody R&D Systems Cat#AF3690; RRID: AB_2232499 Bacterial and Virus Strains NEB5-Alpha Competent E. Coli New England Biolabs Cat# {"type":"entrez-nucleotide","attrs":{"text":"C29871","term_id":"2361667","term_text":"C29871"}} C29871 BL21 Competent E.Coli New England Biolabs Cat#C2530H Chemicals, Peptides, and Recombinant Proteins Leptomycin B Millipore Sigma Cat#L2913 Acti-Stain 670 phalloidin Cytoskeleton Cat#PHDN1-A Bluo-Gal Thermo Fisher Scientific Cat#15519028 L-Glutathione Reduced Millipore Sigma Cat#G4251 SAG Millipore Sigma Cat#566660 DAPI Millipore Sigma Cat#D9542 ATP [γ − 32 P] Perkin Elmer Cat#BLU002A Insulin Millipore Sigma Cat#I6634 Fetal Bovine Serum Millipore Sigma Cat#F4135 Fetal Bovine Serum Millipore Sigma Cat#F0926 Calf Bovine Serum Iron Fortified ATCC Cat#ATCC30-2030 3× Flag Peptide Millipore Sigma Cat#F4799 HA Synthetic Peptide Thermo Fisher Scientific Cat#26184 Casein Kinase I New England Biolabs Cat#P6030 Recombinant Human Sonic Hedgehog Protein (Shh-N) R&D Systems Cat#8908-HSH/CF Lambda Protein Phosphatase New England Biolabs Cat#P0753 Critical Commercial Assays Dual Luciferase Reporter Assay System Promega Cat#E1910 MEGAscript T7 High Yield Transcription Kit Thermo Fisher Scientific Cat#AM1334 pIMAGO-biotin Phosphoprotein Detection Kit Millipore Sigma Cat#67221 Anti-Flag M2 Affinity Gel Millipore Sigma Cat#A2220 Pierce Anti-HA Argarose Thermo Fisher Scientific Cat#26181 Glutathione Sepharose 4B GE Healthcare Cat#17075601 SimplyBlue SafeStain Thermo Fisher Scientific Cat#LC6060 RNeasy Plus Mini Kit Qiagen Cat#74134 High Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat#4368814 Fast SYBR Green Master Mix Applied Biosystems Cat#4385612 CellTiter-Glo Luminescent Cell Viability Assay Promega Cat#G7572 Experimental Models: Cell Lines Mouse: NIH/3T3 ATCC Cat#ATCC CRL-1658 Human: HEK293T/17 ATCC Cat#ATCC CRL-11268 D. melanogaster .

Techniques: Virus, Recombinant, Luciferase, Reporter Assay, Reverse Transcription, SYBR Green Assay, Cell Viability Assay, Knock-In, Software

Journal: Stem cells (Dayton, Ohio)

Article Title: Heparanase Released From Mesenchymal Stem Cells Activates Integrin beta1/HIF-2alpha/Flk-1 Signaling and Promotes Endothelial Cell Migration and Angiogenesis

doi: 10.1002/stem.1995

Figure Lengend Snippet: MSC hpa promotes vascular regeneration in vivo. (A) : Representative Laser-Doppler images (LDI) of hind limbs before, immediately after, and 3, 7, and 14 days after femoral artery occlusion. (B) : Quantitative LDI analysis showing the right-to-left ( R/L ) ratio; n ≥ for each group, * denotes p < .05. (C) : Detection of heparanase protein expression using Western blot in WT MSC, MSC harboring empty vector, and heparanase over-expressed vector. (D) : Detection of heparanase by ELISA in conditioned medium of WT MSC, MSC harboring empty vector, and heparanase over-expressed vector; n = 4 for each group, * denotes p < .05. (E) : HE and immunofluorescent staining of α -SMA and CD31 in muscle tissues from each group; Bar = 100 μm for HE and Bar = 50 μm for immunofluorescent staining. (F, G) : Bar graph showed quantitative analysis of SMA and CD31 positive area density; n = 5 for each group, * denotes p < .05; ** denotes p < .01. Abbreviations: MSC, mesenchymal stem cell; PBS, phosphate buffer saline; SMA, smooth muscle actin.

Article Snippet: The lower chamber was filled with MSC-conditioned medium or control medium as above or with recombinant human active heparanase (100 mg/ml, R&D Systems, Minneapolis, MN, USA).

Techniques: In Vivo, Expressing, Western Blot, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Staining, Saline

Journal: Stem cells (Dayton, Ohio)

Article Title: Heparanase Released From Mesenchymal Stem Cells Activates Integrin beta1/HIF-2alpha/Flk-1 Signaling and Promotes Endothelial Cell Migration and Angiogenesis

doi: 10.1002/stem.1995

Figure Lengend Snippet: Proangiogenic role of MSC hpa in vitro. (A) : Protein expression of heparanase and actin by Western blot in MSC null and MSC hpa-KD . (B) : Detection of heparanase concentration using ELISA in conditioned medium of MSC WT , MSC null , and MSC hpa-KD ; n = 4 for each group, * denotes p < .05. (C) : Representative images showing tube formation of human umbilical vein endothelial cells after cocultured with conditioned medium derived from MSC WT , MSC null , MSC hpa , MSC hpa-KD , respectively; Bar = 50 μm. (D) : Quantification of tube length in each group; n = 3 for each group, ** denotes p < .01. (E) : Aortic ring assay showing sprouting and branching in each group; Bar = 50 μm. (F, G) : Bar graph showed quantitative analysis of sprout length and outgrowth area, respectively; n = 3 for each group, * denotes p < .05, ** denotes p < .01. Abbreviation: MSC, mesenchymal stem cell.

Article Snippet: The lower chamber was filled with MSC-conditioned medium or control medium as above or with recombinant human active heparanase (100 mg/ml, R&D Systems, Minneapolis, MN, USA).

Techniques: In Vitro, Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Aortic Ring Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Heparanase Released From Mesenchymal Stem Cells Activates Integrin beta1/HIF-2alpha/Flk-1 Signaling and Promotes Endothelial Cell Migration and Angiogenesis

doi: 10.1002/stem.1995

Figure Lengend Snippet: HIF-2 α activation by heparanase requires integrin β 1. (A) : Western blot and quantification of integrin β 1 and β 3 expressions in HUVECs treated with conditioned medium from MSC null , MSC hpa , and MSC hpa-KD . (B) : HIF-2 α and integrin β 1 expressions are decreased in integrin β 1 knockdown HUVECs. (C) : Representative images and bar graph of cell migration in HUVECs transfected with vector or integrin β 1 shRNA lentivirus; Bar = 50 μm, n = 3, ** denotes p < .01. (D) : Quantification of VEGF expression in MSC WT , MSC null , MSC hpa-KD , and MSC hpa . (E) : Quantification of VEGF, PDGF-BB, HGF, and TGF- β concentrations by ELISA in conditioned medium of MSC WT , MSC null , MSC hpa-KD , and MSC hpa ; n = 4 for each group. * denotes p < .05 (hpa vs. null) and ## denotes p < .01 (hpa-KD vs. null). (F) : Viability of MSCs in different concentrations of OGT2115 by CCK-8 assay. (G) : Bar graph shows quantitative analysis of cell migration, respectively; n = 3 for each group. * denotes p < .05 (hpa vs. null), ## denotes p < .01 (hpa1OGT2115 vs. hpa). (H) : Detection of heparanase concentration by ELISA in conditioned medium of MSC WT , MSC null , MSC hpa , and MSC hpa + OGT2115; n = 4 for each group, * denotes p < .05 (hpa vs. null), # denotes p < .05 (hpa+OGT2115 vs. hpa). (I) : Detection of VEGF, PDGF-BB, HGF, and TGF- β concentrations by ELISA in conditioned medium of MSC hpa and MSC hpa with OGT2115, n = 4 for each group. (J, K) : Bar graph shows quantitative analysis of cell migration and tube formation in each group; n = 3, ** denotes p < .01, * denotes p < .05. (L) : Quantification of integrin β 1, Flk-1, and HIF-2 α expression in HUVECs after incubated with active heparanase; n = 3. Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; HUVEC, human umbilical vein endothelial cell; MSC, mesenchymal stem cell.

Article Snippet: The lower chamber was filled with MSC-conditioned medium or control medium as above or with recombinant human active heparanase (100 mg/ml, R&D Systems, Minneapolis, MN, USA).

Techniques: Activation Assay, Western Blot, Knockdown, Migration, Transfection, Plasmid Preparation, shRNA, Expressing, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Concentration Assay, Incubation, Modification

Journal: Stem cells (Dayton, Ohio)

Article Title: Heparanase Released From Mesenchymal Stem Cells Activates Integrin beta1/HIF-2alpha/Flk-1 Signaling and Promotes Endothelial Cell Migration and Angiogenesis

doi: 10.1002/stem.1995

Figure Lengend Snippet: Schematic of cell migration and angiogenesis by MSC-secreted heparanase. Abbreviation: MSC, mesenchymal stem cell.

Article Snippet: The lower chamber was filled with MSC-conditioned medium or control medium as above or with recombinant human active heparanase (100 mg/ml, R&D Systems, Minneapolis, MN, USA).

Techniques: Migration