Journal: Oncology Letters

Article Title: Siva 1 inhibits proliferation, migration and invasion by phosphorylating Stathmin in ovarian cancer cells

doi: 10.3892/ol.2017.6307

Figure Lengend Snippet: Sequences of real-time PCR primers.

Article Snippet: Subsequent to blocking with 5% skim milk (YILI, Hohhot, Inner Mongolia, China) at room temperature for 1 h, diluted by TBS Tween (TBST), the PVDF membrane was incubated with the following antibodies at 4°C overnight: rabbit anti-human anti-Siva 1 polyclonal antibody (dilution, 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA, cat. no., sc-48767), rabbit anti-human anti-cleaved caspase-3 polyclonal antibody (dilution, 1:1,000; Abcam, Cambridge, UK, cat. no., ab2302), goat anti-human anti-cleaved caspase-9 polyclonal antibody (dilution, 1:200; Santa Cruz Biotechnology, Inc., cat. no., sc-22182), rabbit anti-human anti-Bcl-2-like protein 4 (Bax) polyclonal antibody (dilution, 1:400; Boster, Hubei, Wuhan, China, cat. no., BA0315), rabbit anti-human anti-Bcl-2 polyclonal antibody (dilution, 1:400; Boster, cat. no., BA0412), rabbit anti-human anti-Stathmin polyclonal antibody (dilution, 1:500; Bioss Antibodies, Beijing, China, cat. no., bs-1902R), rabbit anti-human anti-phosphorylated Stathmin polyclonal antibody (dilution, 1:500; Bioss Antibodies, cat. no., bs-3431R), rabbit anti-human anti-α-tubulin polyclonal antibody (dilution, 1:1,000; Abcam, cat. no., ab178484) or mouse anti-human anti-acetyl-α-tubulin monoclonal antibody (dilution, 1:1,000; Abcam, cat. no., ab24610).

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Oncology Letters

Article Title: Siva 1 inhibits proliferation, migration and invasion by phosphorylating Stathmin in ovarian cancer cells

doi: 10.3892/ol.2017.6307

Figure Lengend Snippet: Siva 1 enhances phosphorylation of Stathmin. (A) Siva 1 does not affect Stathmin mRNA level. (B and C) Overexpression of Siva 1 enhances phosphorylation of Stathmin and acetylation of α-tubulin, but did not markedly affect the expression level of Stathmin and α-tubulin detected by western blot. ***P<0.001 vs. pCMV group; ns, no significance.

Article Snippet: Subsequent to blocking with 5% skim milk (YILI, Hohhot, Inner Mongolia, China) at room temperature for 1 h, diluted by TBS Tween (TBST), the PVDF membrane was incubated with the following antibodies at 4°C overnight: rabbit anti-human anti-Siva 1 polyclonal antibody (dilution, 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA, cat. no., sc-48767), rabbit anti-human anti-cleaved caspase-3 polyclonal antibody (dilution, 1:1,000; Abcam, Cambridge, UK, cat. no., ab2302), goat anti-human anti-cleaved caspase-9 polyclonal antibody (dilution, 1:200; Santa Cruz Biotechnology, Inc., cat. no., sc-22182), rabbit anti-human anti-Bcl-2-like protein 4 (Bax) polyclonal antibody (dilution, 1:400; Boster, Hubei, Wuhan, China, cat. no., BA0315), rabbit anti-human anti-Bcl-2 polyclonal antibody (dilution, 1:400; Boster, cat. no., BA0412), rabbit anti-human anti-Stathmin polyclonal antibody (dilution, 1:500; Bioss Antibodies, Beijing, China, cat. no., bs-1902R), rabbit anti-human anti-phosphorylated Stathmin polyclonal antibody (dilution, 1:500; Bioss Antibodies, cat. no., bs-3431R), rabbit anti-human anti-α-tubulin polyclonal antibody (dilution, 1:1,000; Abcam, cat. no., ab178484) or mouse anti-human anti-acetyl-α-tubulin monoclonal antibody (dilution, 1:1,000; Abcam, cat. no., ab24610).

Techniques: Over Expression, Expressing, Western Blot

Journal: Acta pharmaceutica Sinica. B

Article Title: Rociletinib (CO-1686) enhanced the efficacy of chemotherapeutic agents in ABCG2-overexpressing cancer cells in vitro and in viv o.

doi: 10.1016/j.apsb.2020.01.008

Figure Lengend Snippet: Figure 1 The structure of rociletinib and cytotoxicity of rociletinib. (A) The structure of rociletinib. MTT cytotoxicity assay was conducted in ABCG2 and ABCB1-overexpressing cells and their parental sensitive cells; (B) ABCB1-overexpressing KBv200 cells and their parental drug sensitive KB cells; (C) ABCG2-overexpressing H460/MX20 cells and their parental drug sensitive H460 cells; (D) ABCG2-negative S1 and ABCG2-overexpressing S1-MI-80 cells; (E) ABCB1-negative HEK293/pcDNA3.1 and ABCG2-overexpressing wild type ABCG2-482-R2; (F) ABCG2-negative HEK293/pcDNA3.1 and ABCG2-overexpressing mutant ABCG2-482-T7 cells. Cells were treated with a range of concen- trations of rociletinib for 72 h. Results from three independent experiments are expressed as the mean SD.

Article Snippet: The antibodies used for Western blot analysis to detect AKT, p-AKT, ERK, p-ERK, and ABCG2 were purchased from Santa Cruz Biotechnology Inc.

Techniques: Cytotoxicity Assay, Mutagenesis

Journal: Acta pharmaceutica Sinica. B

Article Title: Rociletinib (CO-1686) enhanced the efficacy of chemotherapeutic agents in ABCG2-overexpressing cancer cells in vitro and in viv o.

doi: 10.1016/j.apsb.2020.01.008

Figure Lengend Snippet: Figure 4 Effect of rociletinib on the efflux of Rho 123, ATPase activity and [125I]-IAAPphotoaffinity labeling of ABCG2. (A) Time course of Rho 123 efflux was measured in S1 and S1-MI-80 cells, with or without 1 mmol/L rociletinib. (B) Effect of rociletinib on ABCG2 ATPase activity. The vanadate-sensitive ABCG2 ATPase activity in the presence of the indicated concentrations of rociletinib was evaluated. The mean SD values from three independent experiments are shown. (C) Rociletinib competed for photolabeling of ABCG2 by [125I]-IAAP. Crude membranes from ABCG2-overexpressing MCF7/FLV1000 cells were incubated with [125I]-IAAP and a range of different concentration (0e10 mmol/L) of rociletinib. The samples were then cross-linked by UV illumination, subjected to SDS-PAGE, and analyzed as described in the method section. A representative autoradiogram from three independent experiments is shown. The relative amount of [125I]-IAAP incorporated was plotted against the concentration of rociletinib used in the competition. 100% incorporation refers to the absence of rociletinib. Data are expressed as mean SD from three independent experiments.

Article Snippet: The antibodies used for Western blot analysis to detect AKT, p-AKT, ERK, p-ERK, and ABCG2 were purchased from Santa Cruz Biotechnology Inc.

Techniques: Activity Assay, Labeling, Incubation, Concentration Assay, SDS Page

Journal: Acta pharmaceutica Sinica. B

Article Title: Rociletinib (CO-1686) enhanced the efficacy of chemotherapeutic agents in ABCG2-overexpressing cancer cells in vitro and in viv o.

doi: 10.1016/j.apsb.2020.01.008

Figure Lengend Snippet: Figure 5 Effect of rociletinib on the expression of ABCG2 in MDR cells. (A) The protein level of ABCG2 on MDR cells after 0, 0.25, 0.5 and 1 mmol/L rociletinib stimulation for 48 h were measured by Western blot analysis. (B) The level of mRNA was measured by PCR (GAPDH as loading control), real time-PCR was further applied to confirm the unaltered mRNA levels in MDR cells. Rociletinib did not alter the mRNA and protein levels in MDR cells in concentration dependent manner. (C) The cell surface expression of ABCG2 was measured by flow cytometry before and after rociletinib stimulation on MDR cells and their parental cells. (D) The internalization of ABCG2 on MDR cells was measured by immunofluorescence confocal microscopy in the presence or absence of 1 mmol/L rociletinib. All experiments were repeated at least three times, and representative images and densitometry results were shown in each panel.

Article Snippet: The antibodies used for Western blot analysis to detect AKT, p-AKT, ERK, p-ERK, and ABCG2 were purchased from Santa Cruz Biotechnology Inc.

Techniques: Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction, Concentration Assay, Cytometry, Confocal Microscopy

Journal: Acta pharmaceutica Sinica. B

Article Title: Rociletinib (CO-1686) enhanced the efficacy of chemotherapeutic agents in ABCG2-overexpressing cancer cells in vitro and in viv o.

doi: 10.1016/j.apsb.2020.01.008

Figure Lengend Snippet: Figure 7 Schematic diagram showing the mechanism of rociletinib. (A) ABCG2 transporters utilize energy derived from the hydrolysis of ATP to efflux its substrates agents across the cell membrane in the absence of rociletinib. (B) Rociletinib may bind to the ATP binding site of ABCG2, thereby blocking the efflux of the transporter substrates and increasing the intracellular accumulation of the substrate drugs. Therefore, rociletinib could increase the efficacy of conventional chemotherapeutic drugs in ABCG2-overexpressing MDR cancer cells.

Article Snippet: The antibodies used for Western blot analysis to detect AKT, p-AKT, ERK, p-ERK, and ABCG2 were purchased from Santa Cruz Biotechnology Inc.

Techniques: Derivative Assay, Membrane, Binding Assay, Blocking Assay

Journal: Circulation journal : official journal of the Japanese Circulation Society

Article Title: Tertiary-butylhydroquinone upregulates expression of ATP-binding cassette transporter A1 via nuclear factor E2-related factor 2/heme oxygenase-1 signaling in THP-1 macrophage-derived foam cells.

doi: 10.1253/circj.cj-12-1616

Figure Lengend Snippet: Figure 1. Effects of tert-butylhydroquinone (tBHQ) on ATP-binding cassette transporter A1 (ABCA1) expression in THP-1 macro- phage-derived foam cells. Foam cells were treated with various concentrations of tBHQ as indicated for 24 h (A,C), or incubated with 100 μmol/L of tBHQ for various time periods (B,D). (A,B) Effects of tBHQ on ABCA1 mRNA levels. Total RNA was extracted and real-time quantitative PCR was performed as described. (C,D) Effects of tBHQ on ABCA1 protein levels. Total proteins were extracted. The protein levels of ABCA1 or β-actin were measured by Western blot assays (see Methods). All results are expressed as mean ± SD from 3 independent experiments. *P<0.05 vs. control group, **P<0.01 vs. control group.

Article Snippet: After blocking in 5% fat-free dry milk, the membranes were incubated with rabbit antibodies against ABCA1 (Abcam, Cambridge, MA, USA), HO-1 (Abcam), Nrf2 (Santa Cruz Biotechnology) and β-actin (Santa Cruz Biotechnology).

Techniques: Binding Assay, Expressing, Derivative Assay, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Circulation journal : official journal of the Japanese Circulation Society

Article Title: Tertiary-butylhydroquinone upregulates expression of ATP-binding cassette transporter A1 via nuclear factor E2-related factor 2/heme oxygenase-1 signaling in THP-1 macrophage-derived foam cells.

doi: 10.1253/circj.cj-12-1616

Figure Lengend Snippet: Figure 3. Effects of tert-butylhydroquinone (tBHQ) on the stability of ATP-binding cassette transporter A1 (ABCA1) protein and calpain activity. (A,B) Effect of tBHQ on ABCA1 stability. Foam cells were preincubated with cycloheximide (CHX, 2 mg/ml) for 1 h, and then treated with tBHQ (100 μmol/L) for the indicated times. Cellular lysates were prepared and the same amount of total proteins was subjected to Western blot assays to determine the protein levels of ABCA1 and β-actin. (C) Effect of tBHQ on calpain activity. Foam cells were treated with the indicated concentrations of tBHQ for 24 h and the calpain activity was determined as described. (D) Effect of tBHQ on calpain-induced ABCA1 degradation. Foam cells were pretreated with 100 μmol/L tBHQ for 12 h and then incubated with and without 0.5 μmol/L μ-calpain following digitonin permeabilization. The protein levels of ABCA1 and β-actin were analyzed by Western blot assays. The data represent the mean ± SD for 3 samples. *P<0.05 vs. control group, **P<0.01 vs. control group.

Article Snippet: After blocking in 5% fat-free dry milk, the membranes were incubated with rabbit antibodies against ABCA1 (Abcam, Cambridge, MA, USA), HO-1 (Abcam), Nrf2 (Santa Cruz Biotechnology) and β-actin (Santa Cruz Biotechnology).

Techniques: Binding Assay, Activity Assay, Western Blot, Incubation, Control

Journal: Circulation journal : official journal of the Japanese Circulation Society

Article Title: Tertiary-butylhydroquinone upregulates expression of ATP-binding cassette transporter A1 via nuclear factor E2-related factor 2/heme oxygenase-1 signaling in THP-1 macrophage-derived foam cells.

doi: 10.1253/circj.cj-12-1616

Figure Lengend Snippet: Figure 4. Increased ATP-binding cas- sette transporter A1 (ABCA1) protein expression by tert-butylhydroquinone (tBHQ) is dependent on the Nrf2/ HO-1 signaling pathway. (A–D) Foam cells were transfected with control or Nrf2 siRNA, and then incubated with tBHQ (100 μmol/L) for 24 h. (A) Pro- tein samples were immunoblotted with anti-Nrf2 or anti-β-actin antibod- ies. (B,C) ABCA1 and HO-1 protein expressions were determined using Western blot assays as shown. All the results are expressed as mean ± SD from 3 independent experiments. *P<0.05 vs. control group, **P<0.01 vs. control group. (D) Calpain activity was determined as described. (E–G) Foam cells were transfected with control or HO-1 siRNA or pretreated with ZnPPIX for 1 h, and then incu- bated with tBHQ (100 μmol/L) for 24 h. (E) Protein samples were immunob- lotted with anti-HO-1 or anti-β-actin antibodies. (F) ABCA1 protein levels were determined using Western blot assays. (G) Calpain activity was de- termined as described. The data rep- resent the mean ± SD for 3 samples. *P<0.05 vs. control group, **P<0.01 vs. control group. HO-1, heme oxy- genase-1; Nrf2, nuclear factor E2- related factor 2; ZnPPIX, zinc proto- porphyrin-IX.

Article Snippet: After blocking in 5% fat-free dry milk, the membranes were incubated with rabbit antibodies against ABCA1 (Abcam, Cambridge, MA, USA), HO-1 (Abcam), Nrf2 (Santa Cruz Biotechnology) and β-actin (Santa Cruz Biotechnology).

Techniques: Binding Assay, Expressing, Transfection, Control, Incubation, Western Blot, Activity Assay

Journal: eLife

Article Title: Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

doi: 10.7554/eLife.72579

Figure Lengend Snippet: ( a ) Depiction of the partial carotid ligation (PCL) surgery and flow-sensitive regions in the aortic arch: right carotid artery (RCA; s-flow ), left carotid artery (LCA; d-flow ), greater curvature (GC: s-flow ), and lesser curvature (LC; d-flow ). Two days following the PCL of C57BL/6J mice, the RCA and LCA were collected for frozen section imaging ( b, c ) and ( d ) endothelial-enriched RNA preparation. ( b ) Confocal images of immunostaining with anti-KLK10 or anti-CD31 antibodies (red) and counterstained with 4',6-diamidino-2-phenylindole (DAPI, blue) are shown. Scale bar = 20 μm. Arrows indicate endothelial cells and L is the lumen. ( c ) Quantification of endothelial KLK10 fluorescence intensity expressed as fold-change normalized to the RCA. N = 4. ( d ) Klk10 mRNA was measured in endothelial-enriched RNA from the carotid arteries by quantitative real-time polymerase chain reaction (qPCR). Data are expressed as fold-change normalized to 18s internal control. N = 3–4. ( e ) Confocal images of en face coimmunostaining of the LC and GC with anti-KLK10 (green) and anti-VE-Cadherin (red) antibody are shown counterstained with DAPI (blue). Scale bar = 10 μm. ( f ) Quantification of endothelial KLK10 fluorescence intensity expressed as fold-change normalized to the GC. N = 5. ( g–j ) Human artery endothelial cells (HAECs) subjected to 24 hr of unidirectional laminar shear (LS; 15 dynes/cm 2 ) or oscillatory shear (OS; ± 5 dynes/cm 2 ) were used to measure expression of KLK10 mRNA by qPCR ( g ), KLK10 protein in cell lysates by western blot ( h, i ), and KLK10 protein secreted to the conditioned media by ELISA ( j ). N = 4–6. All data are represented as mean ± standard error of mean (SEM). Statistical analyses were performed using paired t -test . ( k ) Single-cell RNAseq analysis of Klk10 gene transcripts and ( l ) single-cell ATACseq analysis of Klk10 chromatin accessibility in eight endothelial cell clusters (E1–E8), smooth muscle cells (SMCs), fibroblasts (Fibro), 4 monocytes/macrophages clusters (Mo1–4), dendritic cells (DCs), and T cells (T) in the mouse carotid arteries following 2 days or 2 weeks of the PCL surgery as we recently reported . The published datasets were reanalyzed here for the Klk10 gene. E1–E4 clusters represent ECs exposed to s-flow conditions in the RCA. E5 and E7 clusters represent ECs exposed to acute (2 days) d-flow in the LCA. E6 and E8 clusters represent ECs exposed to chronic (2 weeks) d-flow in the LCA. TSS indicates transcription start site. Figure 1—source data 1. Western blots for KLK10 and GAPDH.

Article Snippet: The aortas were carefully cleaned in situ, and the aortic arches and thoracic aortas were dissected, opened longitudinally, and fixed in 4% paraformaldehyde for 1 hr, permeabilized using 0.1% Triton X-100 in PBS for 15 min, blocked for 2 hr with 10% donkey serum, and incubated with anti-KLK10 (BiossUSA bs-2531R, 1:100), anti-VCAM1 (Abcam ab134047, 1:100), or anti-VE-Cadherin (Santa Cruz sc-9989, 1:100) primary antibodies overnight at 4°C followed by Alexa Fluor-647 secondary antibodies (Thermo Fisher Scientific, 1:500) for 2 hr at room temperature.

Techniques: Ligation, Imaging, Immunostaining, Fluorescence, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

doi: 10.7554/eLife.72579

Figure Lengend Snippet: Violin plots show single-cell expression of ( a ) Klk10 and ( b ) CD31 ( Pecam1 ) gene transcripts in eight endothelial cell clusters (E1–E8), smooth muscle cells (SMCs), fibroblasts (Fibro), 4 monocytes/macrophages clusters (Mo1–4), dendritic cells (DCs), and T cells (T) in the mouse carotid arteries following 2 days or 2 weeks of the PCL surgery as we recently reported . The published scRNAseq data were reanalyzed here for Klk10 and Pecam1 genes. E1–E4 clusters represent ECs exposed to s-flow conditions in the right carotid artery (RCA). E5 and E7 clusters represent ECs exposed to acute (2 days) d-flow in the LCA. E6 and E8 clusters represent ECs exposed to chronic (2 weeks) d-flow in the LCA.

Article Snippet: The aortas were carefully cleaned in situ, and the aortic arches and thoracic aortas were dissected, opened longitudinally, and fixed in 4% paraformaldehyde for 1 hr, permeabilized using 0.1% Triton X-100 in PBS for 15 min, blocked for 2 hr with 10% donkey serum, and incubated with anti-KLK10 (BiossUSA bs-2531R, 1:100), anti-VCAM1 (Abcam ab134047, 1:100), or anti-VE-Cadherin (Santa Cruz sc-9989, 1:100) primary antibodies overnight at 4°C followed by Alexa Fluor-647 secondary antibodies (Thermo Fisher Scientific, 1:500) for 2 hr at room temperature.

Techniques: Expressing

Journal: eLife

Article Title: Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

doi: 10.7554/eLife.72579

Figure Lengend Snippet: The plots display single-cell chromatin accessibility status of ( a ) Klk10 and ( b ) CD31 ( Pecam1 ) genes in eight endothelial cell clusters (E1–E8), smooth muscle cells (SMCs), fibroblasts (Fibro), 4 monocytes/macrophages clusters (Mo1–4), dendritic cells (DCs), and T cells (T) in the mouse carotid arteries following 2 days or 2 weeks of the PCL surgery as we recently reported . The published scATACseq data were reanalyzed here for Klk10 and Pecam1 genes. E1–E4 clusters represent ECs exposed to s-flow conditions in the right carotid artery (RCA). E5 and E7 clusters represent ECs exposed to acute (2 days) d-flow in the LCA. E6 and E8 clusters represent ECs exposed to chronic (2 weeks) d-flow in the LCA.

Article Snippet: The aortas were carefully cleaned in situ, and the aortic arches and thoracic aortas were dissected, opened longitudinally, and fixed in 4% paraformaldehyde for 1 hr, permeabilized using 0.1% Triton X-100 in PBS for 15 min, blocked for 2 hr with 10% donkey serum, and incubated with anti-KLK10 (BiossUSA bs-2531R, 1:100), anti-VCAM1 (Abcam ab134047, 1:100), or anti-VE-Cadherin (Santa Cruz sc-9989, 1:100) primary antibodies overnight at 4°C followed by Alexa Fluor-647 secondary antibodies (Thermo Fisher Scientific, 1:500) for 2 hr at room temperature.

Techniques:

Journal: eLife

Article Title: Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

doi: 10.7554/eLife.72579

Figure Lengend Snippet: ( a ) THP-1 monocyte adhesion assay was carried out in human artery endothelial cells (HAECs) transfected with 0.1 or 0.25 μg of KLK10 plasmid (KLK10-p) or GFP plasmid (GFP-p) for 48 hr followed by TNFɑ treatment (5 ng/ml for 4 hr). Data are represented as percentage of monocyte adhesion normalized to GFP-p control. N = 3. ( b ) THP-1 monocyte adhesion assay was carried out in HAECs treated with rKLK10 (0.1–10 ng/ml) or heat-inactivated rKLK10 (HI-10) for 24 hr followed by TNFɑ treatment (5 ng/ml for 4 hr). Data are represented as percentage of monocyte adhesion normalized to vehicle control. N = 3. ( c–g ) HAECs were treated with rKLK10 (0.1–10 ng/ml for 24 hr) followed by TNFɑ treatment (5 ng/ml for 4 hr) and expression of vascular cell adhesion molecule 1 (VCAM1) and intracellular adhesion molecule 1 (ICAM1) were assessed by quantitative real-time polymerase chain reaction (qPCR) ( c, d ) or western blot ( e–g ). N = 3. Data are represented as fold-change of the vehicle control and normalized to 18s or GAPDH . ( h, i ) THP-1 monocyte adhesion assay was conducted on HAECs subjected to 24 hr of either laminar shear (LS; 15 dynes/cm 2 ) or oscillatory shear (OS; ±5 dynes/cm 2 ) with either ( h ) rKLK10 (100 ng/ml) or ( i ) KLK10 siRNA (50 nM) or a nontargeting siRNA control. Data are represented as percentage of monocyte adhesion normalized to the control OS condition. N = 3–7. ( j ) C57BL/6J mice were injected with rKLK10 (0.6 mg/kg) or a vehicle control by tail vein once every 2 days for 5 days. The aortic arches were en face immunostained and imaged using confocal microscopy with an anti-VCAM1 antibody (red) and DAPI (blue). ( k ) Quantification of endothelial VCAM1 fluorescence intensity represented as fold-change normalized to control LC condition. N = 4–5. Scale bar = 10 μm. All data are represented as mean ± standard error of mean (SEM). Statistical analyses were performed using one-way analysis of variance (ANOVA) with Bonferroni correction for multiple comparisons. Figure 2—source data 1. Western blots for VCAM1, ICAM1, and GAPDH.

Article Snippet: The aortas were carefully cleaned in situ, and the aortic arches and thoracic aortas were dissected, opened longitudinally, and fixed in 4% paraformaldehyde for 1 hr, permeabilized using 0.1% Triton X-100 in PBS for 15 min, blocked for 2 hr with 10% donkey serum, and incubated with anti-KLK10 (BiossUSA bs-2531R, 1:100), anti-VCAM1 (Abcam ab134047, 1:100), or anti-VE-Cadherin (Santa Cruz sc-9989, 1:100) primary antibodies overnight at 4°C followed by Alexa Fluor-647 secondary antibodies (Thermo Fisher Scientific, 1:500) for 2 hr at room temperature.

Techniques: Cell Adhesion Assay, Transfection, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection, Confocal Microscopy, Fluorescence

Journal: eLife

Article Title: Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

doi: 10.7554/eLife.72579

Figure Lengend Snippet: ( a ) Human aortic endothelial cells (HAECs) were transfected with KLK10 plasmid ranging from 0.1 to 1 or 1 μg/ml GFP plasmid for 24 hr and the THP-1 monocyte adhesion assay was performed. N = 3. ( b ) HAECs were treated with 0.5–100 ng/ml rKLK10 and monocyte adhesion assay was performed. N = 4–6. ( c ) HAECs were treated with 100 ng/ml rKLK10 for 24 hr and quantitative real-time polymerase chain reaction (qPCR) was performed to assess mRNA expression of VCAM1 , ICAM1 , and MCP1 . N = 3–5. One-way analysis of variance (ANOVA) with Bonferroni correction for multiple comparisons ( a, b ) or two-way ANOVA with Bonferroni correction for multiple comparisons. ( c ) Mean ± standard error of mean (SEM).

Article Snippet: The aortas were carefully cleaned in situ, and the aortic arches and thoracic aortas were dissected, opened longitudinally, and fixed in 4% paraformaldehyde for 1 hr, permeabilized using 0.1% Triton X-100 in PBS for 15 min, blocked for 2 hr with 10% donkey serum, and incubated with anti-KLK10 (BiossUSA bs-2531R, 1:100), anti-VCAM1 (Abcam ab134047, 1:100), or anti-VE-Cadherin (Santa Cruz sc-9989, 1:100) primary antibodies overnight at 4°C followed by Alexa Fluor-647 secondary antibodies (Thermo Fisher Scientific, 1:500) for 2 hr at room temperature.

Techniques: Transfection, Plasmid Preparation, Cell Adhesion Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: eLife

Article Title: Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

doi: 10.7554/eLife.72579

Figure Lengend Snippet: ( a ) Mice (male, C57BL/6J) were administered 0.006–0.6 mg/kg rKLK10 or vehicle by tail-vein injection and inflammation was assessed by en face immunostaining of VCAM1 at the lesser curvature (LC) and the greater curvature (GC) of the aortic arch. Red = VCAM1, blue = DAPI, green = Elastin. Scale bar = 10 µm. ( b ) Quantification of VCAM1 staining in A normalized to the LC. Shown are mean ± standard error of mean (SEM), N = 3–6. Two-way analysis of variance (ANOVA) with Bonferroni correction for multiple comparisons. Part of results in ( a ) and ( b ) are shown in . ( c ) C57BL/6J mice (8-week-old males, n = 12) were injected with human rKLK10 (0.6 mg/kg) via tail-vein injection in three groups ( n = 4 mice per group) to collect blood via cheek vein at three different time points per group: (1) before injection (Time 0), 1, and 12 hr, (2) 0, 3, and 24 hr, and (3) 0, 6, and 48 hr after the injection. rKLK10 levels in the plasma were determined by human KLK10 ELISA and data were shown as one phase decay plot. t 1/2 of rKLK10 was 4.458 hr. Mean ± SEM is shown.

Article Snippet: The aortas were carefully cleaned in situ, and the aortic arches and thoracic aortas were dissected, opened longitudinally, and fixed in 4% paraformaldehyde for 1 hr, permeabilized using 0.1% Triton X-100 in PBS for 15 min, blocked for 2 hr with 10% donkey serum, and incubated with anti-KLK10 (BiossUSA bs-2531R, 1:100), anti-VCAM1 (Abcam ab134047, 1:100), or anti-VE-Cadherin (Santa Cruz sc-9989, 1:100) primary antibodies overnight at 4°C followed by Alexa Fluor-647 secondary antibodies (Thermo Fisher Scientific, 1:500) for 2 hr at room temperature.

Techniques: Injection, Immunostaining, Staining, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

doi: 10.7554/eLife.72579

Figure Lengend Snippet: ( a ) Bioluminescent imaging of Apoe −/− partial carotid ligation (PCL) mice on a high-fat diet injected with luciferase control plasmid or Klk10 -luciferase plasmid, measured in photons/second. ( b ) Gross plaque images of excised carotid arteries and ( c ) quantification of plaque burden normalized to the percentage of the luciferase control. ( d ) H&E staining of sections from the left carotid artery (LCA) and right carotid artery (RCA) of mice injected with luciferase control plasmid or Klk10 -luciferase plasmid. Scale bar low mag = 250 μm, high mag = 50 μm. ( e ) Quantification of plaque area measured in μm 2 . All data are represented as mean ± standard error of mean (SEM). Statistical analyses were performed using paired t -test. N = 11. ( f ) Sections from the RCA and LCA were coimmunostained with anti-KLK10 (orange) and anti-CD31 (red) antibodies. Blue is DAPI. Arrows indicate the ECs. L is the lumen and Adv is the adventitia. Scale bar = 10 μm. ( g ) Quantification of endothelial KLK10 fluorescent intensity represented as fold-change normalized to luciferase control. ( h ) Western blot analysis of KLK10 expression in lung tissue from mice injected with control luciferase plasmid or Klk10 plasmid . ( i ) Quantification of KLK10 expression normalized to GAPDH and luciferase control. Plasma lipid analysis of ( j ) total cholesterol, ( k ) triglycerides, ( l ) high-density lipoprotein (HDL) cholesterol, ( m ) low-density lipoprotein (LDL) cholesterol, or ( n ) non-HDL cholesterol. All data are represented as mean ± SEM. Statistical analyses were performed using paired t -test. N = 5. ns = not significant. Figure 6—source data 1. Western blots for KLK10 and GAPDH.

Article Snippet: The aortas were carefully cleaned in situ, and the aortic arches and thoracic aortas were dissected, opened longitudinally, and fixed in 4% paraformaldehyde for 1 hr, permeabilized using 0.1% Triton X-100 in PBS for 15 min, blocked for 2 hr with 10% donkey serum, and incubated with anti-KLK10 (BiossUSA bs-2531R, 1:100), anti-VCAM1 (Abcam ab134047, 1:100), or anti-VE-Cadherin (Santa Cruz sc-9989, 1:100) primary antibodies overnight at 4°C followed by Alexa Fluor-647 secondary antibodies (Thermo Fisher Scientific, 1:500) for 2 hr at room temperature.

Techniques: Imaging, Ligation, Injection, Luciferase, Plasmid Preparation, Staining, Western Blot, Expressing

Journal: eLife

Article Title: Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

doi: 10.7554/eLife.72579

Figure Lengend Snippet: KLK10 expression was measured in the plasma from mice overexpressing luciferase (Ctrl) or mouse Klk10 plasmid with ultrasound treatment in the hind-limb as described in . Plasma KLK10 level was determined using mouse KLK10 ELISA (BG-MUS11429). Paired two-tailed t -test was used. Shown are mean ± standard error of mean (SEM), n = 5.

Article Snippet: The aortas were carefully cleaned in situ, and the aortic arches and thoracic aortas were dissected, opened longitudinally, and fixed in 4% paraformaldehyde for 1 hr, permeabilized using 0.1% Triton X-100 in PBS for 15 min, blocked for 2 hr with 10% donkey serum, and incubated with anti-KLK10 (BiossUSA bs-2531R, 1:100), anti-VCAM1 (Abcam ab134047, 1:100), or anti-VE-Cadherin (Santa Cruz sc-9989, 1:100) primary antibodies overnight at 4°C followed by Alexa Fluor-647 secondary antibodies (Thermo Fisher Scientific, 1:500) for 2 hr at room temperature.

Techniques: Expressing, Luciferase, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: eLife

Article Title: Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

doi: 10.7554/eLife.72579

Figure Lengend Snippet: ( a ) Human coronary artery sections with varying degrees of atherosclerotic lesions were stained with anti-KLK10 antibody (red) and DAPI (blue). Scale bar low mag = 500 μm, scale bar; high mag = 50 μm. Arrows indicate endothelial cells. ( b ) Consecutive arterial sections from the same patients were stained with anti-CD31 antibody (red) and DAPI (blue). ( c ) Quantification of endothelial KLK10 fluorescence intensity in lower stage plaques (AHA grades 1–3) and advanced stage plaques (AHA grades 4–6). Data are from 40 different patients. Statistical analyses were performed using unpaired t -test. Mean ± standard error of mean (SEM) .

Article Snippet: The aortas were carefully cleaned in situ, and the aortic arches and thoracic aortas were dissected, opened longitudinally, and fixed in 4% paraformaldehyde for 1 hr, permeabilized using 0.1% Triton X-100 in PBS for 15 min, blocked for 2 hr with 10% donkey serum, and incubated with anti-KLK10 (BiossUSA bs-2531R, 1:100), anti-VCAM1 (Abcam ab134047, 1:100), or anti-VE-Cadherin (Santa Cruz sc-9989, 1:100) primary antibodies overnight at 4°C followed by Alexa Fluor-647 secondary antibodies (Thermo Fisher Scientific, 1:500) for 2 hr at room temperature.

Techniques: Staining, Fluorescence

Journal: eLife

Article Title: Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

doi: 10.7554/eLife.72579

Figure Lengend Snippet: KL10 is upregulated by s-flow and downregulated by d-flow at the genomic and protein levels. Under s-flow conditions when KLK10 is expression is high, KLK10 inhibits NFκB and expression of vascular cell adhesion molecule 1 (VCAM1) and intracellular adhesion molecule 1 (ICAM1), thereby preventing monocyte adhesion. Additionally, KLK10 produced by s-flow protects the endothelial permeability barrier. Together, the anti-inflammatory and barrier-protective effects of KLK10 lead to an overall protection against atherosclerosis.

Article Snippet: The aortas were carefully cleaned in situ, and the aortic arches and thoracic aortas were dissected, opened longitudinally, and fixed in 4% paraformaldehyde for 1 hr, permeabilized using 0.1% Triton X-100 in PBS for 15 min, blocked for 2 hr with 10% donkey serum, and incubated with anti-KLK10 (BiossUSA bs-2531R, 1:100), anti-VCAM1 (Abcam ab134047, 1:100), or anti-VE-Cadherin (Santa Cruz sc-9989, 1:100) primary antibodies overnight at 4°C followed by Alexa Fluor-647 secondary antibodies (Thermo Fisher Scientific, 1:500) for 2 hr at room temperature.

Techniques: Expressing, Produced, Permeability

Journal: eLife

Article Title: Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

doi: 10.7554/eLife.72579

Figure Lengend Snippet: Quantitative real-time polymerase chain reaction (qPCR) primers.

Article Snippet: The aortas were carefully cleaned in situ, and the aortic arches and thoracic aortas were dissected, opened longitudinally, and fixed in 4% paraformaldehyde for 1 hr, permeabilized using 0.1% Triton X-100 in PBS for 15 min, blocked for 2 hr with 10% donkey serum, and incubated with anti-KLK10 (BiossUSA bs-2531R, 1:100), anti-VCAM1 (Abcam ab134047, 1:100), or anti-VE-Cadherin (Santa Cruz sc-9989, 1:100) primary antibodies overnight at 4°C followed by Alexa Fluor-647 secondary antibodies (Thermo Fisher Scientific, 1:500) for 2 hr at room temperature.

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: eLife

Article Title: Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

doi: 10.7554/eLife.72579

Figure Lengend Snippet:

Article Snippet: The aortas were carefully cleaned in situ, and the aortic arches and thoracic aortas were dissected, opened longitudinally, and fixed in 4% paraformaldehyde for 1 hr, permeabilized using 0.1% Triton X-100 in PBS for 15 min, blocked for 2 hr with 10% donkey serum, and incubated with anti-KLK10 (BiossUSA bs-2531R, 1:100), anti-VCAM1 (Abcam ab134047, 1:100), or anti-VE-Cadherin (Santa Cruz sc-9989, 1:100) primary antibodies overnight at 4°C followed by Alexa Fluor-647 secondary antibodies (Thermo Fisher Scientific, 1:500) for 2 hr at room temperature.

Techniques: Mutagenesis, Recombinant, Produced, Plasmid Preparation, Transplantation Assay, Staining, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Software, Confocal Microscopy, Immunostaining, Over Expression, In Vitro

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Selective roles of MAPKs during the macrophage response to IFN-gamma.

doi: 10.4049/jimmunol.180.7.4523

Figure Lengend Snippet: FIGURE 1. Effects of IFN- on MAPK activation and MKP expression. Macrophages were obtained after 7 days of culture in the presence of M- CSF. The cells were rendered quiescent by incubating them in medium supplemented with 10% FCS (without M-CSF) for 18 h before the start of the experiment. To study direct effects of IFN- on MAPK activation, quiescent macrophages were stimulated with IFN- (10 ng/ml; in the ab- sence of M-CSF) during the indicated periods of time. Control cells were treated with vehicle (DMSO). A, p38 activation was determined by West- ern blotting using anti-phospho-p38 Abs. B, p38 isoform expression was determined by Western blotting using Abs specific against each isoform. C, Activation of JNK-1 was studied by immunoprecipitating JNK-1 and then performing an in vitro kinase assay on recombinant c-Jun. D, Activation of ERK-1 and -2 was analyzed by Western blotting using Abs against diphos- pho-ERK-1/2. These experiments were confirmed at least twice. E, Mac- rophages were treated with M-CSF (10 ng/ml), IFN- (10 ng/ml) or vehicle (DMSO) for 30 min (top), 2 h (middle), and 4 h (bottom). MKP expression was analyzed by real-time PCR In all real-time PCR experiments, the ex- pression values were normalized to the expression levels of the ribosomal gene L14. Error bars were determined from two independent experiments.

Article Snippet: We used the following Abs: monoclonal anti-diphospho-ERK-1/2 (clone MAPK-YT; Sigma-Aldrich); rabbit IgG anti-phospho-p38 (Thr180/Tyr182; Cell Signaling Technology); anti-I-A (BD Pharmingen), anti-phosphoStat-1 (Ser727; Cell Signaling Technology); anti-p38 (Cell Signaling Technology); anti-p38 (Zymed); anti-p38 (Cell Signaling Technology); anti-p38 (Cell Signaling Technology); and monoclonal anti-mouse -actin (Sigma-Aldrich).

Techniques: Activation Assay, Expressing, Control, Western Blot, In Vitro, Kinase Assay, Recombinant, Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Selective roles of MAPKs during the macrophage response to IFN-gamma.

doi: 10.4049/jimmunol.180.7.4523

Figure Lengend Snippet: FIGURE 2. Effects of MAPK inhibition on IFN- -mediated chemokine and cytokine expression. A, Macrophages were preincubated for 1 h with inhib- itors of MAPK signaling: PD98059 (50 M; to block ERK activation), SB203580 (5 M) (to in- hibit p38), or vehicle (DMSO, as control). The cells were then stimulated for 2 or 6 h with IFN-. B, Macrophages derived from WT or myeloid-specific p38-deficient mice (p38/) were stimulated for 6 h with IFN-. C, Macrophages derived from WT or JNK-1-deficient mice (JNK1/) were stimulated for 6 h with IFN-. B and C, control cells from each genotype were left untreated. In all these experi- ments, changes in gene expression were monitored by real-time PCR and normalized to the expression values of the ribosomal gene L14. A and C, error bars were determined from three independent exper- iments. B, duplicate experiments were analyzed. For statistical determinations, a nonparametric Wilcoxon test for paired differences (17) was used. , p 0.05; , p 0.01.

Article Snippet: We used the following Abs: monoclonal anti-diphospho-ERK-1/2 (clone MAPK-YT; Sigma-Aldrich); rabbit IgG anti-phospho-p38 (Thr180/Tyr182; Cell Signaling Technology); anti-I-A (BD Pharmingen), anti-phosphoStat-1 (Ser727; Cell Signaling Technology); anti-p38 (Cell Signaling Technology); anti-p38 (Zymed); anti-p38 (Cell Signaling Technology); anti-p38 (Cell Signaling Technology); and monoclonal anti-mouse -actin (Sigma-Aldrich).

Techniques: Inhibition, Expressing, Blocking Assay, Activation Assay, Control, Derivative Assay, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Selective roles of MAPKs during the macrophage response to IFN-gamma.

doi: 10.4049/jimmunol.180.7.4523

Figure Lengend Snippet: FIGURE 5. Effects of MAPKs on mRNA stability of IFN--induced genes. A, Macrophages were stimulated with IFN- for 2 h in the presence of inhibitors of the MEK-ERK cascade (PD98059, 50 M) or p38 (SB203580, 5 M). The cells were then treated for the indicated periods of time with a combination of RNA synthesis inhibitors, actinomycin D (Act D; 5 g/ml) and DBR (20 g/ml). B, Macrophages from WT or JNK-1-deficient mice were incubated with IFN- for 6 h. Inhibition of RNA synthesis was performed as described in A. In all these experiments, the levels of gene expression were evaluated by real-time PCR. Expression of acidic ribosomal phosphoprotein P0 (36B4) was used for normalization. To evaluate the rate of mRNA degradation, the remaining mRNA after treatment with inhibitors of RNA synthesis was calculated as a percentage of the expression levels of that gene in cells stimulated with IFN- (inhibitors of MAPK signaling; in the absence of RNA synthesis inhibitors). Therefore, the graphics do not reflect differences in gene expression between treatments before the addition of the RNA synthesis inhibitors.

Article Snippet: We used the following Abs: monoclonal anti-diphospho-ERK-1/2 (clone MAPK-YT; Sigma-Aldrich); rabbit IgG anti-phospho-p38 (Thr180/Tyr182; Cell Signaling Technology); anti-I-A (BD Pharmingen), anti-phosphoStat-1 (Ser727; Cell Signaling Technology); anti-p38 (Cell Signaling Technology); anti-p38 (Zymed); anti-p38 (Cell Signaling Technology); anti-p38 (Cell Signaling Technology); and monoclonal anti-mouse -actin (Sigma-Aldrich).

Techniques: Incubation, Inhibition, Gene Expression, Real-time Polymerase Chain Reaction, Expressing

Journal: PLoS Genetics

Article Title: Aurora-A-Dependent Control of TACC3 Influences the Rate of Mitotic Spindle Assembly

doi: 10.1371/journal.pgen.1005345

Figure Lengend Snippet: (A) The domain structure of human TACC3 is shown with conserved regions marked: N-terminal region (NTR, residues 1–108, coloured medium grey), Clathrin Interaction Domain (CID, residues 522–577, marked below), TACC domain (residues 636–838, coloured dark grey). Aurora-A phosphorylation sites are marked in bold italics. Known protein binding regions are marked below. (B) Co-precipitation assay between GST-AurA 1–129, GST-AurA-DN and TACC3-H6c. GST was used as a binding control. Reactions were analysed by SDS-PAGE (top panel). Binding of TACC3-H6c was confirmed by Western blot using an α-His 6 antibody (bottom panel). (C) In vitro kinase activity assay of Aurora-A 122–403 in the presence of TACC3-H6c and TACC3 fragments. The known Aurora-A activator, TPX2 1-43 was used as a positive control. Incorporation of 32 P radioisotope into MBP was quantified by scintillation counting. Error bars represent the standard error for two independent reactions. ** = P<0.01, *** = P<0.001 and **** = P<0.0001 using one-way ANOVA with Dunnett's post-hoc test compared to the MBP only reaction. SDS-PAGE analysis of TACC3 proteins used in this assay is shown in . (D) Stimulation of Aurora-A 122–403 autophosphorylation by TPX2 1-43 and TACC3act. Total Aurora-A is shown in the SDS-PAGE gel (top panel). Levels of phosphorylation were observed by Western blot using an antibody specific to Aurora-A phosphorylated on Thr288 (bottom panel). (E) Protection of Aurora-A 122–403 from dephosphorylation by PP1 in the presence of TPX2 1-43 and TACC3act. Aurora-A, TPX2 1-43 and TACC3act were resolved by SDS-PAGE (top panel). Aurora-A phosphorylation was detected by Western blot using a α-phosphoThr288 Aurora-A antibody (bottom panel).

Article Snippet: Primary antibodies used in this study were anti-CDK5RAP2 [ ], anti-TACC3 against aa 126–442 of Gallus gallus TACC3 [ ], anti-phospho-S558-TACC3/P-TACC3 (Cell Signaling), anti-ch-TOG (QED Bioscience, 34032), anti-Clathrin Heavy Chain (Abcam; 21679 and BD Biosciences, 610500), anti-Aurora-A (35C1; Sigma), anti-α-tubulin (Dm1α; Sigma), anti-α-tubulin-FITC (Sigma), anti-γ-tubulin (GTU88; Sigma) anti-phospho-Histone H3 (Millipore), anti-BubR1 (kind gift of W. Earnshaw) and phospho-Aurora-A/B/C (Cell Signaling).

Techniques: Protein Binding, Binding Assay, SDS Page, Western Blot, In Vitro, Kinase Assay, Positive Control, De-Phosphorylation Assay

Journal: PLoS Genetics

Article Title: Aurora-A-Dependent Control of TACC3 Influences the Rate of Mitotic Spindle Assembly

doi: 10.1371/journal.pgen.1005345

Figure Lengend Snippet: (A) In vitro kinase activity assay of Aurora-A 122–403 in the presence of TACC3act, TACC3-H6c and TACC3 mutants. ΔΔ, TACC3-H6c Δ519–546 and Δ564–629. Stimulation of Aurora-A catalytic activity by TACC3 was determined by incorporation of 32 P into MBP and quantified by scintillation counting. TPX2 1-43 was used as a positive control for Aurora-A activation. Error bars represent the standard error for two independent reactions. ** = P<0.01, *** = P<0.001 and **** = P<0.0001 using one-way ANOVA with Dunnett's post-hoc test compared to the MBP only reaction (left) and compared to the WT reaction (right). (B) Co-precipitation assay to assess binding between GST-AurA-DN and TACC3act-H6c (WT) and the point mutant, TACC3act-H6c F525A (F525A). GST was used as a binding control. (C) Binding affinities of Aurora-A 122–403 C290A, C393A for TACC3act-H6c WT and F525A were determined using microscale thermophoresis. Data were fitted to . Error bars represent the standard deviation of 3 measurements. (D) Multiple sequence alignment of TACC3 homologues within the minimal Aurora-A binding region. Asterisks above the alignment mark Aurora-A phosphorylation sites. Sequence conservation is shown below the alignment: ‘*’ indicates identical residues, ‘:’ identifies conservative substitutions and ‘.’ represents semi-conserved substitutions. Conserved aromatic residues are marked with black boxes. Residue F525 is marked with an arrow.

Article Snippet: Primary antibodies used in this study were anti-CDK5RAP2 [ ], anti-TACC3 against aa 126–442 of Gallus gallus TACC3 [ ], anti-phospho-S558-TACC3/P-TACC3 (Cell Signaling), anti-ch-TOG (QED Bioscience, 34032), anti-Clathrin Heavy Chain (Abcam; 21679 and BD Biosciences, 610500), anti-Aurora-A (35C1; Sigma), anti-α-tubulin (Dm1α; Sigma), anti-α-tubulin-FITC (Sigma), anti-γ-tubulin (GTU88; Sigma) anti-phospho-Histone H3 (Millipore), anti-BubR1 (kind gift of W. Earnshaw) and phospho-Aurora-A/B/C (Cell Signaling).

Techniques: In Vitro, Kinase Assay, Activity Assay, Positive Control, Activation Assay, Binding Assay, Mutagenesis, Microscale Thermophoresis, Standard Deviation, Sequencing

Journal: PLoS Genetics

Article Title: Aurora-A-Dependent Control of TACC3 Influences the Rate of Mitotic Spindle Assembly

doi: 10.1371/journal.pgen.1005345

Figure Lengend Snippet: (A) Ser558 phosphorylation of TACC3-H6c WT, F525A, ΔΔ (TACC3-H6c Δ519–546 and Δ564–629) and S558A by Aurora-A was measured by quantitative immunofluorescent blotting using an antibody specific to phosphorylated Ser558 in TACC3 (middle, the scanned blot was converted to black and white). The coomassie-stained gel is shown above. Quantification of phosphorylation is shown below. Error bars represent the standard error for two independent reactions. *** = P<0.001 using one-way ANOVA with Dunnett's post-hoc test compared to the WT reaction. (B) Total phosphorylation of TACC3-H6c WT, ΔΔ and point mutants by Aurora-A was monitored by incorporation of 32 P-labelled ATP (bottom). Phospho-null (SA) has three mutations: S34A, S552A, S558A. Coomassie-stained gels are shown above. (C) Co-precipitation assay to assess binding between GST-AurA-DN and TACC3. The assay used wild-type, phospho-null (SA) and phospho-mimic (SE) TACC3-H6c as prey proteins. GST was used as a binding control. (D) Activation of Aurora-A by TACC3-H6c WT, SA and SE was monitored by in vitro kinase activity assay. The catalytic activity of Aurora-A was determined by incorporation of 32 P into MBP and quantified by scintillation counting. TPX2 1-43 was used as a positive control for Aurora-A activation. Error bars represent the standard error for two independent reactions. ** = P<0.01 and *** = P<0.001 using one-way ANOVA with Dunnett's post-hoc test compared to the WT reaction.

Article Snippet: Primary antibodies used in this study were anti-CDK5RAP2 [ ], anti-TACC3 against aa 126–442 of Gallus gallus TACC3 [ ], anti-phospho-S558-TACC3/P-TACC3 (Cell Signaling), anti-ch-TOG (QED Bioscience, 34032), anti-Clathrin Heavy Chain (Abcam; 21679 and BD Biosciences, 610500), anti-Aurora-A (35C1; Sigma), anti-α-tubulin (Dm1α; Sigma), anti-α-tubulin-FITC (Sigma), anti-γ-tubulin (GTU88; Sigma) anti-phospho-Histone H3 (Millipore), anti-BubR1 (kind gift of W. Earnshaw) and phospho-Aurora-A/B/C (Cell Signaling).

Techniques: Staining, Binding Assay, Activation Assay, In Vitro, Kinase Assay, Activity Assay, Positive Control

Journal: PLoS Genetics

Article Title: Aurora-A-Dependent Control of TACC3 Influences the Rate of Mitotic Spindle Assembly

doi: 10.1371/journal.pgen.1005345

Figure Lengend Snippet: (A) Graphical illustration of domain organization, numbering and properties of key residues in Homo sapiens (Hs) and Gallus gallus (Gg) TACC3 proteins. Framed area below shows properties of mutant TACC3 protein products expressed in F543A, S574A and DEL cells, respectively. (B) Growth curves are shown for WT and mutant cell lines. n = 3 technical replicates, error bars represent standard deviation. (C) Measurement of 5-hydroxymethylcytosine (hmC) by tandem liquid–chromatography mass spectrometry in cells. hmC levels are expressed as parts per million (ppm) of total cytosines or ‘C’. Note that hmC levels inversely correlate with proliferation rate . n = 3 technical replicates. Statistical significance was assessed using t-test (**** P < 0.0001). (D) Western blot shows TACC3 protein levels in the DT40 cell lines with genotypes as indicated. The p150 subunit of dynactin serves as loading control. (E) TACC3 localisation to the mitotic spindle is impaired in all three mutant DT40 cell lines. In merged images α-tubulin is green, TACC3 is red and DNA is blue. Scale bar = 5 μm. Box plot on right depicts intensity of TACC3 staining on the mitotic spindle. TACC3 signal intensity was quantified in mitotic spindle volumes defined by α-tubulin staining. A minimum of 60 cells was scored per genotype. Statistical significance was assessed using t-test (**** P < 0.0001).

Article Snippet: Primary antibodies used in this study were anti-CDK5RAP2 [ ], anti-TACC3 against aa 126–442 of Gallus gallus TACC3 [ ], anti-phospho-S558-TACC3/P-TACC3 (Cell Signaling), anti-ch-TOG (QED Bioscience, 34032), anti-Clathrin Heavy Chain (Abcam; 21679 and BD Biosciences, 610500), anti-Aurora-A (35C1; Sigma), anti-α-tubulin (Dm1α; Sigma), anti-α-tubulin-FITC (Sigma), anti-γ-tubulin (GTU88; Sigma) anti-phospho-Histone H3 (Millipore), anti-BubR1 (kind gift of W. Earnshaw) and phospho-Aurora-A/B/C (Cell Signaling).

Techniques: Mutagenesis, Standard Deviation, Liquid Chromatography, Mass Spectrometry, Western Blot, Staining

Journal: PLoS Genetics

Article Title: Aurora-A-Dependent Control of TACC3 Influences the Rate of Mitotic Spindle Assembly

doi: 10.1371/journal.pgen.1005345

Figure Lengend Snippet: (A) Western blots show immunoprecipitation of TACC3 from DT40 cell extracts. Cells were blocked in mitosis by nocodazole (+Noc) for 16 hours or released from nocodazole block into MG132 for 1 hour (+MG132). Genotypes are as indicated. Antibodies against TACC3 or random rabbit IgG (Con) were used for immunoprecipitation (IP). Inputs represent cytoplasmic extracts. Blots were probed with anti-TACC3 or anti-phospho-S574-TACC3 (P-TACC3) antibodies. The percentages below the plots refer to P-TACC3 signal levels in the mutants relative to WT. Band intensities were analysed on films with ImageJ; P-TACC3 signal was normalized against total TACC3 (inputs) or total immunoprecipitated TACC3 for each genotype. (B) Western blots show co-immunoprecipitation of clathrin and TACC3 from DT40 cell extracts. Genotypes are as indicated. Antibodies against clathrin heavy chain (CHC) or random rabbit IgG (con) were used for immunoprecipitation (IP). Inputs represent cytoplasmic extracts. Blots were probed with anti-CHC or anti-TACC3 antibodies, as indicated. The percentages below the plots refer to TACC3 signal levels in F543A relative to WT. Band intensities were analysed on films with ImageJ and TACC3 signal was normalized against total immunoprecipitated clathrin for each genotype. (C) Western blots show co-immunoprecipitation of clathrin with P-TACC3 from WT and F543A DT40 cell extracts. Antibodies against clathrin heavy chain (CHC) or random rabbit IgG (con) were used for immunoprecipitation (IP). Inputs represent cytoplasmic extracts. Blots were probed with anti-CHC, anti-TACC3 or anti P-TACC3 antibodies, as indicated. (D) Localisation of clathrin in mitotic cells is shown on left. In merged images α-tubulin is green, clathrin (CHC) is red and DNA is blue. Scale bar = 5 μm. Box plot on right depicts mean intensity of clathrin staining on the mitotic spindle. Signal intensity of clathrin (CHC) antibody was quantified in mitotic spindle volumes defined by α-tubulin-FITC staining. A minimum of 25 cells was scored per genotype. Statistical significance was assessed using t-test (***P = 0.0001, **** P < 0.0001). (E) Western blots of sucrose gradient centrifugation from WT and F543A cytoplasmic cell extracts. Note different fractionation patterns between genotypes: in F543A cells fraction 13 contains increased amounts of ch-TOG, TACC3 and Aurora-A, but not clathrin. TPX2 and EB1 display similar fractionation patterns between WT and F543A cells. Position of molecular weight markers (BSA: 4.4S; Apoferritin: 17.6S; Thyroglobulin: 19.4S) on the gradient is shown.

Article Snippet: Primary antibodies used in this study were anti-CDK5RAP2 [ ], anti-TACC3 against aa 126–442 of Gallus gallus TACC3 [ ], anti-phospho-S558-TACC3/P-TACC3 (Cell Signaling), anti-ch-TOG (QED Bioscience, 34032), anti-Clathrin Heavy Chain (Abcam; 21679 and BD Biosciences, 610500), anti-Aurora-A (35C1; Sigma), anti-α-tubulin (Dm1α; Sigma), anti-α-tubulin-FITC (Sigma), anti-γ-tubulin (GTU88; Sigma) anti-phospho-Histone H3 (Millipore), anti-BubR1 (kind gift of W. Earnshaw) and phospho-Aurora-A/B/C (Cell Signaling).

Techniques: Western Blot, Immunoprecipitation, Blocking Assay, Staining, Gradient Centrifugation, Fractionation, Molecular Weight

Journal: PLoS Genetics

Article Title: Aurora-A-Dependent Control of TACC3 Influences the Rate of Mitotic Spindle Assembly

doi: 10.1371/journal.pgen.1005345

Figure Lengend Snippet: (A) Spindle length measured in 3D using Volocity. Number of cells analysed is indicated in graph (n). Student t-test (**** P < 0.0001). (B) Mitotic spindle morphologies (as described in main text) observed during time-lapse microscopy of GFP-tubulin-expressing TACC3 mutant cell lines. Representative still frames are shown on left. Scale bar = 5 μm. (C) Durations of NEBD to anaphase onset obtained from time-lapse microscopy performed on GFP-tubulin-expressing TACC3 mutant cell lines. F543A_1 and F543A_2 cells represent independently derived clones. Note that we observed no correlation between GFP levels and mitotic timing, or between mitotic timing and time of NEBD with respect to duration of filming in these experiments. Box plot shows 10–90 percentiles for each genotype. Number of cells analysed is indicated in graph (n). Mann Whitney nonparametric t-test (**** P < 0.0001, *** P < 0.001 and n.s. stands for ‘no significance’). (D) Durations of NEBD to anaphase onset obtained from time-lapse microscopy performed on EB3-GFP-expressing cells. Box plot shows 10–90 percentiles. Number of cells analysed is indicated in the graph (n). Mann Whitney nonparametric t-test. (**** P < 0.0001). (E) Analysis of chromosome segregation. Top table shows percentage of lagging chromatids seen during anaphase in fixed cells. Bottom table shows results from metaphase chromosome (chr) spreads. Number of autosomes 1, 2, 3, 4 and the sex chromosome, Z was analysed in cells. Frequencies of loss or gain of a single copy of individual chromosomes are indicated. Note that WT DT40 cells are trisomic for chromosome 2, whilst all the TACC3 mutants are diploid. Therefore, cells with two or three copies of chromosome 2’s are marked with ‘*’ or ‘#’, respectively. (F) Plot depicts time between NEBD and bipolar spindle appearance in time-lapse microscopy from Fig 7B. Cells with multipolar spindles and cells in which one of the two spindle poles was outside of the Z stack range for these time points were excluded from analysis. Number of cells analysed is indicated in graph (n). Mann Whitney nonparametric t-test (* P = 0.04, **** P < 0.0001).

Article Snippet: Primary antibodies used in this study were anti-CDK5RAP2 [ ], anti-TACC3 against aa 126–442 of Gallus gallus TACC3 [ ], anti-phospho-S558-TACC3/P-TACC3 (Cell Signaling), anti-ch-TOG (QED Bioscience, 34032), anti-Clathrin Heavy Chain (Abcam; 21679 and BD Biosciences, 610500), anti-Aurora-A (35C1; Sigma), anti-α-tubulin (Dm1α; Sigma), anti-α-tubulin-FITC (Sigma), anti-γ-tubulin (GTU88; Sigma) anti-phospho-Histone H3 (Millipore), anti-BubR1 (kind gift of W. Earnshaw) and phospho-Aurora-A/B/C (Cell Signaling).

Techniques: Time-lapse Microscopy, Expressing, Mutagenesis, Derivative Assay, Clone Assay, MANN-WHITNEY

Journal: PLoS Genetics

Article Title: Aurora-A-Dependent Control of TACC3 Influences the Rate of Mitotic Spindle Assembly

doi: 10.1371/journal.pgen.1005345

Figure Lengend Snippet: (A) MT regrowth assays in DT40 cells with genotypes as indicated. MTs were depolymerized by nocodazole and then allowed to recover for 3 or 15 minutes. Note that the mutant TACC3 products accumulate on nascent MTs. Scale bar = 5 μm. (B) Graph depicts percentages of mitotic cells with discernible bipolar spindles after 15 minutes of MT regrowth. n = 3 independent experiments; minimum of 100 cells scored per genotype per experiment. Student t-test (* P = 0.03).

Article Snippet: Primary antibodies used in this study were anti-CDK5RAP2 [ ], anti-TACC3 against aa 126–442 of Gallus gallus TACC3 [ ], anti-phospho-S558-TACC3/P-TACC3 (Cell Signaling), anti-ch-TOG (QED Bioscience, 34032), anti-Clathrin Heavy Chain (Abcam; 21679 and BD Biosciences, 610500), anti-Aurora-A (35C1; Sigma), anti-α-tubulin (Dm1α; Sigma), anti-α-tubulin-FITC (Sigma), anti-γ-tubulin (GTU88; Sigma) anti-phospho-Histone H3 (Millipore), anti-BubR1 (kind gift of W. Earnshaw) and phospho-Aurora-A/B/C (Cell Signaling).

Techniques: Mutagenesis

Journal: PLoS Genetics

Article Title: Aurora-A-Dependent Control of TACC3 Influences the Rate of Mitotic Spindle Assembly

doi: 10.1371/journal.pgen.1005345

Figure Lengend Snippet: (A) Table summarises phenotypes observed in the DT40 TACC3 mutants. Cell doubling time of F543A is similar to WT, but hmC levels indicate faster proliferation, hence the n.e./+ description. (B) Model illustrates roles of different TACC3 pools during mitosis. Briefly, in prometaphase TACC3 is phosphorylated on S558 by TPX2-bound Aurora-A, and P-TACC3 enhances MT polymerase activity of ch-TOG both near centrosomes and chromatin. As kinetochore MTs become established, TACC3 recruits clathrin to these MTs, but the complex is initially unstable. Long-lived kinetochore MTs allow local activation of Aurora-A by TACC3 and phosphorylation at S558 stabilises the TACC3-clathrin complex, which in turn crosslinks these MTs.

Article Snippet: Primary antibodies used in this study were anti-CDK5RAP2 [ ], anti-TACC3 against aa 126–442 of Gallus gallus TACC3 [ ], anti-phospho-S558-TACC3/P-TACC3 (Cell Signaling), anti-ch-TOG (QED Bioscience, 34032), anti-Clathrin Heavy Chain (Abcam; 21679 and BD Biosciences, 610500), anti-Aurora-A (35C1; Sigma), anti-α-tubulin (Dm1α; Sigma), anti-α-tubulin-FITC (Sigma), anti-γ-tubulin (GTU88; Sigma) anti-phospho-Histone H3 (Millipore), anti-BubR1 (kind gift of W. Earnshaw) and phospho-Aurora-A/B/C (Cell Signaling).

Techniques: Activity Assay, Activation Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Human airway smooth muscle cells secrete amphiregulin via bradykinin/COX-2/PGE 2 , inducing COX-2, CXCL8, and VEGF expression in airway epithelial cells

doi: 10.1152/ajplung.00390.2014

Figure Lengend Snippet: Bradykinin (BK) induced amphiregulin mRNA accumulation and protein secretion from airway smooth muscle cells. Three human airway smooth muscle (HASM) cell lines were treated with BK (1 × 10−5 M) in a time course of up to 24 h and the culture supernatants were analyzed by ELISA for amphiregulin (A). Three HASM cell lines were treated with BK (1 × 10−5 M) in a time course of up to 24 h and the accumulation of amphiregulin mRNA analyzed by quantitative PCR (QPCR) (B). β2-MG, β2-microglobulin. All measurements represent means ± SE of 3 independent experiments.

Article Snippet: Recombinant amphiregulin was purchased from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Human airway smooth muscle cells secrete amphiregulin via bradykinin/COX-2/PGE 2 , inducing COX-2, CXCL8, and VEGF expression in airway epithelial cells

doi: 10.1152/ajplung.00390.2014

Figure Lengend Snippet: Bradykinin-induced amphiregulin mRNA accumulation and protein secretion from HASM cells (HASMC) is COX-2, PGE2, and EP2/EP4 receptor dependent. COX-2 inhibitors block BK-induced amphiregulin protein secretion and mRNA accumulation. Three HASM cell lines were treated with indomethacin (1 × 10−6 M) or NS398 (1 × 10−6 M) for 30 min prior to treatment with BK for 24 h. Culture supernatants were analyzed by ELISA for amphiregulin (A). Three HASM cell lines were treated with indomethacin (1 × 10−6 M) or NS398 (1 × 10−6 M) for 30 min prior to treatment with BK for 8 h with amphiregulin mRNA accumulation analyzed by QPCR (B). Three HASM cell lines were incubated with control siRNA (SC and solid bars) or COX-2 specific siRNA (I and open bars) for 48 h prior to treatment with BK (1 × 10−5 M) in a time course of up to 24 h. Protein samples were analyzed by Western blot to confirm COX-2 protein knockdown (C) and parallel RNA samples analyze by RT-QPCR for amphiregulin mRNA accumulation at 0 and 4 h post-BK addition. Each cell line was also incubated with amphiregulin siRNA (shaded bars) (D). PGE2 induces amphiregulin mRNA accumulation and amphiregulin secretion from HASM cells. Three HASM cell lines were treated with PGE2 (1 × 10−6 M) for up to 24 h and the accumulation of amphiregulin mRNA analyzed by QPCR (E); the experiment was repeated and amphiregulin secretion from HASM cells was monitored by amphiregulin ELISA (F). PGE2-induced amphiregulin secretion is not COX-2 dependent. Three HASM cell lines were treated with indomethacin (1 × 10−6 M) or NS398 (1 × 10−6 M) for 30 min prior to PGE2 addition (1 × 10−6 M) for 24 h followed by an amphiregulin ELISA of the culture supernatants (G). Selective EP2/EP4 receptor agonists-induced amphiregulin secretion from HASMC. Three HASM cell lines were treated with ONO AEI 329 (EP4), ONO AEI 259 (EP2), ONO AEI 248 (EP3), or ONO AEI D1 004 (EP1) for 24 h followed by an amphiregulin ELISA (H). All measurements represent means ± SE of 3 independent experiments. ***P < 0.001.

Article Snippet: Recombinant amphiregulin was purchased from R&D Systems.

Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay, Incubation, Control, Western Blot, Knockdown, Quantitative RT-PCR

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Human airway smooth muscle cells secrete amphiregulin via bradykinin/COX-2/PGE 2 , inducing COX-2, CXCL8, and VEGF expression in airway epithelial cells

doi: 10.1152/ajplung.00390.2014

Figure Lengend Snippet: Recombinant amphiregulin and bradykinin conditioned cell culture medium from airway smooth muscle cells can induce CXCL8 mRNA accumulation and CXCL8 secretion from human airway epithelial cells. HASMC conditioned medium-induced CXCL8 expression in human bronchial epithelial cells (HBEC) is amphiregulin dependent. HBEC were treated with recombinant amphiregulin (10 ng/ml) for 24 h followed by a CXCL8 ELISA of the culture supernatant (A). HBEC were treated with recombinant amphiregulin for 24 h and CXCL8 mRNA accumulation analyzed by QPCR (B). Cell culture medium from HASMC treated for 24 h with BK (1 × 10−6 M) (BK con med) or a vehicle control (control con med) were added to HBEC for 24 h and the resulting HBEC supernatants and HASMC conditioned media were analyzed by CXCL8 ELISA, with HASMC conditioned medium CXCL8 levels subtracted from HBEC medium levels (C). HASMC BK conditioned medium and control conditioned media were used to treat HBEC for a time course of up to 24 h. CXCL8 mRNA accumulation was analyzed in HBEC by QPCR (D). HBEC were treated with BK conditioned HASMC medium (BK con med) for 24 h with either anti-amphiregulin blocking antibody (10 μg/ml) (anti-AR) or mouse IgG1 (10 μg/ml) (mouse IgG1) and CXCL8 mRNA levels analyzed by QPCR (E). HBEC were treated with HASMC control conditioned medium (con med) or HASMC BK conditioned medium (BK con med) for 24 h with either anti-amphiregulin blocking antibody (anti-AR) at 10 μg/ml or mouse IgG1 (anti-mouse) at 10 μg/ml, and secreted CXCL8 protein levels were analyzed by ELISA (F). HASM cells were transfected with control scrambled siRNA (solid bars) or amphiregulin siRNA (open bars) for 48 h, treated with BK for 24 h and the conditioned medium transferred to HBEC cells for 24 h, CXCL8 mRNA levels were analyzed by QPCR (G). All measurements represent means ± SE of 3 independent experiments. **P = 0.001–0.01; ***P < 0.001.

Article Snippet: Recombinant amphiregulin was purchased from R&D Systems.

Techniques: Recombinant, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Control, Blocking Assay, Transfection

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Human airway smooth muscle cells secrete amphiregulin via bradykinin/COX-2/PGE 2 , inducing COX-2, CXCL8, and VEGF expression in airway epithelial cells

doi: 10.1152/ajplung.00390.2014

Figure Lengend Snippet: Amphiregulin and HASMC conditioned medium induction of CXCL8 mRNA expression and CXCL8 protein secretion in HBEC were blocked by EGFR inhibition with AG1478. Amphiregulin, BK conditioned HASMC medium, or control conditioned HASMC medium increased EGFR phosphorylation in HBEC was inhibited by AG1478. HBEC were assayed for CXCL8 mRNA (QPCR) after 2-h addition of amphiregulin (10 ng/ml) (solid bars) with or without pretreatment with AG1478 (1 × 10−6 M) (A). HBEC supernatants were assayed for CXCL8 protein by ELISA 24 h after addition of amphiregulin (10 ng/ml) (solid bars) with or without pretreatment with AG1478 (1 × 10−6 M) (B). HBEC were assayed for CXCL8 mRNA (QPCR) after incubation with BK conditioned HASMC medium (BK con med) or HASMC control conditioned medium (con med) (all solid bars) after 8 h, with or without pretreatment with AG1478 (1 × 10−6 M) (open bars are treated 0-h controls) (C). HBEC culture supernatants were assayed by ELISA for CXCL8 protein after pretreatment with AG1478 followed by treatment with HASMC control conditioned medium (con med) or BK conditioned medium (BK con med) with or without AG1478 (1 × 10−6 M) (D). Recombinant amphiregulin was added to HBEC in a time course, cell lysates were analyzed by Western blot for EGFR tyrosine-845 phosphorylation (EGFR-PY845), and blots were striped and reprobed with a “total” EGFR antibody (EGFR) (E). HBEC were treated with HASMC cell culture supernatants previously either untreated (con med) or treated with BK for 24 h (BK con med). Total cell lysates were analyzed by Western blot for EGFR tyrosine-845 phosphorylation (EGFR-PY845), then blots were striped and reprobed with anti-EGFR antibody (EGFR) (F). All measurements represent means ± SE of 3 independent experiments. Western blots are representative of 3 independent experiments. ***P < 0.001.

Article Snippet: Recombinant amphiregulin was purchased from R&D Systems.

Techniques: Expressing, Inhibition, Control, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant, Western Blot, Cell Culture

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Human airway smooth muscle cells secrete amphiregulin via bradykinin/COX-2/PGE 2 , inducing COX-2, CXCL8, and VEGF expression in airway epithelial cells

doi: 10.1152/ajplung.00390.2014

Figure Lengend Snippet: Recombinant amphiregulin and BK conditioned medium from airway smooth muscle cells increased COX-2 mRNA accumulation and COX-2 protein expression in HBEC. HBEC were treated with amphiregulin (10 ng/ml) in a time course of up to 24 h and assayed for COX-2 mRNA by QPCR (A). HBEC were treated with amphiregulin (10 ng/ml) in a time course of up to 24 h and the total protein extracts analyzed by Western blot for COX-2 protein (B). HBEC were treated with HASMC culture supernatants conditioned for 24 h with BK (▲, BK con med) or HASMC culture supernatants conditioned with a vehicle control (■, control con med) in a time course of up to 24 h. COX-2 mRNA levels were quantified by QPCR (C), COX-2 protein levels were quantified by Western blot (D). Blots were stripped and reprobed for GAPDH. Western blots are representative of 3 independent experiments.

Article Snippet: Recombinant amphiregulin was purchased from R&D Systems.

Techniques: Recombinant, Expressing, Western Blot, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Human airway smooth muscle cells secrete amphiregulin via bradykinin/COX-2/PGE 2 , inducing COX-2, CXCL8, and VEGF expression in airway epithelial cells

doi: 10.1152/ajplung.00390.2014

Figure Lengend Snippet: Amphiregulin blocking antibodies and amphiregulin siRNA decrease COX-2 mRNA accumulation and protein expression in HBEC exposed to BK treated airway smooth muscle cell conditioned medium. HBEC were cultured for 4 h with control HASMC conditioned medium (Control con med), or BK HASMC conditioned medium (BK con med) with either anti-amphiregulin antibody (anti-AR) or control mouse IgG1 antibody (anti-mouse). COX-2 mRNA expression was quantified by QPCR (A) and COX-2 protein by Western blot (B). Western blots were reprobed with antisera to GAPDH. Western blots are representative of 3 independent experiments. HASM cells were transfected with control scrambled siRNA (SC; solid bars) or amphiregulin siRNA (AREG siRNA; open bars) for 48 h and treated with BK for 24 h; the conditioned media were transferred to HBEC cells for 24 h, and COX-2 mRNA accumulation in HBEC was analyzed by QPCR (C). All other measurements represent means ± SE of 3 independent experiments. *P = 0.01–0.05; ***P < 0.001.

Article Snippet: Recombinant amphiregulin was purchased from R&D Systems.

Techniques: Blocking Assay, Expressing, Cell Culture, Control, Western Blot, Transfection

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Human airway smooth muscle cells secrete amphiregulin via bradykinin/COX-2/PGE 2 , inducing COX-2, CXCL8, and VEGF expression in airway epithelial cells

doi: 10.1152/ajplung.00390.2014

Figure Lengend Snippet: The EGFR inhibitor AG1478 blocks amphiregulin and BK conditioned HASMC medium induction of COX-2 mRNA and COX-2 protein accumulation in airway epithelial cells. HBEC were treated with amphiregulin (AR) for 4 h (solid bars) with or without AG1478 (1 × 10−6 M). COX-2 mRNA levels were quantified by QPCR (A). HBEC were treated with 10 ng/ml of AR in a time course of up to 24 h with or without AG1478 (1 × 10−6 M). Total protein extracts were analyzed by Western blot for COX-2 expression (B). HBEC were treated for 4 h with HASMC control conditioned medium (control con med) or BK conditioned (BK con med) smooth muscle medium with or without AG1478 (1 × 10−6 M) followed by QPCR for COX-2 mRNA (C). Total protein extracts from the same treatments at 4 and 8 h were Western blotted for COX-2 protein (D). All blots were reprobed with GAPDH antisera. Western blots are representative of 3 independent experiments. All other measurements represent means ± SE of 3 independent experiments. ***P < 0.001.

Article Snippet: Recombinant amphiregulin was purchased from R&D Systems.

Techniques: Western Blot, Expressing, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Human airway smooth muscle cells secrete amphiregulin via bradykinin/COX-2/PGE 2 , inducing COX-2, CXCL8, and VEGF expression in airway epithelial cells

doi: 10.1152/ajplung.00390.2014

Figure Lengend Snippet: Recombinant amphiregulin and bradykinin conditioned cell culture medium from airway smooth muscle cells can induce VEGF165 mRNA accumulation and VEGF165 secretion from human airway epithelial cells. HASMC conditioned medium induced VEGF165 expression in HBEC is amphiregulin dependent. HBEC were treated with recombinant amphiregulin (10 ng/ml) for 24 h followed by a VEGF165 ELISA of the culture supernatant (A). HBEC were treated with recombinant amphiregulin for 24 h and VEGF165 mRNA accumulation was analyzed by QPCR (B). Cell culture medium from HASMC treated for 24 h with BK (1 × 10−6 M) (BK con med) or a vehicle control (control con med) were added to HBEC for 24 h and the resulting HBEC supernatants and HASMC conditioned medium were analyzed by VEGF165 ELISA, with HASMC conditioned medium VEGF165 levels subtracted from HBEC medium levels (C). HASMC BK conditioned medium and control conditioned media were used to treat HBEC for a time course of up to 24 h, VEGF165 mRNA accumulation was analyzed in HBEC by QPCR (D). HBEC were treated with BK conditioned HASMC medium (BK con med) for 24 h with either anti-amphiregulin blocking antibody (10 μg/ml) (anti-AR) or mouse IgG1 (10 μg/ml) and VEGF165 mRNA levels were analyzed by QPCR (E). HBEC were treated with HASMC control conditioned medium (con med) or HASMC BK con med for 24 h with either anti-AR at 10 μg/ml or mouse IgG1 (anti-mouse) at 10 μg/ml, and secreted VEGF165 protein levels were analyzed by ELISA (F). HASM cells were transfected with control scrambled siRNA (solid bars) or amphiregulin siRNA (open bars) for 48 h and treated with BK for 24 h, the conditioned media were transferred to HBEC cells for 24 h, and VEGF165 mRNA levels were analyzed by QPCR (G). All measurements represent means ± SE of 3 independent experiments. *P = 0.01–0.05; ***P < 0.001.

Article Snippet: Recombinant amphiregulin was purchased from R&D Systems.

Techniques: Recombinant, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Control, Blocking Assay, Transfection

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Human airway smooth muscle cells secrete amphiregulin via bradykinin/COX-2/PGE 2 , inducing COX-2, CXCL8, and VEGF expression in airway epithelial cells

doi: 10.1152/ajplung.00390.2014

Figure Lengend Snippet: Amphiregulin-treated HBEC supernatants stimulate airway smooth muscle amphiregulin mRNA accumulation. Amphiregulin links reciprocal COX-2 in airway epithelial/smooth muscle inflammation. Culture supernatants from HBEC, untreated (HBEC con med) treated with amphiregulin (10 ng/ml) (AR con HBEC med) with indomethacin (1 × 10−6 M) (AR con med + indo), or serum free media only (CM−) for 8 h were then added to HASMC for 4 h, the HASMC RNA were assayed for amphiregulin expression by QPCR (A), and 10 ng/ml of recombinant amphiregulin was added as a control directly to the HASMC (AR direct). All other measurements represent means ± SE of 3 independent experiments. Reciprocal, COX-2-dependent, relationship between HASMC and HBEC: BK-induced HASMC amphiregulin acts as a proinflammatory stimulus resulting in COX-2, CXCL8, and VEGF production from HBEC and HBEC stimulation of the HASMC leading to amphiregulin growth factor production (B). BK1 and BK2, bradykinin receptors 1 and 2; AREG, amphiregulin; EP, EP class G-protein coupled receptors. ***P < 0.001.

Article Snippet: Recombinant amphiregulin was purchased from R&D Systems.

Techniques: Expressing, Recombinant, Control

Journal: bioRxiv

Article Title: DDR1-INDUCED NEUTROPHIL EXTRACELLULAR TRAPS DRIVE PANCREATIC CANCER METASTASIS

doi: 10.1101/2020.08.05.238097

Figure Lengend Snippet: DDR1 Induces liver metastasis in pancreatic cancer. A DDR1 and DDR2 expression were analyzed by western blotting in 2 fibroblasts, 14 primary PDAC cell lines and 2 of metastatic PDAC cell lines. B DDR1 were observed at PDX tumors derived from metastatic or primary human PDAC tumors by immunohistochemical staining using anti- human DDR1 antibody and identified using PE Vectra3. Scale bar, 50 μm. The H-score of DDR1 quantification was displayed as DBA signals by inform software. C Cell invasion assay in MDA-PATC 148 cells with knockdown or re-expression DDR1 were used by matrigel transwell chamber. The invading cells in each chamber were counted under a fluorescence microscope after culture 18 hours, and the average number of cells was calculated based on the number of cells found in six fields per chamber. D , E , F Mice were orthotopically injected with MDA-PATC 148 (control, DDR1-deficient or DDR1-reexpression clones) cells for 9 weeks. D H&E staining of pancreas and liver section. Arrow : region of tumor (10 mice per group), Scale bar, 50 μm. E Tumors size measurement in pancreas. F The numbers of liver-met calculation ( n = 10 for each group).

Article Snippet: Sections were treated with 3% H 2 O 2 , blocked with Fc Receptor blocker (Innovex), and incubated with 1x blocking buffer (5% BSA in PBS) for 1 h. Then the tissue sections were incubated overnight at 4°C with antibodies against human DDR1 (R&D Systems, AF2396), CXCL5 (Abcam, ab9802), Ly6G (BD Pharmingen, 551459) antibodies.

Techniques: Expressing, Western Blot, Derivative Assay, Immunohistochemical staining, Staining, Software, Invasion Assay, Knockdown, Fluorescence, Microscopy, Injection, Control, Clone Assay

Journal: bioRxiv

Article Title: DDR1-INDUCED NEUTROPHIL EXTRACELLULAR TRAPS DRIVE PANCREATIC CANCER METASTASIS

doi: 10.1101/2020.08.05.238097

Figure Lengend Snippet: DDR1 Induces CXCL5 production in pancreatic cancer cells. A chemokine array analysis in cell lysate and supernatant of MDA-PATC 148 cells with knockdown DDR1. B and C MDA-PATC 148 cells with knockdown or re-express DDR1 were treated with collagen I for 3 hours. B CXCL5 mRNA level by using real-time PCR. C Protein level by using ELISA. D overexpressed DDR1 in 5 pancreatic cancer cell lines, upper: DDR1 level were checked by western; middle: CXCL5 level were detected by real-time PCR; bottom: . CXCL5 level were analyzed by ELISA. E , F and G Mice were orthotopically injected with MDA-PATC 148 (control, DDR1-deficient or DDR1-reexpression clones) cells for 9 weeks. E Immunohistochemical staining with anti-DDR1 (upper panel) and anti-CXCL5 (bottom panel) antibodies in pancreas ( n = 10 for each group). F ELISA showed CXCL5 level in plasma harvest from mice ( n = 10 for each group). G Left: FACS by using anti-CD11b and anti-Ly6G antibodies to determine the presence of CD11b + Ly6G + neutrophils infiltration in pancreas; right . The data were collected from three independent experiments ( n = 5 for each group).

Article Snippet: Sections were treated with 3% H 2 O 2 , blocked with Fc Receptor blocker (Innovex), and incubated with 1x blocking buffer (5% BSA in PBS) for 1 h. Then the tissue sections were incubated overnight at 4°C with antibodies against human DDR1 (R&D Systems, AF2396), CXCL5 (Abcam, ab9802), Ly6G (BD Pharmingen, 551459) antibodies.

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Injection, Control, Clone Assay, Immunohistochemical staining, Staining, Clinical Proteomics

Journal: bioRxiv

Article Title: DDR1-INDUCED NEUTROPHIL EXTRACELLULAR TRAPS DRIVE PANCREATIC CANCER METASTASIS

doi: 10.1101/2020.08.05.238097

Figure Lengend Snippet: The correlation of DDR1, CXCL5 and neutrophils infiltration at TMA in PDX tumors. A immunohistochemical staining showed DDR1, CXCL5 and Ly6G + neutrophils infiltration at PDX tumors and identified using PE Vectra3. Scale bar, 50 μm. B, C, D The Pearson correlation showed relationship of DDR1, CXCL5 and Ly6G by using H-score which quantified the DBA signals by inform software.

Article Snippet: Sections were treated with 3% H 2 O 2 , blocked with Fc Receptor blocker (Innovex), and incubated with 1x blocking buffer (5% BSA in PBS) for 1 h. Then the tissue sections were incubated overnight at 4°C with antibodies against human DDR1 (R&D Systems, AF2396), CXCL5 (Abcam, ab9802), Ly6G (BD Pharmingen, 551459) antibodies.

Techniques: Immunohistochemical staining, Staining, Software

Journal: bioRxiv

Article Title: DDR1-INDUCED NEUTROPHIL EXTRACELLULAR TRAPS DRIVE PANCREATIC CANCER METASTASIS

doi: 10.1101/2020.08.05.238097

Figure Lengend Snippet: DDR1-positive pancreatic cancer cells mediated NET formation from neutrophils and enhanced cancer cell invasion. A-G Human neutrophils were co-culture with DDR1-knockdown or re-expression of MDA-PATC 148 or BxPC-3 by matrigel transwell chamber, with or without NADPH oxidase inhibitor, PDA4 inhibitor, NE inhibitor and Dase I treatment, for 18 hours. A and E NET structures were analyzed by immunofluorescence staining using DAPI (blue) , anti-NE ( red) and anti-histone (green) mAbs. Scale bar , 50 μm. B and F The NET quantification is displayed as NET histone area (μm 2 ) /per filed. C Cit-histone H3 expression were analyzed by western blotting. D and G The number of invaded cells analyzed by immunofluorescence staining using DAPI and calculated based on the number of cells found in six fields /per chamber.

Article Snippet: Sections were treated with 3% H 2 O 2 , blocked with Fc Receptor blocker (Innovex), and incubated with 1x blocking buffer (5% BSA in PBS) for 1 h. Then the tissue sections were incubated overnight at 4°C with antibodies against human DDR1 (R&D Systems, AF2396), CXCL5 (Abcam, ab9802), Ly6G (BD Pharmingen, 551459) antibodies.

Techniques: Co-Culture Assay, Knockdown, Expressing, Immunofluorescence, Staining, Western Blot

Journal: bioRxiv

Article Title: DDR1-INDUCED NEUTROPHIL EXTRACELLULAR TRAPS DRIVE PANCREATIC CANCER METASTASIS

doi: 10.1101/2020.08.05.238097

Figure Lengend Snippet: CXCL5 involved in DDR1 mediated NET formation and cancer cell invasion. A-F Human neutrophils were co-culture with DDR1-knockdown or re-expression of MDA-PATC 148 or BxPC-3 by matrigel transwell chamber, with or without anti-CXCL5 neutralized antibody or recombinant CXCL5 treatment, for 18 hours. A NET structures were analyzed by immunofluorescence staining using DAPI (blue) , anti-NE ( red) and anti-histone (green) mAbs. Scale bar , 50 μm. B and C The NET quantification is displayed as NET histone area (μm 2 ) /per filed. D and E Cit-histone H3 expression were analyzed by western blotting. F and G The number of invaded cells were analyzed by immunofluorescence staining using DAPI and calculated based on the number of cells found in six fields/per chamber. H Neutrophils Elastase activity were showed in human neutrophils with CCM treatment for 18 hours. G The number of invaded cells were showed in MDA-PATC 148 cells with heat-treated NCCM treatment for 18 hours.

Article Snippet: Sections were treated with 3% H 2 O 2 , blocked with Fc Receptor blocker (Innovex), and incubated with 1x blocking buffer (5% BSA in PBS) for 1 h. Then the tissue sections were incubated overnight at 4°C with antibodies against human DDR1 (R&D Systems, AF2396), CXCL5 (Abcam, ab9802), Ly6G (BD Pharmingen, 551459) antibodies.

Techniques: Co-Culture Assay, Knockdown, Expressing, Recombinant, Immunofluorescence, Staining, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: DDR1-INDUCED NEUTROPHIL EXTRACELLULAR TRAPS DRIVE PANCREATIC CANCER METASTASIS

doi: 10.1101/2020.08.05.238097

Figure Lengend Snippet: The correlation of DDR1, CXCL5 and NET-like structure in PADC patient samples. A upper and middle panel: immunohistochemical staining showed DDR1, CXCL5 expression in PDAC patient samples and identified using PE Vectra3. Scale bar, 3mm and 50 μm. bottom panel: NET-like structures were analyzed by immunofluorescence staining using DAPI (blue) , anti-CK19 (white), anti-MPO (green) , anti-citrullinated histone H3 ( red) mAbs in PDAC patient samples. Scale bar , 20 μm. B The Pearson correlation showed relationship of DDR1 and CXCL5 by using H-score which quantified the DBA signals by inform software.

Article Snippet: Sections were treated with 3% H 2 O 2 , blocked with Fc Receptor blocker (Innovex), and incubated with 1x blocking buffer (5% BSA in PBS) for 1 h. Then the tissue sections were incubated overnight at 4°C with antibodies against human DDR1 (R&D Systems, AF2396), CXCL5 (Abcam, ab9802), Ly6G (BD Pharmingen, 551459) antibodies.

Techniques: Immunohistochemical staining, Staining, Expressing, Immunofluorescence, Software

Journal: bioRxiv

Article Title: DDR1-INDUCED NEUTROPHIL EXTRACELLULAR TRAPS DRIVE PANCREATIC CANCER METASTASIS

doi: 10.1101/2020.08.05.238097

Figure Lengend Snippet: PKCq-SYK-NFkB pathway involved in DDR1 induced CXCL5 production, NET formation form neutrophils and enhanced cancer cell invasion. A qPCR results were used to quantify enrichment of NF-kB P65 at the CXCL5 promoter using chromatin immunoprecipitation (ChIP) assay in MDA-PATC 148 cells with DDR1 knockdown. B activated NF-kB were analyzed by western blotting using nuclear fraction in MDA-PATC 148 cells with DDR1 knockdown. C Phospho-NF-kB P65, phospho-PKCq and phospho-SYK were analyzed by western blotting in MDA-PATC 148 cells with DDR1 knockdown. D and E CXCL5 level were analyzed by ELISA D in MDA-PATC 148 cells with IkB superrepressor mutation. E in MDA-PATC 148 and BxPC-3 cells with or without SYK inhibitor and PKC inhibitor pretreatment. F Phospho-NF-kB P65, phospho-PKCq and phospho-SYK were analyzed by western blotting in MDA-PATC 148 cells with or without SYK inhibitor and PKC inhibitor pretreatment. G and H NET structures were analyzed by immunofluorescence staining using DAPI (blue) , anti-NE ( red) and anti-histone (green) mAbs G in MDA-PATC 148 cells with IkB superrepressor mutation. H in MDA-PATC 148 cells with or without SYK inhibitor and PKC inhibitor pretreatment.. Scale bar , 50 μm. The NET quantification is displayed as NET histone area (μm 2 ) /per filed. I and J The number of invaded cells were analyzed by immunofluorescence staining using DAPI and calculated based on the number of cells found in six fields /per chamber. I in MDA-PATC 148 cells with NCCM from MDA-PATC 148 cells with IkB superrepressor mutation/neutrophils/collagen I, treatment for 18 hours. J in MDA-PATC 148 cells with NCCM from MDA-PATC 148/ collagenl/SYK or PKC inhibitor, treatment for 18 hours.

Article Snippet: Sections were treated with 3% H 2 O 2 , blocked with Fc Receptor blocker (Innovex), and incubated with 1x blocking buffer (5% BSA in PBS) for 1 h. Then the tissue sections were incubated overnight at 4°C with antibodies against human DDR1 (R&D Systems, AF2396), CXCL5 (Abcam, ab9802), Ly6G (BD Pharmingen, 551459) antibodies.

Techniques: Chromatin Immunoprecipitation, Knockdown, Western Blot, Enzyme-linked Immunosorbent Assay, Mutagenesis, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: DDR1-INDUCED NEUTROPHIL EXTRACELLULAR TRAPS DRIVE PANCREATIC CANCER METASTASIS

doi: 10.1101/2020.08.05.238097

Figure Lengend Snippet: 7rh treatment reduced NET formation through inhibition of DDR1-PKCq-SYK-CXCL5 axis and reduced cancer cell invasion. A and B MDA-PATC 148 cells and BxPC-3 were pretreated with 7rh for 30 min and then with collagen I for 3 hours. A Phospho-NF-kB P65, phospho-PKCq and phospho-SYK were analyzed by western blotting. B CXCL5 level were analyzed by ELISA. C-F human neutrophils were co-culture with MDA-PATC 148 and BxPC-3 cells by matrigel transwell chamber for 18 hours. C NET structures were analyzed by immunofluorescence staining using DAPI (blue) , anti-NE (red) and anti-histone (green) mAbs. Scale bar , 50 μm. D The NET quantification is displayed as NET histone area (μm 2 ) /per filed. E Cit-histone H3 expression were analyzed by western blotting. F The number of invaded cells analyzed by immunofluorescence staining using DAPI and calculated based on the number of cells found in six fields /per chamber.

Article Snippet: Sections were treated with 3% H 2 O 2 , blocked with Fc Receptor blocker (Innovex), and incubated with 1x blocking buffer (5% BSA in PBS) for 1 h. Then the tissue sections were incubated overnight at 4°C with antibodies against human DDR1 (R&D Systems, AF2396), CXCL5 (Abcam, ab9802), Ly6G (BD Pharmingen, 551459) antibodies.

Techniques: Inhibition, Western Blot, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Immunofluorescence, Staining, Expressing