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: Breast Cancer Research : BCR

Article Title: Vascular endothelial growth factor C promotes breast cancer progression via a novel antioxidant mechanism that involves regulation of superoxide dismutase 3

doi: 10.1186/s13058-014-0462-2

Figure Lengend Snippet: Vascular endothelial growth factor C regulates Sod3 expression in 66 cl4 mammary carcinoma cells. (A) Total RNA from 66 cl4-scramble and 66 cl4-VEGF-C KD2 cells was converted to cDNA and used to perform a mouse oxidative stress PCR array. Nine candidate genes of the eighty-four examined were identified in the PCR array with more than a twofold change in response to VEGF-C KD. (B) Sod3 mRNA expression was determined using a real-time PCR SYBR Green assay on 66 cl4-scram and 66 cl4-VEGF-C KD1 and KD2 cells (top). Western blot analysis of Sod3 expression in 66 cl4-scram and VEGF-C KD1 and KD2 cells (bottom). (C) Sod3 mRNA expression was determined by real-time PCR SYBR Green assay on three pairs of 66 cl4-scram and VEGF-C KD1 or KD2 tumors (each pair was derived from the same animal).

Article Snippet: VEGF-C and Sod3 antibodies (sc-25783 and sc-67089, respectively) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, SYBR Green Assay, Western Blot, Derivative Assay

Journal: Breast Cancer Research : BCR

Article Title: Vascular endothelial growth factor C promotes breast cancer progression via a novel antioxidant mechanism that involves regulation of superoxide dismutase 3

doi: 10.1186/s13058-014-0462-2

Figure Lengend Snippet: Restoration of Sod3 in 66 cl4-VEGF-C KD cells partially rescues resistance to oxidative stress and tumor progression. (A) Expression of Sod3 in 66 cl4-VEGF-C KD cells (KD2 in Figure ). Empty vector was also introduced into 66 cl4-scram and 66 cl4-VEGF-C KD2 cells as a control. Sod3 expression was assessed in each cell line using a real-time PCR SYBR Green assay (top). Expression of secreted Sod3 in the media of 66 cl4-scram, 66 cl4-VEGF-C KD and 66 cl4-VEGF-C KD + Sod3 cells was measured by Western blot analysis (bottom). (B) Flow cytometry was performed to measure cell death induced by H 2 O 2 under each condition shown. Three independent experiments were performed, and the data were combined for quantitation. (C) Cells from the 66 cl4-scram, VEGF-C KD and VEGF-C KD + Sod3 lines were injected into the fourth mammary fat pad of female BALB/c mice. Tumor growth in the mice was measured using calipers and calculated using the formula V = 1/2(W)(W)(L) (top). A representative picture of tumors from each group shows that restoration of Sod3 in VEGF-C KD cells partially rescues the size of tumors compared to scramble control tumors (bottom). (D) Representative in vivo image of 66 cl4-scram, VEGF-C KD and VEGF-C KD + Sod3 groups at day 60 after injection (top). Quantitation of bioluminescence imaging (in photons per second) emanating from the region surrounding the lungs (bottom). Mice that did not develop primary tumors were excluded from the quantitation. (E) Incidence of tumor formation and metastasis in the groups of mice injected with 66 cl4-scram, VEGF-C KD or VEGF-C KD + Sod3 cells. Restoration of Sod3 expression in the VEGF-C KD cells increased the number of mice that developed primary tumors and metastases, although not to the levels observed in the scramble control group. Fisher's exact test (two-sided) results indicated a significant increase in the number of mice that developed metastases when Sod3 expression was restored in the VEGF-C KD tumors.

Article Snippet: VEGF-C and Sod3 antibodies (sc-25783 and sc-67089, respectively) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction, SYBR Green Assay, Western Blot, Flow Cytometry, Quantitation Assay, Injection, In Vivo, Imaging

Journal: Breast Cancer Research : BCR

Article Title: Vascular endothelial growth factor C promotes breast cancer progression via a novel antioxidant mechanism that involves regulation of superoxide dismutase 3

doi: 10.1186/s13058-014-0462-2

Figure Lengend Snippet: Neuropilin 2-knockdown in 66 cl4 mouse mammary carcinoma cells decreases superoxide dismutase 3 expression and sensitizes cells to oxidative stress. (A) NRP2 and VEGFR3 mRNA expression in human breast cancer cell lines. Box plots of NRP2 (left) or VEGFR3 ( FLT4 ) (right) gene expression across 51 previously reported breast cancer cell lines grouped into basal A, basal B and luminal subgroups. NRP2 and VEGFR3 gene expression in human breast cancer cell lines was assessed using GOBO . (B) Two different short-hairpin RNAs (shRNAs 1 and 2) were used to knock down neuropilin 2 (Nrp2) in 66 cl4 cells. Expression of Nrp2 in 66 cl4-scram and 66 cl4-Nrp2 knockdown (KD) cells was determined by Western blot analysis (left). Decreased expression of superoxide dismutase 3 (Sod3) was observed in 66 cl4-Nrp2 KD cells compared to 66 cl4-scram cells (right). Whole-cell lysates (top) or media (bottom) from 66 cl4-scram and 66 cl4-Nrp2 KD cells were collected for the detection of Sod3 by Western blotting. (C) Viability of 66 cl4-scram and 66 cl4-Nrp2 KD cells treated with increasing doses of H 2 O 2 . A CellTiter-Glo assay was used to measure viable cells. Three independent experiments were performed and combined for quantification.

Article Snippet: VEGF-C and Sod3 antibodies (sc-25783 and sc-67089, respectively) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Knockdown, Expressing, Gene Expression, Western Blot, Glo Assay

Journal: Breast Cancer Research : BCR

Article Title: Vascular endothelial growth factor C promotes breast cancer progression via a novel antioxidant mechanism that involves regulation of superoxide dismutase 3

doi: 10.1186/s13058-014-0462-2

Figure Lengend Snippet: Expression of VEGFC and SOD3 in human cancers. (A) VEGFC and SOD3 expression values were retrieved from an Oncomine microarray data set (as indicated in the figure) and were plotted by expression value. Statistical analysis was performed using Pearson r correlation (two-tailed). (B) Proposed model for the function of vascular endothelial growth factor C (VEGF-C) in breast cancer progression. Expression of VEGF-C in a subset of tumor cells confers the ability to resist oxidative stress generated during tumor growth, and this ability is partially mediated by Sod3. However, VEGF-C mediates other pathways that are important in conferring resistance to chemotherapies, thus contributing to tumor recurrence. Blocking VEGF-C signaling would therefore be expected to sensitize breast cancers to chemotherapies that induce oxidative stress, to contribute to a reduction in tumor-initiating cells (TICs) and to decrease lymphangiogenesis, thus likely improving survival and prevent recurrence.

Article Snippet: VEGF-C and Sod3 antibodies (sc-25783 and sc-67089, respectively) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Microarray, Two Tailed Test, Generated, Blocking Assay

Journal:

Article Title: Laminin-511 is an epithelial message promoting dermal papilla development and function during early hair morphogenesis

doi: 10.1101/gad.1689908

Figure Lengend Snippet: Cooperation of laminin-511, Shh, and PDGFrα in mesenchymal development. (A, right) Real-time RT–PCR-evaluated mRNA levels of DP markers Cspg2 (versican), nestin, nexin, snail2, and Wnt5a in E16.5 wild-type (WT) and lama5−/− (null) skin. (Left) Real-time RT–PCR evaluated the mRNA level of major developing DP cell surface receptors, Fgfr2, Pdgfrα, and Tgfrb1. (B) Functional blocking anti-PDGFrα antibody-treated lama5+/− skin. E16.5 lama5+/− skin was incubated overnight in anti-PDGFrα antibody or control goat IgG and analyzed 9 d following grafting to nude mice. Quantification of hair follicle formation is shown in right panel. (C) IF microscopy of E16.5 wild-type skin before (left panel) or after dispase digestion (middle and right panels), using CD133, type VII collagen (ColVII), and laminin-511 (Lm10) antibodies. Note loss of laminin-511 but preservation of CD133-expressing DP cells following dispase digestion. (DP) Developing DP; (BMZ) basement membrane zone marked by white dots; (Epi) epidermis; (Der) dermis. (D) Quantitative-PCR-evaluated induction of noggin mRNA expression in dispase-treated E16.5 mesenchyme under five conditions: laminin-111 + PDGF-AA, laminin-511 + PDGF-AA, laminin-111 + Shh + PDGF-AA, laminin-511 + Shh + PDGF-AA, and laminin-511 + PDGF-AA.

Article Snippet: The following antibodies were used: anti-laminin α5 chain rabbit pAb ( Patton et al. 1999 ); rat anti-e-cadherin and p-cadherin mAbs (Zymed); rabbit anti-collagen VII pAb ( Ortiz-Urda et al. 2005 ); rabbit anti-β-catenin, PECAM, PDGFRα, and p-PDGFRα Tyr720/754 and goat anti-Lef-1 pAbs (Santa Cruz Biotechnologies); rat anti-noggin mAb RP57-16 (Regeneron); rat anti-perlecan mAb (Chemicon); rabbit anti-smad 1/5/8 pAb (Cell Signaling); hamster anti-integrin β1 blocking IgM mAb ( Mendrick and Kelly 1993 ) (Ha2/5; BD Pharmingen); hamster IgM (G235-1; BD Biosciences); β3 integrin blocking antibody ( Ashkar et al. 2000 ) (2C9.G2; BD Biosciences); rat anti-CD133 mAb (eBioscience); rabbit anti-keratin 17 pAb (DAKO); rabbit anti-Ki67 pAb (Neomarkers); mouse anti-acetylated α-tubulin mAb and rabbit anti-β-actin pAb (Sigma); and rabbit anti-polaris pAb ( Taulman et al. 2001 ).

Techniques: Quantitative RT-PCR, Functional Assay, Blocking Assay, Incubation, Control, Microscopy, Preserving, Expressing, Membrane, Real-time Polymerase Chain Reaction

Journal:

Article Title: Laminin-511 is an epithelial message promoting dermal papilla development and function during early hair morphogenesis

doi: 10.1101/gad.1689908

Figure Lengend Snippet: Schematic of laminin-511’s role in hair development. Laminin-511 is secreted by the follicular epithelium into basement membrane separating follicular epithelium and DP, where it interacts with β1 integrin receptors on the DP cell surface. This leads to primary cilia development, which in turn amplifies Shh signaling, through Shh effector proteins including patched (PTCH), smoothened (SMO), and Gli. Epithelilal derived PDGF interacts with PDGFRα on DP primary cilia, which, together with Shh, promotes the expression and secretion of noggin in mesenchymal cells. Noggin promotes epithelial BMP inhibition leading to Lef-1 expression, which, coupled with Wnt/β-catenin-induced activation, drives further Shh expression. Red arrows indicate a Shh-noggin-positive amplification loop that laminin-511 facilitates.

Article Snippet: The following antibodies were used: anti-laminin α5 chain rabbit pAb ( Patton et al. 1999 ); rat anti-e-cadherin and p-cadherin mAbs (Zymed); rabbit anti-collagen VII pAb ( Ortiz-Urda et al. 2005 ); rabbit anti-β-catenin, PECAM, PDGFRα, and p-PDGFRα Tyr720/754 and goat anti-Lef-1 pAbs (Santa Cruz Biotechnologies); rat anti-noggin mAb RP57-16 (Regeneron); rat anti-perlecan mAb (Chemicon); rabbit anti-smad 1/5/8 pAb (Cell Signaling); hamster anti-integrin β1 blocking IgM mAb ( Mendrick and Kelly 1993 ) (Ha2/5; BD Pharmingen); hamster IgM (G235-1; BD Biosciences); β3 integrin blocking antibody ( Ashkar et al. 2000 ) (2C9.G2; BD Biosciences); rat anti-CD133 mAb (eBioscience); rabbit anti-keratin 17 pAb (DAKO); rabbit anti-Ki67 pAb (Neomarkers); mouse anti-acetylated α-tubulin mAb and rabbit anti-β-actin pAb (Sigma); and rabbit anti-polaris pAb ( Taulman et al. 2001 ).

Techniques: Membrane, Derivative Assay, Expressing, Inhibition, Activation Assay, Amplification

Journal: Nature Communications

Article Title: PD-1/PD-L1 checkpoint blockade harnesses monocyte-derived macrophages to combat cognitive impairment in a tauopathy mouse model

doi: 10.1038/s41467-019-08352-5

Figure Lengend Snippet: Early treatment with anti-PD-1 antibody delays cognitive loss and rescues neurons in 5XFAD mice. 5XFAD mice were treated with either PD-1–specific antibody or IgG control starting at an average age of 3.5 months, and continued once a month (total of three injections). Experimental design is presented in ( a ). Black arrows indicate time points of treatment. Time points of cognitive testing are also indicated. b RAWM performance of 5.5-month-old anti-PD-1-treated mice ( n = 7), control antibody (IgG)-treated 5XFAD mice ( n = 9), and wild-type (WT) animals ( n = 8). c RAWM performance of the same mice one month later (6.5-month-old). (Two-way repeated-measures ANOVA and Dunnett’s post-hoc test for multiple comparisons between the two treated groups.) d Comparison of the performance of anti-PD-1 and IgG-treated groups at 5.5 and 6.5 months; values indicating the number of errors for each mouse, taken from the last measurement on the second day of the test. e , f Representative Cresyl violet-stained images of the cortex and subiculum, and g , h quantitative analysis of IgG-treated 5XFAD mice ( n = 9) as compared to anti-PD-1-treated 5XFAD mice ( n = 7); the WT ( n = 7) controls that were analyzed were used to calculate the percentage of surviving neurons. i , j Images and quantitative analysis of Neu-N + neurons in the subiculum of anti-PD-1-treated 5XFAD mice ( n = 7), and IgG-treated 5XFAD mice ( n = 9), respectively; the WT controls ( n = 7) that were analyzed were used to calculate the percentage of surviving neurons. Neuronal survival is presented relative to the number of the pyramidal neurons in the age-matched WT littermates. k , l Representative immunofluorescence images and quantitative analysis of Neu-N + neurons (green) in the subiculum, showing increased immunoreactivity of Activated Caspase-3 (red) in IgG-treated 5XFAD mice ( n = 9), in comparison to anti-PD-1-treated 5XFAD mice ( n = 7) (Student’s t test). Scale bars, 50 μm ( e , f ), 100 μm ( i ), and 25 μm ( k ). Data are presented as mean ± s.e.m.; * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For PD-1 blockade, PD-1-specific blocking antibody (anti-PD-1; rat IgG2a isotype; clone RPM1-14; BIOXCELL) and isotype control (anti-trinitrophenol; clone 2A3, BIOXCELL) were administered i.p. For PD-L1 blockade, throughout the entire study, PD-L1-blocking antibody directed to mouse PD-L1 was used (anti-PD-L1 antibody; rat IgG2b isotype; clone 10F.9G2; BIOXCELL) and isotype control (anti-keyhole limpet hemocyanin; clone LTF-2; BIOXCELL) was administered i.p.

Techniques: Control, Comparison, Staining, Immunofluorescence

Journal: Nature Communications

Article Title: PD-1/PD-L1 checkpoint blockade harnesses monocyte-derived macrophages to combat cognitive impairment in a tauopathy mouse model

doi: 10.1038/s41467-019-08352-5

Figure Lengend Snippet: Blockade of PD-L1 overcomes loss of cognitive performance and reduces cerebral pathology in 5XFAD mice. 5XFAD mice were treated with either anti-PD-1, anti-PD-L1 or IgG. Experimental design is presented in a . b RAWM performance of WT littermates ( n = 10), and of mice treated with 0.1 mg/mouse ( n = 8), 0.5 mg/mouse ( n = 9), or 1.5 mg/mouse ( n = 9), of anti-PD-L1 antibody, or with 1.5 mg/mouse IgG ( n = 10). c RAWM performance of WT ( n = 19), and of 5XFAD mice treated with either 0.5 mg of anti-PD-1 antibody ( n = 14), or with 0.5 mg of anti-PD-L1 antibody ( n = 7); IgG control antibody ( n = 6 IgG2a; n = 9 IgG2b). Two-way repeated-measures ANOVA and Dunnett’s post-hoc test for multiple comparisons between each anti-PD-1-treated group and the IgG-treated groups (* P < 0.05, ** P < 0.01, *** P < 0.001) and between Anti-PD-L1 and IgG-treated groups ( # P < 0.05, ## P < 0.01, ### P < 0.001) . Results shown are pooled from two experiments. d – g Analysis of disease pathology from a single experiment; one hemisphere of each brain was taken for histology and one for quantitative measurements of mRNA. d Representative immunofluorescence images of brains stained for Aβ (in red), GFAP (in green) and DAPI nuclear staining (scale bars, 100 μm). e , f Quantitative analysis of Aβ in anti-PD-1-treated mice ( n = 9), anti-PD-L1-treated mice ( n = 7), and IgG-treated ( n = 9) mice. g GFAP in anti-PD-1-treated ( n = 9), anti-PD-L1-treated mice ( n = 7), IgG-treated ( n = 8) mice, and WT ( n = 6). Plaque area and numbers were quantified in the dentate gyrus (DG) and in the cerebral cortex (layer V), and GFAP immunoreactivity was measured in the hippocampus (one-way ANOVA and Fisher’s exact test). h , i Representative immunofluorescence images, and quantitative analysis of synaptophysin in the hippocampal CA3 and in the DG, in the brains of anti-PD-1-treated mice ( n = 6), anti-PD-L1-treated 5XFAD mice ( n = 7), and IgG-treated mice ( n = 9) (one-way ANOVA and Fisher’s exact test). Scale bars, 50 μm ( h ). j , k RT-qPCR, in hippocampal tissue isolated from mice treated with IgG control ( n = 5), anti-PD-1 antibody ( n = 5), or anti-PD-L1 antibody ( n = 5) (one-way ANOVA and Fisher’s exact test). l Experimental design of the mice studied in m and n . m RAWM performance before ( n = 13) treatment, and of the same mice ( n ) following their treatment either with 1.5 mg/mouse ( n = 7) anti-PD-L1, or with IgG control ( n = 6). WT littermates were tested twice ( n = 7) (two-way repeated-measures ANOVA and Dunnett’s post-hoc test for multiple comparisons between anti-PD-L1 IgG-treated groups). b , c , e – g , i – k mean ± s.e.m.; * P < 0.05, ** P < 0.01, *** P < 0.001. DAPI: 4′,6-diamidino-2-phenylindole, RT-qPCR: real-time quantitative PCR

Article Snippet: For PD-1 blockade, PD-1-specific blocking antibody (anti-PD-1; rat IgG2a isotype; clone RPM1-14; BIOXCELL) and isotype control (anti-trinitrophenol; clone 2A3, BIOXCELL) were administered i.p. For PD-L1 blockade, throughout the entire study, PD-L1-blocking antibody directed to mouse PD-L1 was used (anti-PD-L1 antibody; rat IgG2b isotype; clone 10F.9G2; BIOXCELL) and isotype control (anti-keyhole limpet hemocyanin; clone LTF-2; BIOXCELL) was administered i.p.

Techniques: Control, Immunofluorescence, Staining, Quantitative RT-PCR, Isolation, Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: PD-1/PD-L1 checkpoint blockade harnesses monocyte-derived macrophages to combat cognitive impairment in a tauopathy mouse model

doi: 10.1038/s41467-019-08352-5

Figure Lengend Snippet: Blockade of the PD-1/PD-L1 axis in DM-hTAU mice reduces cognitive deficits and cerebral pathology. DM-hTAU mice (8-month-old) were treated with anti-PD antibody, anti-PD-L1 antibody, or IgG control antibody (0.5 mg/mouse); experimental design is presented in a . b Representative heat-map plots of the time spent in the distinct arms, by the three tested groups. c Effect of anti-PD-1/PD-L1 antibodies on performance in T-maze. DM-hTAU mice treated with anti-PD-1 antibody ( n = 10) or anti-PD-L1 antibody ( n = 7) exhibited increased novel arm preference, compared to IgG controls ( n = 11); age-matched WT littermates ( n = 10) were also tested. d Y-maze performance of male DM-hTAU mice treated with anti-PD-1 antibody ( n = 6), anti-PD-L1 antibody ( n = 4), or IgG isotype control ( n = 6), and age-matched WT ( n = 5). e – h Hippocampi were isolated from one hemisphere of the brains of the animals in d for quantitative RT-qPCR; IgG control ( n = 6), anti-PD-1 antibody ( n = 6), or anti-PD-L1antibody ( n = 4) (one-way ANOVA and Fisher exact test). i Dose-dependent effect of anti-PD-L1 antibody on T-maze performance of DM-hTAU mice (male and female; 9-month-old) (0.1 mg/mouse ( n = 7), 0.5 mg/mouse ( n = 10), or 1.5 mg/mouse ( n = 9), and IgG control at 1.5 mg/mouse ( n = 10)), WT ( n = 10). Results are expressed as the time spent in the novel arm (one-way ANOVA and Fisher’s exact test). j Representative images of IL-1β immunoreactivity in mice treated with anti-PD-L1 antibody or with IgG control. k Representative orthogonal projection of confocal z -axis stacks, showing colocalization of IL-1β (green) with GFAP + astrocytes (red), but not with IBA-1 + microglia/macrophages (white), in the dentate gyrus. Cell nuclei (DAPI, blue). Scale bar, 100 μm. l Immunoassay of hippocampal IL-1β protein levels in untreated mice ( n = 3), mice treated with anti-PD-L1 antibody ( n = 6) or IgG ( n = 6) and WT littermates ( n = 6), normalized to mg protein in each homogenate. m Correlation between cognitive performance in the T-maze, shown in Supplementary Fig. , and IL-1β protein levels for all groups. n T-maze of DM-hTAU mice (9-month-old, male and female) treated with either 1.5 mg of anti-mouse PD-L1 antibody ( n = 11), or with anti-human PD-L1 antibody ( n = 13), 1.5 mg/mouse of IgG control ( n = 14), and compared to untreated DM-hTAU mice ( n = 12) and WT littermates ( n = 7). One-way ANOVA and Fisher exact test. In all panels, error bars represent mean ± s.e.m.; * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For PD-1 blockade, PD-1-specific blocking antibody (anti-PD-1; rat IgG2a isotype; clone RPM1-14; BIOXCELL) and isotype control (anti-trinitrophenol; clone 2A3, BIOXCELL) were administered i.p. For PD-L1 blockade, throughout the entire study, PD-L1-blocking antibody directed to mouse PD-L1 was used (anti-PD-L1 antibody; rat IgG2b isotype; clone 10F.9G2; BIOXCELL) and isotype control (anti-keyhole limpet hemocyanin; clone LTF-2; BIOXCELL) was administered i.p.

Techniques: Control, Isolation, Quantitative RT-PCR

Journal: Nature Communications

Article Title: PD-1/PD-L1 checkpoint blockade harnesses monocyte-derived macrophages to combat cognitive impairment in a tauopathy mouse model

doi: 10.1038/s41467-019-08352-5

Figure Lengend Snippet: Blocking PD-1/PD-L1 pathway reduces tau hyperphosphorylation in DM-tau mice. Immunostaining of tau-phosphorylation in brains of 8-month-old male DM-hTAU mice excised 1 month after treatment with anti-PD-1, anti-PD-L1, or isotype-matched control antibody (IgG). The analyzed mice were taken from the experiment in Fig. ( a – d ). a Representative immunofluorescence images, and b quantitative analysis of AT-100. Immunoreactivity of AT-100 was measured in the hippocampal CA1 and CA3 regions of DM-hTAU + IgG ( n = 11), DM- hTAU + anti-PD-1 ( n = 10), and DM-tau + anti-PD-L1 ( n = 4). c , d Representative immunofluorescence images, and quantitative analysis of AT-180 staining. Immunoreactivity of AT-180 was measured in the hippocampal CA1 and CA3 regions of the following treatment groups DM-hTAU + IgG ( n = 11), DM-hTAU + anti-PD-1 ( n = 10), DM-hTAU + anti-PD-L1 ( n = 4) (one-way ANOVA and Fisher’s exact test). Scale bars, 100 μm ( a , c ). e Hippocampal and cortical levels of aggregated tau, measured in the protein extracts of the mice shown in Supplementary Fig. . Results are normalized to mg protein, and are expressed relative to untreated DM-hTAU; n = 6 per group (Student’s t test). f , g Representative Cresyl violet-stained images of the cortex and quantification of cortical layer five neurons, of male and female DM-hTAU mice (average cohorts aged 9 months) treated with anti-PD-L1 ( n = 6) compared to IgG control group ( n = 7); the wild-type ( n = 6) controls that were analyzed were used to determine the percentage of surviving neurons. (Student’s t test). Scale bars, 100 μm. In all panels, error bars represent mean ± s.e.m.; * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For PD-1 blockade, PD-1-specific blocking antibody (anti-PD-1; rat IgG2a isotype; clone RPM1-14; BIOXCELL) and isotype control (anti-trinitrophenol; clone 2A3, BIOXCELL) were administered i.p. For PD-L1 blockade, throughout the entire study, PD-L1-blocking antibody directed to mouse PD-L1 was used (anti-PD-L1 antibody; rat IgG2b isotype; clone 10F.9G2; BIOXCELL) and isotype control (anti-keyhole limpet hemocyanin; clone LTF-2; BIOXCELL) was administered i.p.

Techniques: Blocking Assay, Immunostaining, Phospho-proteomics, Control, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: PD-1/PD-L1 checkpoint blockade harnesses monocyte-derived macrophages to combat cognitive impairment in a tauopathy mouse model

doi: 10.1038/s41467-019-08352-5

Figure Lengend Snippet: Monocyte-derived macrophages uniquely affect disease modification in PD-L1 blockade in DM-hTAU mice. a , b Flow cytometry of splenocytes, CD44 + CD62L low effector memory T (T EM ) cells, versus CD44 + CD62L high central memory T (T CM ) cells in DM-hTAU mice, treated with 0.5 mg of anti-PD-L1 ( n = 10) or IgG ( n = 11) (one-way ANOVA, Fisher’s exact test). c , d Flow cytometry of brains from anti-PD-L1-treated mice ( n = 10), and IgG-treated mice ( n = 16) analyzed for CD45 high CD11b high , pooled from two experiments. e – g Repeated experiment as in a , b using GFP-BM-chimeric DM-hTAU mice. e Flow cytometry of GFP-labeled cells gated from CD45 high CD11b high cells, expressing Ly6C. f Quantitation of the number of GFP + CD45 high CD11b high cells in anti-PD-L1 ( n = 4), relative to IgG-treated mice ( n = 6). g Representative projections of confocal z -axis stacks, showing colocalization of GFP + cells (green) with IBA-1 (blue), in the cortex of DM-hTAU GFP/+ mice, treated with anti-PD-L1 antibody (arrowheads). Scale bar: 100 μm. h Representative confocal z -axis stacks, showing colocalization of GFP + cells (green), IBA-1 (blue), and IL-10 (red) in the brains of anti-PD-L1-treated DM-hTAU GFP/+ mice. Scale bar: 50 μm. i Sorted CD45 high CD11b high from DM-hTAU mice treated with anti-PD-L1, analyzed by single-cell RNASeq (Supplementary Fig. ). tSNE plot depicting 899 cells. Clusters indicated by color and number. j Average Unique Molecular Identifier counts for selected genes across the 12 clusters. k , l Representative projections of confocal z -axis stacks, showing colocalization of GFP + cells (green) with MSR1 (red) and IBA-1 (blue) in the cortex ( k ), and of GFP + cells (green) with MSR1 (red) in the hippocampus of DM-hTAU GFP/+ mice treated with anti-PD-L1 antibody ( l ). Scale bars: 25 and 50 μm. m , n BM-chimeric DM-hTAU and WT mice (male and female) prepared using WT or MSR1 −/− mice as BM donors. m T-maze task, 2 weeks after BM transplant, of WT > WT ( n = 4), MSR1 −/− > WT ( n = 5), WT > DM-hTAU ( n = 8) and MSR1 −/− > DM-hTAU ( n = 8) chimeric mice. n The same mice were treated after the behavioral assessment in m with 1.5 mg of anti-PD-L1 antibody or IgG control antibody, and were tested again 1 month later for their performance in T-maze; nonchimeric IgG-treated DM-hTAU littermates were used as additional controls. Improved performance of WT > DM-hTAU treated with anti-PD-L1 ( n = 5) versus IgG-treated WT > DM-hTAU ( n = 3) and IgG-treated nonchimeric DM-hTAU mice ( n = 6). MSR1 −/− > DM-hTAU mice failed to show beneficial effect following treatment with anti-PD-L1 ( n = 5), performing similarly to MSR1 −/− > DM-hTAU treated with IgG ( n = 3). In all panels, error bars represent mean ± s.e.m.; * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way ANOVA and Fisherʼs exact test)

Article Snippet: For PD-1 blockade, PD-1-specific blocking antibody (anti-PD-1; rat IgG2a isotype; clone RPM1-14; BIOXCELL) and isotype control (anti-trinitrophenol; clone 2A3, BIOXCELL) were administered i.p. For PD-L1 blockade, throughout the entire study, PD-L1-blocking antibody directed to mouse PD-L1 was used (anti-PD-L1 antibody; rat IgG2b isotype; clone 10F.9G2; BIOXCELL) and isotype control (anti-keyhole limpet hemocyanin; clone LTF-2; BIOXCELL) was administered i.p.

Techniques: Derivative Assay, Modification, Flow Cytometry, Labeling, Expressing, Quantitation Assay, Control