Journal: Oncotarget

Article Title: Role of BCL9L in transforming growth factor-β (TGF-β)-induced epithelial-to-mesenchymal-transition (EMT) and metastasis of pancreatic cancer

doi: 10.18632/oncotarget.12455

Figure Lengend Snippet: E-cadherin, α-catenin, BCL9L and α-tubulin (loading control) protein levels were determined in stably shRNA-transduced Panc-1 cells ( A ) as well as S2-007, S2-028 and Panc-1 cells transiently transfected with siRNA ( B ). Knockdown of BCL9L by both shRNA (shBCL9L) and siRNA (siRNA1, siRNA2, siRNA pool) induced an up-regulation of E-cadherin protein in all cell lines compared to controls (Vector control, siCTR). Shown are representative images of 3 western blot experiments. qRT-PCR analyses of BCL9L and E-cadherin mRNA expression levels in Panc-1 ( C ), S2-007 ( D ), and S2-028 ( E ) cells transfected with control siRNA or BCL9L-targeting siRNA showed a significant up-regulation of E-cadherin expression in response to knockdown of BCL9L. Data represent mean ± s.e.m. of three independent experiments. ** p ≤ 0.01, *** p ≤ 0.001 (Student's t-test ). ( F ) Untransduced (UNT), Vector control and BCL9L-shRNA transduced Panc-1 cells were treated or not with 10 μM CHIR and subjected to immunofluorescence co-staining of E-cadherin (left panel; visualized by Alexa 555-tagged secondary antibody) and β-catenin (middle panel; visualized by Alexa 488-tagged secondary antibody), respectively. Irrespective of CHIR treatment BCL9L-knockdown cells show increased retention of E-cadherin and β-catenin staining at the plasma membrane compared to controls (see arrows, bottom right panel). ( G ) Quantification of CHIR-induced nuclear translocation of β-Catenin in samples from (F). Data shown represent mean and standard deviation of mean grey values from nuclear ROIs of each 20 cells quantified as described in Materials and Methods. ( H ) β-catenin was immunoprecipitated from total cell lysates of Panc-1 BCL9L-knockdown and control cells treated or not with CHIR inhibitor. Western Blot analysis of β-catenin, E-cadherin and GAPDH (control) protein levels in input (IP) and bound (B) fractions of the immunoprecipitation revealed increased binding of E-cadherin to β-catenin protein in presence and absence of CHIR in BCL9L knockdown cells compared to controls. Shown is a representative image of 2 experiments.

Article Snippet: The following antibodies were used in this study: BCL9L (sheep polyclonal, # AF4967, R&D Systems; HPA 049370, Atlas Antibodies), α-tubulin (mouse monoclonal, B-5-1-2, #T5168, Sigma Aldrich), β-catenin (rabbit polyclonal, H102, #sc-7199, Santa Cruz), E-cadherin (goat polyclonal, #AF648, R&D Systems), α-catenin (mouse monoclonal, 6D202, #C2069-44H, USBiological).

Techniques: Control, Stable Transfection, shRNA, Transfection, Knockdown, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Clinical Proteomics, Membrane, Translocation Assay, Standard Deviation, Immunoprecipitation, Binding Assay

Journal: Oncotarget

Article Title: Role of BCL9L in transforming growth factor-β (TGF-β)-induced epithelial-to-mesenchymal-transition (EMT) and metastasis of pancreatic cancer

doi: 10.18632/oncotarget.12455

Figure Lengend Snippet: ( A ) Panc-1 cells stably transduced with control vector or BCL9L shRNA (shBCL9L) were treated with 5 ng/ml TGF-β or left untreated for 72 h and subsequently visualized using a phase-contrast microscope. Control cells responded to TGF-β treatment by adopting a mesenchymal, spindle-like phenotype whereas BCL9L-knockdown cells largely retained the cobblestone-like epithelial morphology. ( B ) Western Blot analysis of E-cadherin and α-tubulin (loading control) protein levels in Panc-1 cells treated with 5 ng/ml TGF-β for 72 h. shRNA- and siRNA-mediated knockdown of BCL9L (shBCL9L, siRNA1, siRNA2, siRNA pool) induced an upregulation of E-cadherin expression in comparison to controls (Vector ctrl, siCTR). Shown are representative images of 2 experiments. mRNA expression levels of epithelial (CDH1) and mesenchymal (SNAI2, VIM) genes were quantified in BCL9L-knockdown and control Panc-1 cells treated or not with TGF-β for 24 h ( C ), 48 h ( D ), 72 h ( E ) and 96 h ( F ), respectively. Data shown represent mean and standard deviation of 3 independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (Student's t-test ).

Article Snippet: The following antibodies were used in this study: BCL9L (sheep polyclonal, # AF4967, R&D Systems; HPA 049370, Atlas Antibodies), α-tubulin (mouse monoclonal, B-5-1-2, #T5168, Sigma Aldrich), β-catenin (rabbit polyclonal, H102, #sc-7199, Santa Cruz), E-cadherin (goat polyclonal, #AF648, R&D Systems), α-catenin (mouse monoclonal, 6D202, #C2069-44H, USBiological).

Techniques: Stable Transfection, Transduction, Control, Plasmid Preparation, shRNA, Microscopy, Knockdown, Western Blot, Expressing, Comparison, Standard Deviation

Journal: Discover. Oncology

Article Title: Veillonella parvula promotes the proliferation of lung adenocarcinoma through the nucleotide oligomerization domain 2/cellular communication network factor 4/nuclear factor kappa B pathway.

doi: 10.1007/s12672-023-00748-6

Figure Lengend Snippet: Fig. 1 Correlation between Veillonella parvula and NSCLC. A Bar graph of colony composition; B Analysis of significant difference test between groups; C Correlation of V. parvula abundance with NSCLC and metastasis; D Differences in the abundance of V. parvula in LUAD and squamous lung cancer, *p < 0.05, **p < 0.01, ***p < 0.001. Control (benign lung disease group, n = 29); non-small cell lung carcinoma (NSCLC, n = 46); non-small cell lung adenocarcinoma (LUAD, n = 25); non-small cell squamous lung cancer (LSCC, n = 21). Metastasis (n = 23) and non-metastasis (n = 23)

Article Snippet: Veillonella parvula ATCC®10790TM (#0867) was purchased from ATCC, and Escherichia coli MG1655 (700926) was a gift from the Department of Microbiology, School of Public Health, Southern Medical University (Guangzhou, China).

Techniques: Control

Journal: Discover. Oncology

Article Title: Veillonella parvula promotes the proliferation of lung adenocarcinoma through the nucleotide oligomerization domain 2/cellular communication network factor 4/nuclear factor kappa B pathway.

doi: 10.1007/s12672-023-00748-6

Figure Lengend Snippet: Fig. 4 Veillonella parvula promotes the proliferation of lung adenocarcinoma cells in vitro. A Representative TEM images of V. parvula and E. coli (MG1655) co-culture with A549 or LLC cells. E. coli MG1655 is a negative control. The red arrows indicate V. parvula. Scale bars = 1 μM in V. parvula and E. coli MG1655 (right two panels); 2 μM for the remaining panels; B The level of attachment of V. parvula or E. coli MG1655 on A549, LLC, and BEAS-2B cell lines, MOI = 100 for 1 h; C Co-culture V. parvula (MOI = 100) or V. parvula -Heat (MOI = 100) with A549, LLC, and BEAS-2B cell lines. The proliferation of cells was measured by CCK8 assay; D Colony formation of A549, LLC, and BEAS-2B cells co-culture with V. parvula or V. parvula-Heat at MOI = 100, PBS is the control. p < 0.05 indicates significance

Article Snippet: Veillonella parvula ATCC®10790TM (#0867) was purchased from ATCC, and Escherichia coli MG1655 (700926) was a gift from the Department of Microbiology, School of Public Health, Southern Medical University (Guangzhou, China).

Techniques: In Vitro, Co-Culture Assay, Negative Control, CCK-8 Assay, Control

Journal: Discover. Oncology

Article Title: Veillonella parvula promotes the proliferation of lung adenocarcinoma through the nucleotide oligomerization domain 2/cellular communication network factor 4/nuclear factor kappa B pathway.

doi: 10.1007/s12672-023-00748-6

Figure Lengend Snippet: Fig. 5 Veillonella parvula promotes CCN4 expression in lung adenocarcinoma cells. A Effect of PBS, V. parvula-Heat, and V. parvula on the growth of LLC xenografts, PBS-treated is the control group. B-C Up/downregulated genes co-regulated between V. parvula vs. V. parvula- Heat, V. parvula vs. PBS, B Venn diagram, C heatmap; D Analysis of CCN4 expression in unpaired (left) and paired (right) LUAD and normal tissue samples based on TCGA data, Normal = 46, LUAD = 521; E Correlation of CCN4 expression with OS and PFS in LUAD; F Expression of CCN4 mRNA by qRT-PCR; G Expression of CCN4 protein by western blotting

Article Snippet: Veillonella parvula ATCC®10790TM (#0867) was purchased from ATCC, and Escherichia coli MG1655 (700926) was a gift from the Department of Microbiology, School of Public Health, Southern Medical University (Guangzhou, China).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: Discover. Oncology

Article Title: Veillonella parvula promotes the proliferation of lung adenocarcinoma through the nucleotide oligomerization domain 2/cellular communication network factor 4/nuclear factor kappa B pathway.

doi: 10.1007/s12672-023-00748-6

Figure Lengend Snippet: Fig. 6 CCN4 involved in regulating the promotion of lung adenocarcinoma cell proliferation by Veillonella parvula. A EdU cell proliferation assay, DAPI-labelled nuclei (Blue), EdU-labelled cells for DNA replication (Red), Scale Bar: 100 μM; B Co-culture V. parvula (MOI = 100) with Lv-NC and Lv-CCN4-RNAi from day 0 to 4, respectively, cell proliferation was measured by CCK8 assay; C Western blot detection of CCN4, PCNA protein expression; D Representative TEM images of V. parvula co-culture with Lv-NC, Lv-CCN4-RNAi cells, red arrows indicate V. paru- vula, scale Bar: 2 μM; E Bacterial adhesion assay detects the ability of V. parvula to adhere to Lv-NC, Lv-CCN4-RNAi cells. p < 0.05 indicate statistical significance

Article Snippet: Veillonella parvula ATCC®10790TM (#0867) was purchased from ATCC, and Escherichia coli MG1655 (700926) was a gift from the Department of Microbiology, School of Public Health, Southern Medical University (Guangzhou, China).

Techniques: Proliferation Assay, Co-Culture Assay, CCK-8 Assay, Western Blot, Expressing, Cell Adhesion Assay

Journal: Discover. Oncology

Article Title: Veillonella parvula promotes the proliferation of lung adenocarcinoma through the nucleotide oligomerization domain 2/cellular communication network factor 4/nuclear factor kappa B pathway.

doi: 10.1007/s12672-023-00748-6

Figure Lengend Snippet: Fig. 7 Veillonella parvula activates Nod2 and NF-κB signaling pathways. A KEGG enrichment analysis of differential genes from transcrip- tome sequencing; B Western blot detects the expression of CDH1, p-β-catenin, TLR4, MYD88, and GAPDH in LLC and A549 cells treated with V. parvula at different MOI at 50 and 100; C Western blot detects the expression of Nod1, Nod2, NF-κB p65, p-NF-κB p65(Ser536), and GAPDH in LLC and A549 cells treated with V. parvula at different MOI at 50 and 100; D Immunofluorescence staining assay showed that p65 translo- cation to the nucleus was significantly increased in LLC and A549 cells treated with V. parvula. scale bar, 25 μM

Article Snippet: Veillonella parvula ATCC®10790TM (#0867) was purchased from ATCC, and Escherichia coli MG1655 (700926) was a gift from the Department of Microbiology, School of Public Health, Southern Medical University (Guangzhou, China).

Techniques: Protein-Protein interactions, Sequencing, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Discover. Oncology

Article Title: Veillonella parvula promotes the proliferation of lung adenocarcinoma through the nucleotide oligomerization domain 2/cellular communication network factor 4/nuclear factor kappa B pathway.

doi: 10.1007/s12672-023-00748-6

Figure Lengend Snippet: Fig. 9 Diagram of Veillonella parvula promoting lung adenocarcinoma progression. V. parvula is enriched in lung adenocarcinoma and pro- motes lung adenocarcinoma progression in vivo by inhibiting T-lymphocyte infiltration and in vitro by adhesion to invade lung adenocarci- noma cells via Nod2/CCN4 signalling to activate the NF-κB pathway

Article Snippet: Veillonella parvula ATCC®10790TM (#0867) was purchased from ATCC, and Escherichia coli MG1655 (700926) was a gift from the Department of Microbiology, School of Public Health, Southern Medical University (Guangzhou, China).

Techniques: In Vivo, In Vitro

Journal: Nature communications

Article Title: A novel Nrf2-miR-29-desmocollin-2 axis regulates desmosome function in keratinocytes.

doi: 10.1038/ncomms6099

Figure Lengend Snippet: Figure 5 | miR-29s regulate the expression of DSC2 in human keratinocytes. (a) Levels of DSC2 mRNA in human primary keratinocytes (HPK) and HaCaT cells transfected with miR-29 mimics or non-specific (scrambled) oligonucleotides (ns). mRNA levels were measured by qRT-PCR and calculated as fold change over control (N ¼ 3). (b) Protein levels of DSC2 in corresponding HaCaT cells transfected with miR-29s or scrambled oligonucleotides were analysed by Western blot. (c,d) Levels of DSC2 mRNA (c) and protein (d) on transfection of HaCaT keratinocytes with antagomiRs targeting miR-29a, miR-29b and miR-29c (combined transfection) or scrambled (non-specific; ns) sequences. Cells were collected at indicated time points post transfection; ns set as 1 for each time point (N ¼ 3). (e) DSC2 mRNA was quantified in HaCaT cells transfected with antigomiR-29s and non-specific antagomiRs before treatment with compound 1a (25 mM) to activate NRF2 (N ¼ 3). (f) Human embryonic kidney cells were transfected with plasmids harbouring the full-length wild-type DSC2 30UTR or the 30UTR with a mutation in the miR-29 binding site downstream of the luciferase coding region together with miR-29 mimics or non-specific controls. Lysates were analysed for luciferase activity (N ¼ 3). Error bars represent s.d., P value *o0.05; **o0.01. RT–PCR, reverse transcriptase-PCR.

Article Snippet: Frozen sections of mouse tail skin (7 mm) were incubated with antibodies against mouse Dsc2 (R&D Systems, Minneapolis, MN) or desmoplakin52 together with Hoechst to counterstain nuclei.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Control, Western Blot, Mutagenesis, Binding Assay, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription

Journal: Nature communications

Article Title: A novel Nrf2-miR-29-desmocollin-2 axis regulates desmosome function in keratinocytes.

doi: 10.1038/ncomms6099

Figure Lengend Snippet: Figure 6 | Nrf2 regulates the expression of DSC2 in the epidermis and controls formation of hyper-adhesive desmosomes. (a) Levels of Dsc2 mRNA in total skin from K5Cre-CMVcaNrf2 mice and control littermates (N ¼ 3) at P2.5 and P32. Levels in control mice were set as 1 for each time point (dashed line). (b) Levels of Dsc2 mRNA in primary keratinocytes from K5Cre-CMVcaNrf2 mice and control littermates (N ¼ 4) isolated from epidermis at P4.5. (c) Tail skin of K5Cre and K5Cre-CMVcaNrf2 mice was stained with antibodies against Dsc2 (green; left and right panel) and desmoplakin (Dsp, red, left panel). Yellow colour indicates co-localization of Dsc2 and Dsp in desmosomes at the cell–cell border in K5Cre epidermis. Scale bars, 20 mm. (d) HPKs were transfected with DSC2-YFP or GFP (control) expression vectors and exposed to 2 mM CaCl2 for 1–3 h to induce the formation of desmosomes. After removal of CaCl2, the remaining hyper-adhesive, calcium-insensitive desmosomes were stained with a desmoplakin (DSP) antibody. The percentage of keratinocytes connected by calcium-insensitive, hyper-adhesive desmosomes among all keratinocytes was determined. Ten fields per dish with at least 100 cells per field were counted in three independent dishes for every time point. Representative images of keratinocytes 2 h after exposure to high CaCl2 concentrations stained with a DSP antibody. DSP in hyper-adhesive desmosomes is indicated with arrowheads. (e) DSC2-YFP (green) co-localizes with DSP (red) at the site of desmosome formation (arrowheads). Note that transfection with a GFP expression vector does not affect formation/function of desmosomes and that GFP does not co-localize with DSP at the cell membrane (arrow). (f) Formation or hyper-adhesive desmosomes by HaCaT cells under high calcium concentrations following transfection with scrambled (Scr) or DSC2 siRNA. Desmoplakin staining indicates hyper-adhesive desmosomes (arrowheads). Arrows point to single cells disconnected from neighbouring cells as a result of loss of hyper- adhesive desmosomes. (g) Quantification of disconnected cells following transfection with Scr or DSC2 siRNA and after treatment with ethylene glycol tetraacetic acid. At least 2,000 cells were counted in three independent dishes. (h) Total number of cells transfected with Scr or DSC2 siRNA and incubated in high or low calcium medium. Error bars represent s.d., t-test P value *o0.05; **o0.01, ***o0.001.

Article Snippet: Frozen sections of mouse tail skin (7 mm) were incubated with antibodies against mouse Dsc2 (R&D Systems, Minneapolis, MN) or desmoplakin52 together with Hoechst to counterstain nuclei.

Techniques: Expressing, Adhesive, Control, Isolation, Staining, Transfection, Plasmid Preparation, Membrane, Incubation

Journal: Nature communications

Article Title: A novel Nrf2-miR-29-desmocollin-2 axis regulates desmosome function in keratinocytes.

doi: 10.1038/ncomms6099

Figure Lengend Snippet: Figure 7 | Regulation and function of the Nrf2-miR-29-Dsc2 axis in the skin. Nrf2 directly induces expression of miR-29a and miR-29b in keratinocytes. This results in the suppression of Dsc2 expression and impairments in the formation of hyperactive desmosomes, which affects the skin barrier. In contrast, DNA methylation of the promoter of the lncRNA excludes functional binding of Nrf2 and likely prevents Nrf2- mediated activation of the miR29b2c cluster.

Article Snippet: Frozen sections of mouse tail skin (7 mm) were incubated with antibodies against mouse Dsc2 (R&D Systems, Minneapolis, MN) or desmoplakin52 together with Hoechst to counterstain nuclei.

Techniques: Expressing, DNA Methylation Assay, Functional Assay, Binding Assay, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) Experimental approach used to conduct the microarray analysis from TRPV1 neurons, 72 hours after i.pl. CFA. ( B ) FACS isolation of TRPV1-pHluorin neurons. Representative FACS plot of GFP + population in WT (top) and TRPV1-pHluorin (bottom) mice. SSC-A side scatter area; FSCA- forward scatter area. ( C ) Scatter plot representation of genes regulated in CFA conditions. Genes that passed a threshold of log 2 fold change in differential expression analysis are represented as green when downregulated and red when upregulated. All genes are listed in . ( D ) qRT-PCR assessment of ALKAL2 upregulation in the DRG ipsilateral to the CFA injection (Ipsi) ( n = 9), compared with the contralateral side (Contra) ( n = 9) and naive control ( n = 8). Statistical analysis was performed using Kruskal-Wallis followed by Dunn’s post hoc test. * P < 0.05; *** P < 0.001. ( E ) Representative Western blot of ALKAL2 in the DRG ipsilateral to the CFA injection compared with the contralateral side. ( F ) Quantification of ALKAL2 protein level from Western blot experiments. Each dot represents a sample collected from a different animal ( n = 6 per group). Statistical analysis was performed by unpaired t test ( F ). **** P < 0.0001. Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: Microarray, Isolation, Expressing, Quantitative RT-PCR, Injection, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) Heatmap of the expression of ALKAL2 and selected population markers on the 17 populations of sensory neurons from DRG described in Zeisel et al. . ( B ) Representative confocal images of coimmunostaining for ALKAL2 and TRPV1, IB4, GFRα3, and NF200 in DRG neurons. Scale bars: 50 μm. ( C ) Dot plot summarizing the results in B : 77.64% ± 2.95% of TRPV1, 77.64% ± 3.06% of GFRα3, 41.65% ± 7.08% of IB4, and 36.46% ± 2.64% of NF200-positive neurons express ALKAL2 (each symbol represents a DRG section from n = 4 individual animals). Statistical analysis was performed using 1-way ANOVA followed by Tukey’s post hoc test. **** P < 0.001. ( D ) Representative confocal images of coimmunostaining for ALKAL2 and NF200, IB4, and GFRα3 in the sciatic nerve. Scale bars: 50 μm. Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) Representative RNAScope image showing expression of ALKAL2 (light blue) in human DRG neurons coexpressing Nav1.8 (pink). ( B ) Bar graph summarizing the results (each symbol represents an individual patient, n = 3). Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) Schematic illustrating the coculture system used to chronically expose DRG neurons to ALKAL2. HEK cells were plated into the upper chamber of a Transwell and then transfected with ALKAL2 plasmid for 16 hours. Cells were washed, and DRG neurons were plated in the lower chamber of the Transwell for another 16 hours of coculture and then immunostained for Tuj1. Scale bars: 50 μm. ALKAL2 induces a significant increase of total neurites ( B ), number of branch points per neuron ( C ), and total neurite length per neuron ( D ) (<20 μm). Control, n = 19; ALKAL2, n = 12; ALKAL2+lorlatinib, n = 18. Statistical analysis was performed using Kruskal-Wallis followed by Dunn’s post hoc test. * P < 0.05; ** P < 0.01; **** P < 0.001. ( E ) Representative confocal images illustrating the TRPV1-GFP innervation of the skin paw following i.pl. injection of CFA (3 days). Scale bars: 50 μm. ( F ) CFA-induced sprouting is reversed by daily administration of lorlatinib (1 mg/kg). Control, n = 5; CFA+vehicle, n = 5; CFA+lorlatinib, n = 5. Statistical analysis was performed using 1-way ANOVA followed by Tukey’s post hoc test. * P < 0.05; ** P < 0.01. Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: Transfection, Plasmid Preparation, Injection

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) Resting membrane potential (RMP) of small DRG neurons in control (–56.06 ± 1.03 mV, n = 19), ALKAL2 (–55.15 ± 2.21 mV, n = 13), or ALKAL2+lorlatinib (–54.5 ± 1.53 mV, n = 13) groups. Statistical analysis was performed using 1-way ANOVA followed by Tukey’s post hoc test. ( B ) AP threshold in control (–35.59 ± 2.26 mV, n = 16), ALKAL2 (–40.57 ± 2.88 mV, n = 11), and ALKAL2+lorlatinib (–31.59 ± 1.76 mV, n = 9) groups. Statistical analysis was performed using 1-way ANOVA followed by Tukey’s post hoc test. ( C ) Representative AP discharge evoked by 100, 200, 300, and 400 pA current injections (1 s) in control, ALKAL2- (10 nM), and ALKAL2+lorlatinib-treated (10 nM+1 μM) DRG neurons. ( D ) Measure of AP frequency evoked by current injection in the different groups represented in E (control, n = 13; ALKAL2 [10 nM], n = 11; ALKAL2+lorlatinib treated [10 nM+1 μM], n = 9). Statistical analysis was performed using 2-way ANOVA followed by Tukey’s post hoc test. * P < 0.05; *** P < 0.001; **** P < 0.0001 versus control. $ P < 0.05; $$$$ P < 0.0001 versus ALKAL2+lorlatinib. Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: Injection

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) Administration i.t. of ALKAL2 (1 μM) promotes thermal hyperalgesia, which is reversed by the administration of lorlatinib (ALKAL2, n = 9; ALKAL2+lorlatinib, n = 8). Statistical analysis was performed using 2-way ANOVA followed by Bonferroni’s post hoc test. * P < 0.05; *** P < 0.001. ( B ) Representative confocal images illustrating pALK induction in the lamina I and II of the spinal dorsal horn following i.t. infusion of ALKAL2. Activation of pALK is reversed by administration of lorlatinib prior to ALKAL2 administration. Scale bars: 100 μm. ( C ) Bar graph of the pALK signal intensity represented in B in the spinal dorsal horn (control, n = 7; ALKAL2, n = 8; ALKAL2+lorlatinib, n = 6; 1 hour). Statistical analysis was performed using 1-way ANOVA followed by Tukey’s post hoc test. * P < 0.05. Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: The neuronal tyrosine kinase receptor ligand ALKAL2 mediates persistent pain

doi: 10.1172/JCI154317

Figure Lengend Snippet: ( A ) Schematic illustrating the experimental protocol of ALKAL2 oligodeoxynucleotide injection in the CFA pain model. ( B ) Representative confocal images of ALKAL2 immunostaining in DRG sections from control, ALKAL2, or scrambled ODN–treated animals. Scale bars: 50 μm. ( C ) Western blot of ALKAL2 in lumbar DRG lysates at D9 following injection of ALKAL2 or scrambled ODN, compared with naive control mice. ( D ) Bar graph illustrating the reduction in ALKAL2 protein expression in the ODN-treated animals ( n = 4–6 mice per group). Statistical analysis was performed using 1-way ANOVA followed by Tukey’s post hoc test. * P < 0.05; *** P < 0.001. ( E ) Measure of thermal withdrawal latency in contralateral and ipsilateral hind paws of CFA-injected animals that received saline control ( n = 8), scrambled ( n = 7), or ALKAL2 ODN ( n = 8). Statistical analysis was performed using 2-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 ****P < 0.0001 ODN ipsi vs Control ipsi; $$P< 0.01, $$$P < 0.001, $$$$P < 0.0001 ODN ipsi vs sODN ipsi. Data are represented as mean ± SEM.

Article Snippet: Then, tissues were incubated overnight in either PBS 3% BSA or 3% FBS, 0.01% Triton-X 100 at 4°C with either polyclonal chicken anti-GFP (1:500, Invitrogen, catalog A10262), polyclonal rabbit anti-GFP (1:500, Chromotek, catalog PABG1) polyclonal rabbit anti-TRPV1 (1:500, Alomone, catalog ACC-030), polyclonal rabbit anti-CGRP (1:1000, Sigma-Aldrich, catalog PC205L), anti–IB4-coupled Alexa Fluor 594 (1:1000, Invitrogen, catalog I21412), polyclonal sheep anti-TH (1:500, Millipore, catalog AB1542), polyclonal goat anti-GFRα3 (1:500, R&D Systems, catalog VFU021721), monoclonal mouse anti-NF200 (1:500, Sigma-Aldrich, catalog N5389), or rabbit anti-ALKAL2 (1:1000, New England Peptide, project 4633, Seq: BU01949).

Techniques: Injection, Immunostaining, Western Blot, Expressing

Journal: PLoS ONE

Article Title: An Evolutionary-Conserved Function of Mammalian Notch Family Members as Cell Adhesion Molecules

doi: 10.1371/journal.pone.0108535

Figure Lengend Snippet: (A and B) Flow cytometric analysis of the expression of (A) Dll1, Dll3, Dll4, Jag1, and Jag2 on OP9 stromal cells transduced with each Notch ligand gene, and (B) Notch receptors on MCs after staining with specific mAbs (open histograms) or isotype-matched control mAbs (filled histograms). (C) Total RNA was analyzed by RT-PCR for the expression of Notch receptors in MCs and OP9-Ctrl cells. (D) Relative expression levels of Notch1 and Notch2 to Gapdh in MCs were analyzed by quantitative RT-PCR. Data represent the mean ± SEM of 3 independent experiments. (E and F) An adhesion assay for MCs on each OP9 cell (E) in a 48-well plate for 60 min and (F) in 96-well plates with serial incubation times of 5, 15, 30, 60, and 120 min. Data represent the percentages of non-adherent MCs (mean ± SEM of triplicate cultures) (*p<0.05 significantly different from OP9-Ctrl at each time point, the Student’s t- test).

Article Snippet: Sheep anti-mouse Notch1 (AF5267) or Notch2 (AF5196) polyclonal IgGs (which showed less than 1% cross-reactivity with recombinant mouse Notch2 or Notch1, respectively, according to the manufacturer’s instructions) and sheep control IgG (5-001-A) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Transduction, Staining, Control, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Cell Adhesion Assay, Incubation

Journal: PLoS ONE

Article Title: An Evolutionary-Conserved Function of Mammalian Notch Family Members as Cell Adhesion Molecules

doi: 10.1371/journal.pone.0108535

Figure Lengend Snippet: MCs were analyzed 48 hours after transfection with siRNA against Notch2 or control siRNA. (A) Relative expression levels of Notch1 and Notch2 to Gapdh in MCs transfected with each siRNA were analyzed by quantitative RT-PCR. Data represent the mean ± SEM of three independent experiments. (*p<0.05 significantly different from the control siRNA treatment, the Student’s t- test) (B) Flow cytometric analysis of the expression of Notch2 and Kit on MCs transfected with each siRNA after staining with specific mAbs (open histograms) or isotype-matched control mAbs (filled histograms). Representative histograms from one of three independent experiments are shown. Numbers indicate the relative mean fluorescence intensities (MFIs) of specific mAbs relative to that of the control siRNA treatment (MFIs of specific mAbs were normalized by the MFIs of control mAbs) (mean ± SEM of three independent experiments). (C) An adhesion assay (60 min) for MCs transfected with each siRNA on each OP9 cell in a 96-well plate. Data represent the percentages of non-adherent MCs (mean ± SEM of triplicate cultures) (*p<0.05, the Student’s t- test).

Article Snippet: Sheep anti-mouse Notch1 (AF5267) or Notch2 (AF5196) polyclonal IgGs (which showed less than 1% cross-reactivity with recombinant mouse Notch2 or Notch1, respectively, according to the manufacturer’s instructions) and sheep control IgG (5-001-A) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Control, Expressing, Quantitative RT-PCR, Staining, Fluorescence, Cell Adhesion Assay

Journal: PLoS ONE

Article Title: An Evolutionary-Conserved Function of Mammalian Notch Family Members as Cell Adhesion Molecules

doi: 10.1371/journal.pone.0108535

Figure Lengend Snippet: An adhesion assay (60 min) for MCs on each OP9 cell in a 96-well plate (A and B) with or without 10 µg/ml of the indicated polyclonal Ab (pAb) and (C) with control pAb (20 µg/ml), anti-Notch2 pAb (10 µg/ml) plus control pAb (10 µg/ml) or anti-Notch2 pAb (10 µg/ml) plus anti-Notch1 pAb (10 µg/ml). Data represent the percentages of non-adherent MCs (mean ± SEM of triplicate cultures) (*p<0.05, the Student’s t- test). Cultures with pAbs contained (A and B) 1.0% PBS (vol/vol) and 38.5 µM NaN 3 and (C) 2.0% PBS (vol/vol) and 76.9 µM NaN 3 , which had no effect on the adhesion of MCs.

Article Snippet: Sheep anti-mouse Notch1 (AF5267) or Notch2 (AF5196) polyclonal IgGs (which showed less than 1% cross-reactivity with recombinant mouse Notch2 or Notch1, respectively, according to the manufacturer’s instructions) and sheep control IgG (5-001-A) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Cell Adhesion Assay, Control