Journal: bioRxiv

Article Title: The Specificity of IQGAP1 Toward the PI3K-Akt Pathway is Dependent on the IQ3 motif

doi: 10.1101/250936

Figure Lengend Snippet: ( a ) IQGAP1 WT and ΔIQ3 mutant associated cell surface receptors were analyzed by immunoprecipitation. IQGAP1 WT and ΔIQ3 mutant interact with EGFR, Integrin α3 and Integrin β4 at similar levels. HEK293FT cells post 24 h of transient transfection with Myc-tagged IQGAP1 WT or Myc-tagged IQGAP1 ΔIQ3 were starved for 24 h and then treated with 10 ng/ml EGF for 15 min as indicated. The whole cell lysates were collected and processed for immunoprecipitation using anti-Myc antibody conjugated Protein A/G agarose beads. ( b-d ) Quantification of immunoblots in a . n=3. Error bars denote SD.

Article Snippet: Antibodies for immunoblotting, immunofluorescent (IF) staining and immunoprecipitation were from Cell Signaling Technology, including, pAkt (Ser473, 4058), ERK (4695), pErk (Thr202/Tyr204, 4370), PDK1 (13037), p85α (4292), PIPK1α (9693), Santa Cruz Biotechnology, including IQGAP1(H-109) and EGFR (SC-03), Abcam, including Akt (ab126811), Merck Millipore, Myc-tag (05-724), Sigma-Aldrich, including Myc-tag (C3956), Novus Biologicals, including integrin α3 (NBP2-48514) and R&D Systems, including integrin β4 (MAB4060).

Techniques: Mutagenesis, Immunoprecipitation, Transfection, Western Blot

Journal: bioRxiv

Article Title: The Specificity of IQGAP1 Toward the PI3K-Akt Pathway is Dependent on the IQ3 motif

doi: 10.1101/250936

Figure Lengend Snippet: PLA demonstrated the interaction between IQGAP1 WT/ ΔIQ3 mutant and EGFR/Integrin α3β1/Integrin α6β4/PIPK1α/p85α. HEK293FT cells post 24 h of transient transfection with Myctagged IQGAP1 WT or Myc-tagged IQGAP1I ΔIQ3 were starved for 24 h and then treated with 10 ng/ml EGF for 15 min. The cells were fixed and processed for PLA to determine the direct interaction between the Myc-tagged IQGAP1 WT/ΔIQ3 mutant and EGFR/Integrin α3/Integrin β4/PIPK1α/p85α. ⋆⋆P<0.01, n=10. Error bars denote SD. Scale bar, 5 μm.

Article Snippet: Antibodies for immunoblotting, immunofluorescent (IF) staining and immunoprecipitation were from Cell Signaling Technology, including, pAkt (Ser473, 4058), ERK (4695), pErk (Thr202/Tyr204, 4370), PDK1 (13037), p85α (4292), PIPK1α (9693), Santa Cruz Biotechnology, including IQGAP1(H-109) and EGFR (SC-03), Abcam, including Akt (ab126811), Merck Millipore, Myc-tag (05-724), Sigma-Aldrich, including Myc-tag (C3956), Novus Biologicals, including integrin α3 (NBP2-48514) and R&D Systems, including integrin β4 (MAB4060).

Techniques: Mutagenesis, Transfection

Journal: Marine drugs

Article Title: Unusual Tetrahydropyridoindole-Containing Tetrapeptides with Human Nicotinic Acetylcholine Receptors Targeting Activity Discovered from Antarctica-Derived Psychrophilic Pseudogymnoascus sp. HDN17-933.

doi: 10.3390/md20100593

Figure Lengend Snippet: Figure 4. Docking models of compound 2 with the crystal structure of hα4β2 (PDB code: 5kxi). (A) H-bonding interactions of compound 2 with hα4β2. (B) 2D schematic diagram of compound 2 with hα4β2. The H-bonds and H-pi stacked bonds are shown in yellow dashed lines and blue dashed lines, respectively.

Article Snippet: The human muscle nAChR clones (α1, β1, γ, δ and ε) were purchased from Integrated DNA Technologies (Coralville, IA, USA), whereas the human α3, α9, α10, β2, and β4 clones were purchased from OriGene (Rockville, MD, USA), and all were subsequently inserted into the pT7TS vector.

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: CD73 + Epithelial Progenitor Cells That Contribute to Homeostasis and Renewal Are Depleted in Eosinophilic Esophagitis

doi: 10.1016/j.jcmgh.2022.01.018

Figure Lengend Snippet: The CD73 + CD104 + population is depleted in the esophagus of active EoE patients and a murine model of EoE. ( A ) Immunofluorescence staining for CD73 and CD104 on biopsy specimens from non-EoE controls and patients with active EoE. Representative images are shown. Dotted line represents the basement membrane. Scale bars : 100 μm. ( B–E ) Biopsy specimens from non-EoE controls (n = 10), and patients with active (n = 10) or inactive EoE (n = 10) were dissociated for flow cytometry. The representative flow cytometry plots are shown in panel B . ( C ) The percentage of CD73 + CD104 + or CD73 - CD104 + cells in non-EoE controls (n = 10), and patients with active (n = 10) or inactive EoE (n = 10). ( D and E ) Pearson correlation coefficient analyses comparing the percentage of CD73 + CD104 + cells detected by flow cytometry and the number of eosinophils (eos/hpf) in ( D ) H&E-stained biopsy specimens or ( E ) CD45 + cells detected by flow cytometry. ( F and G ) After BALB/c mice were treated with the EoE protocol, their esophagi were resected for histologic sections or flow cytometry. ( F ) Histologic sections (H&E staining) of the esophagus from control (nontreated [NT]) or EoE protocol–treated mice. Arrowheads identify tissue-infiltrating eosinophils. ( G ) The percentage of CD73 + cells in esophageal epithelium of EoE mice (n = 3), compared with NT mice (n = 3). The data are shown as means ± SD. A 2-tailed Student t test or analysis of variance was performed for comparing 2 or multiple comparisons, respectively. ∗ P < .05.

Article Snippet: IHC of 3D organoids for CD73 and CD104 was performed by BOND RXm (Leica Biosystems, Nusslock, Germany) with anti-CD73 antibody (rabbit polyclonal, ab175396, 1:200; Abcam) or anti-CD104 monoclonal antibody (NBP2-37392, 1:1000, Novus Biologicals, Englewood, CA).

Techniques: Immunofluorescence, Staining, Membrane, Flow Cytometry, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: CD73 + Epithelial Progenitor Cells That Contribute to Homeostasis and Renewal Are Depleted in Eosinophilic Esophagitis

doi: 10.1016/j.jcmgh.2022.01.018

Figure Lengend Snippet: Th2 cytokines shift the basaloid population from CD73 + to CD73 - in human esophageal 3D organoids. PDOs from non-EoE controls (n = 6) or patients with active EoE (n = 6), or immortalized esophageal epithelial (EPC2-hTERT) organoids were treated with IL4 (10 ng/mL) or IL13 (10 ng/mL) from day 7 to day 11. Nontreated (NT) organoids were used as control. ( A ) Representative flow cytometry plots showing the percentage of CD73 + CD104 + cells in PDOs from non-EoE controls or patients with active EoE, or EPC2-hTERT organoids, treated with IL4 or IL13. ( B ) The percentage of CD73 + CD104 + cells in each PDO treated with IL4 or IL13, paired with NT control. The data are shown as means ± SD. ∗∗∗ P < .001, ∗∗∗∗ P < .0001, 2-tailed Student t test. ( C ) The representative images of H&E staining, and IHC for CD73 or CD104 in EPC2-hTERT organoids treated with IL4 or IL13. Scale bars : 100 μm. ( D ) CD73 , CD104 , SOX2 , and IVL gene expression in EPC2-hTERT organoids treated with IL4 or IL13 were validated by qRT-PCR. The relative expression over NT is represented. The data are shown as means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, and ∗∗∗∗ P < .0001, analysis of variance.

Article Snippet: IHC of 3D organoids for CD73 and CD104 was performed by BOND RXm (Leica Biosystems, Nusslock, Germany) with anti-CD73 antibody (rabbit polyclonal, ab175396, 1:200; Abcam) or anti-CD104 monoclonal antibody (NBP2-37392, 1:1000, Novus Biologicals, Englewood, CA).

Techniques: Control, Flow Cytometry, Staining, Gene Expression, Quantitative RT-PCR, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: CD73 + Epithelial Progenitor Cells That Contribute to Homeostasis and Renewal Are Depleted in Eosinophilic Esophagitis

doi: 10.1016/j.jcmgh.2022.01.018

Figure Lengend Snippet: Single-cell RNA sequencing analysis of PDOs from 4 non-EoE patients. PDOs from 4 non-EoE patients were treated with IL13 (10 ng/mL) or vehicle (phosphate-buffered saline) from day 7 to day 11, and harvested and dissociated for single-cell RNA sequencing analysis on day 11. ( A ) Seurat’s Uniform Manifold Approximation and Projection (UMAP) visualization of 4 classified cell groups, basal-1, basal-2, suprabasal, and superficial, within PDOs from non-EoE controls grown with or without IL13 (10 ng/mL). ( B ) Average normalized gene expression values of MKI67 , CD73 , basal markers ( TP63 , NGFR ), and differentiated markers ( IVL , KRT13 , FLG ) in each cell cluster. Circle size reflects the percentage of cells with a nonzero expression level for the indicated genes. Color intensity reflects the average expression level across all cells within each cluster. ( C ) Expression z-scores for the top 8 up-regulated genes in each cluster. ( D ) The proportions of each cell cluster were compared in PDOs (n = 4) with IL13 or vehicle. ( E–G ) The normalized expression of ( E ) IVL and FLG , ( F ) SOX2 , and ( G ) CD73 in nontreated (NT) PDOs was compared with that in PDOs treated with IL13 for each cluster. ∗∗∗∗ P < .0001, Wilcoxon signed-rank test. ( H ) GO analysis of differentially expressed gene profiles of CD73 + CD104 + cells, compared with CD73 - CD104 + cells in NT PDOs.

Article Snippet: IHC of 3D organoids for CD73 and CD104 was performed by BOND RXm (Leica Biosystems, Nusslock, Germany) with anti-CD73 antibody (rabbit polyclonal, ab175396, 1:200; Abcam) or anti-CD104 monoclonal antibody (NBP2-37392, 1:1000, Novus Biologicals, Englewood, CA).

Techniques: RNA Sequencing, Saline, Gene Expression, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: CD73 + Epithelial Progenitor Cells That Contribute to Homeostasis and Renewal Are Depleted in Eosinophilic Esophagitis

doi: 10.1016/j.jcmgh.2022.01.018

Figure Lengend Snippet: CD73 + CD104 + cells represent a less-differentiated and slow-cycling cell population within the basal zone. ( A and B ) EPC2-hTERT organoids were harvested on day 11 and dissociated for FACS. ( A ) The cells in EPC2-hTERT organoids were sorted based on CD73 and CD104 expression. ( B ) CD73 , CD104 , IVL , and FLG relative gene expression in CD73 + CD104 + and CD73 + CD104 - populations was validated by qRT-PCR. ( C ) EPC2-hTERT cells in 2-dimensional culture (organoid day 0) and EPC2-hTERT organoid (day 7 and day 11) were harvested for flow cytometry and the representative flow cytometry plots for CD73 and CD104 expression are shown. ( D ) Representative IHC images for CD73 staining in EPC2-hTERT organoids harvested on day 5, day 7, day 9, and day 11. Scale bars : 100 μm. ( E–G ) EPC2-hTERT organoids were harvested on day 11 after incubation in culture medium with EdU for 2 hours, and dissociated for flow cytometry. ( E ) A representative FACS dot plot of cells from EPC2-hTERT organoids subjected to EdU, with the frequencies of each population according to CD73 and CD104 expression, is shown. ( F ) Representative histograms of EdU staining in each cell population. The percentage of EdU + cells in each population is shown. ( G ) Percentages of EdU + cells in each population. All data are shown as means ± SD (n = 3). A 2-tailed Student t test or analysis of variance were performed for comparing 2 or multiple comparisons, respectively. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, and ∗∗∗∗ P < .0001.

Article Snippet: IHC of 3D organoids for CD73 and CD104 was performed by BOND RXm (Leica Biosystems, Nusslock, Germany) with anti-CD73 antibody (rabbit polyclonal, ab175396, 1:200; Abcam) or anti-CD104 monoclonal antibody (NBP2-37392, 1:1000, Novus Biologicals, Englewood, CA).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Flow Cytometry, Staining, Incubation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: CD73 + Epithelial Progenitor Cells That Contribute to Homeostasis and Renewal Are Depleted in Eosinophilic Esophagitis

doi: 10.1016/j.jcmgh.2022.01.018

Figure Lengend Snippet: CD73 + cells mediate epithelial renewal in human esophageal epithelium. ( A–D ) PDOs or EPC2-hTERT organoids were harvested on day 11 (1° organoids) and dissociated for FACS. The cells were sorted based on CD73 and CD104 expression and resuspended in Matrigel for organoid establishment and growth. OFR and organoid size (mean area) of 2° organoids were ( B and C ) quantified on a Celigo image cytometer and ( D ) harvested on day 11 and again dissociated for flow cytometry. ( A ) Representative phase-contrast images of organoids formed by corresponding cell populations. ( B ) OFR and ( C ) mean size were compared among organoids from the 3 populations. ( D ) The scheme of this experiment and the representative flow cytometry plots of 2° organoids are shown. The percentage of CD73 + cells in EPC2-hTERT were compared. The data are shown as means ± SD (n = 3). ( E–H ) EPC2-hTERT cells were treated for 48 hours with CD73 siRNA or nonsilencing (scramble) siRNA in 2D culture, followed by seeding into organoid cultures. ( E ) CD73 silencing efficiency was evaluated by flow cytometry. ( F ) Representative phase-contrast images of organoids formed by corresponding cells. ( G ) OFR and ( H ) mean size of organoids from EPC2-hTERT cells transfected with CD73 or scramble siRNA. The data are shown as means ± SD (n = 10). Two-tailed Student t test or analysis of variance were performed for single or multiple comparisons, respectively. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, and ∗∗∗∗ P < .0001.

Article Snippet: IHC of 3D organoids for CD73 and CD104 was performed by BOND RXm (Leica Biosystems, Nusslock, Germany) with anti-CD73 antibody (rabbit polyclonal, ab175396, 1:200; Abcam) or anti-CD104 monoclonal antibody (NBP2-37392, 1:1000, Novus Biologicals, Englewood, CA).

Techniques: Expressing, Cytometry, Flow Cytometry, Transfection, Two Tailed Test

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: CD73 + Epithelial Progenitor Cells That Contribute to Homeostasis and Renewal Are Depleted in Eosinophilic Esophagitis

doi: 10.1016/j.jcmgh.2022.01.018

Figure Lengend Snippet: The therapeutic potential of STAT6 inhibition in EoE. ( A ) IHC staining for phosphorylated STAT6 was performed, with representative images shown. Scale bars : 100 μm. ( B ) The positive rate of phosphorylated STAT6 in esophageal epithelium are compared. The data are shown as means ± SD (n = 5). ( C ) Relative expression of CD73 gene in EPC2-hTERT organoids treated with STAT6 inhibitor (AS151749, 400 nmol/L), IL4 (10 ng/mL), or IL13 (10 ng/mL). ( D ) The frequencies of CD73 + CD104 + cells in organoids treated with AS151749 or dimethyl sulfoxide (DMSO) under the presence of Th2 cytokines. STAT6 silencing efficiency was evaluated by ( E ) qRT-PCR and ( F ) Western blot. ( G ) CD73 gene expression in EPC2-hTERT organoids treated with STAT6 siRNA or nonsilencing (scramble) siRNA with or without IL13. The data are shown as means ± SD (n = 3). Analysis of variance was performed with multiple comparisons. ∗∗ P < .01, ∗∗∗∗ P < .0001.

Article Snippet: IHC of 3D organoids for CD73 and CD104 was performed by BOND RXm (Leica Biosystems, Nusslock, Germany) with anti-CD73 antibody (rabbit polyclonal, ab175396, 1:200; Abcam) or anti-CD104 monoclonal antibody (NBP2-37392, 1:1000, Novus Biologicals, Englewood, CA).

Techniques: Inhibition, Immunohistochemistry, Expressing, Quantitative RT-PCR, Western Blot, Gene Expression

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: CD73 + Epithelial Progenitor Cells That Contribute to Homeostasis and Renewal Are Depleted in Eosinophilic Esophagitis

doi: 10.1016/j.jcmgh.2022.01.018

Figure Lengend Snippet: Omeprazole treatment prevents Th2-mediated depletion of CD73 + CD104 + population. ( A ) EPC2-hTERT organoids were treated with omeprazole (50 μmol/L), IL4 (10 ng/mL), or IL13 (10 ng/mL). The frequencies of CD73 + CD104 + cells in organoids are shown as means ± SD (n = 3). ( B and C ) The treated EPC2-hTERT organoids were harvested on day 11 and dissociated into single cells, followed by seeding in Matrigel to assess organoid formation capacity. ( B ) OFR was evaluated manually on day 11. ( C ) Representative phase-contrast images of organoids from corresponding groups. The data are shown as means ± SD (n = 6). Analysis of variance was performed with multiple comparisons. ∗∗∗∗ P < .0001. DMSO, dimethyl sulfoxide.

Article Snippet: IHC of 3D organoids for CD73 and CD104 was performed by BOND RXm (Leica Biosystems, Nusslock, Germany) with anti-CD73 antibody (rabbit polyclonal, ab175396, 1:200; Abcam) or anti-CD104 monoclonal antibody (NBP2-37392, 1:1000, Novus Biologicals, Englewood, CA).

Techniques:

Journal: Coatings (Basel, Switzerland)

Article Title: Surface Immobilization Chemistry of a Laminin-Derived Peptide Affects Keratinocyte Activity

doi: 10.3390/coatings10060560

Figure Lengend Snippet: Oral keratinocyte hemidesmosome formation [total collagen XVII intensity ( A ), collagen XVII intensity normalized to number of cells ( B ), total integrin β4 intensity ( C ), and integrin β4 intensity normalized to number of cells ( D )] of coated and non-coated, control surfaces at 1 day. Differences in mean between groups were assessed with a one-way analysis of variance (ANOVA) table followed by a Tukey’s HSD (honest significant difference) post hoc test. A p value of <0.05 was considered statistically significant. Dissimilar letters denote statistically significant differences between groups.

Article Snippet: Disks laden with cells were immersed in 5% bovine serum albumin (BSA) in PBS and then probed with a primary mouse monoclonal antibody (paraformaldehyde fixed) for integrin β4 [critical early marker for HD assembly [ ]) NB10065599; Novus Biologicals, Littleton, CO, USA; 1:500] or primary rabbit polyclonal antibody (methanol fixed) for collagen XVII [(important late marker for HD assembly [ ]) ab28440; Abcam, Cambridge, UK; 1:500] for 1 h at room temperature.

Techniques: Control

Journal: American Journal of Cancer Research

Article Title: LPPR4 promotes peritoneal metastasis via Sp1/integrin α/FAK signaling in gastric cancer

doi:

Figure Lengend Snippet: LPPR4 activates transcription of integrin α through Sp1. A. Classical members of integrin family proteins and corresponding downstream genes were assessed by Western blotting in HGC27 and MKN74 cells transfected with siLPPR4 and in MGC803 cells infected with LPPR4 overexpression plasmid. B. Immunoprecipitation using a LPPR4 antibody showed LPPR4 and ITGA1, ITGA2, ITGA5, ITGA6, ITGA7 association. C. Correlation between LPPR4 and Sp1, as well as Sp1 and ITGA1, ITGA2, ITGA5, ITGA6, ITGA7 was performed in human GC tissues based on TCGA-STAD dataset. D. Sp1 mRNA expression levels were tested by qRT-PCR in HGC-27 and MKN74 cells after transfected with siLPPR4. E. Sp1 protein expression levels were detected by Western blotting analysis in HGC27 and MKN74 cells after transfected with siLPPR4. F, G. HGC27 and MKN74 cells were transfected with NC-siRNA and Sp1-siRNA. Sp1 expression levels were detected by qRT-PCR and Western blotting. ITGA1, ITGA2, ITGA5, ITGA6 and ITGA7 protein expression levels were detected by Western blotting analysis in HGC27 and MKN74 cells after transfected with siSp1. β-actin was used as a loading control in Western blotting. Each experiment was repeated at least three times. All the data were expressed as mean ± SD, ***P < 0.001, based on Student’s t-test.

Article Snippet: Mouse anti-integrin β4 (NBP2-37392) was from Novus Biologicals (Shanghai, China).

Techniques: Western Blot, Transfection, Infection, Over Expression, Plasmid Preparation, Immunoprecipitation, Expressing, Quantitative RT-PCR

Journal: Cell Stem Cell

Article Title: Constitutively Active SMAD2/3 Are Broad-Scope Potentiators of Transcription-Factor-Mediated Cellular Reprogramming

doi: 10.1016/j.stem.2017.10.013

Figure Lengend Snippet:

Article Snippet: The following antibodies were used with indicated dilution: ICAM1-biotin conjugate (1:100, eBioscience, #13-0541), CD44-allophycocyanin (APC) conjugate (1:300, eBioscience, #17-0441), E-CADHERIN-eFluor 660 (1:300, eBioscience, #50-3249-82), MEFSK4-biotin conjugate (1:100, Miltenyi, 130-101-875), CD47-biotin conjugate (1:100, BioLegend, 127505), CD73-Alexa Fluor 647 (1:300, BD Biosciences, 561543) and CD104-APC (1:300, Miltenyi, 130-106-924) and Strepdavidin PE-Cy7 (1:1500, eBioscience, #25-4317).

Techniques: Recombinant, Software