Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 7. Validation of compound-target interactions through molecular docking and protein-small molecule binding assays. (A) Chemical structure of EGCG and molecular docking models showing its interactions with NOX2 and p47phox. (B) Chemical structure of quercetin and molecular docking models illustrating its interactions with NOX2 and p47phox. (C) An SPR sensorgram displaying the binding affinity of the NOX2-EGCG interaction. (D) An SPR sensorgram showing the binding affinity of the NOX2-quercetin interaction. (E) A BLI sensorgram illustrating the binding affinity of the p47phox-EGCG interaction. (F)A BLI sensorgram depicting the binding affinity of the p47phox-quercetin interaction. Abbreviations: BLI, Bio-layer interferometry; EGCG, epigallocatechin-3-gallate; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; Que, quercetin; SPR, surface plasmon resonance.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques: Biomarker Discovery, Binding Assay, SPR Assay

Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 8. QKPC and its components, EGCG and quercetin, reduce NOX2-p47phox/ROS signaling in vivo and in vitro. (A) Protein levels of NOX2 and p47phox in human neutrophils were assessed by Western blotting analysis, with relative density quantification shown as the ratio of NOX2 or p47phox to GAPDH (n = 3). (B) Protein levels of NOX2 and p47phox in lung tissues of mice were determined by Western blotting analysis and presented as the ratio of NOX2 or p47phox to GAPDH (n = 3). (C) Cellular ROS levels were measured using the oxidant-sensing probe DCFH-DA, and fluorescence intensity was recorded for each group. EGCG, quercetin, and the positive drugs NAC (20 μM, a ROS scavenger), DPI (50 μM, a general NADPH oxidase inhibitor), and GSK (50 μM, a NOX2-specific inhibitor) effectively suppressed ROS levels elevated by the combined CSE and LPS exposure (n = 6–8). (D) MDA levels in lung tissues were measured with an MDA assay kit. Data are presented as mean ± SD, with statistical significance set at p < 0.05. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Abbreviations: CS, cigarette smoke; CSE, cigarette smoke extract; DPI, Diphenyleneiodonium chloride; EGCG, epigallocatechin-3-gallate; GSK, GSK2795039; LPS, lipopolysaccharide; MDA, malondialdehyde; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; QKPC, Qingke Pingchuan; Que, quercetin; ROS, reactive oxygen species.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques: In Vivo, In Vitro, Western Blot, Fluorescence, Multiple Displacement Amplification

Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 9. Proposed mechanisms of QKPC action against ECOPD. Abbreviations: COPD, chronic obstructive pulmonary disease; EGCG, epigallocatechin-3-gallate; LPS, lipopolysaccharide; MPO, myeloperoxidase; NE, neutrophil Elastase; NET, neutrophil extracellular trap; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; QKPC, Qingke Pingchuan; ROS, reactive oxygen species.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques:

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: Human TMEM30a partially reconstitutes phospholipid import in ⊗Lem3 S. cerevisiae

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques:

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) ΔLem3 S. cerevisiae transformed with empty vector or two isolates transformed with human TMEM30a were grown on glucose or galactose to induce TMEM30a expression. NBD-phosphatidylcholine uptake was determined by flow cytometry. (B) Concentration dependent effect of Edelfosine on colony growth of serially diluted wild-type S. cerevisiae or ΔLem3 transformed with empty vector or two ΔLem3 isolates transformed with human TMEM30a.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Flow Cytometry, Concentration Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake determined by flow cytometry for wild-type S. cerevisiae transformed with empty vector or ΔLem3 transformed with Lem3, TMEM30a or a chimera (Table 1) of Lem3 and TMEM30a. (B) Quantitation (n=3) of NBD-phosphatidylcholine uptake by ΔLem3 transformed with Lem3-TMEM30a (LT; see Table 1 for sequence), TMEM30a-Lem3 (TL), or TMEM30a-Lem3-TMEM30a (TLT) chimeras. Western blot (top) for V5 antigen contained in sequences encoding TMEM30a and its chimeras isolated from protein extracts of S. cerevisiae grown in galactose to induce insert expression or non-inducing glucose. (C) Concentration dependent effect of Edelfosine on colony formation on glucose or galactose plates for wild-type S. cerevisiae or ΔLem3 transformed with galactose induced human, yeast or chimeric constructs. (D) Effect of Edelfosine on ΔLem3 viability after introduction of human TMEM30a, yeast Lem3p, or chimeras formed from them. Cell number (OD600) in liquid culture of wildtype or ΔLem3 transformed with the stated vectors at defined concentrations (left) or 12.5 μg/ml (right).

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Flow Cytometry, Transformation Assay, Plasmid Preparation, Quantitation Assay, Sequencing, Western Blot, Isolation, Expressing, Concentration Assay, Construct

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask™ Orange Plasma Membrane to mark the plasma membrane (top) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum (row 2), or Golgi (row 3). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria (bottom). (B) Western blot for GFP or plasma membrane Na/K ATPase in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Stable Transfection, Transfection, Staining, Confocal Microscopy, Expressing, Labeling, Western Blot, Flow Cytometry, Incubation

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake by CHO cells transfected with empty vector or a TMEM30a vector assessed by confocal microscopy (40X). Inset, 60X. (B) Uptake of [3H]PAF by CHO cells expressing TMEM30a containing a GFP or Lumio tag (n=3). (C) Phosphatidylserine surface expression is not reduced in TMEM30a transfected CHO cells. Surface phosphatidylserine was detected (n=3) by flow cytometry with annexin V conjugated with Alexa647 as described in “Methods.”

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Expressing, Flow Cytometry

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Quantitative PCR for TMEM30a mRNA after transfection by empty vector or one containing TMEM30a shRNA (n=3). (B) Jurkat viability to Edelfosine exposure after transfection with an empty vector or TMEM30a shRNA (n=3). (C) Jurkat cell uptake of fluorescent NBD-phosphatidylcholine (upper) or NBD-phosphatidylethanolamine (lower) by cells expressing TMEM30a shRNA or its vector (n=3). (D) Quantitation of NBD-phosphatidylcholine accumulation by Jurkat cells expressing TMEM30a shRNA or empty vector (n=3). (E) Uptake of [3H]PAF by Jurkat cells is reduced by TMEM30a shRNA knockdown (n=4). All quantitative measures used triplicate determinations in each experiment.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Expressing, Quantitation Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Flow cytometric analysis of JC-1 green fluorescence (FL1, x axis) and orange/red fluorescence (FL2, y axis) in the presence of the stated azelaoyl lysoPAF concentration in vector and TMEM30a shRNA transfected Jurkat cells. The cationic dye JC1 in functional, polarized mitochondria is aggregated and fluoresces red/orange, while monomeric dye free in the cytoplasm fluoresces green. (B) Flow cytometric analysis of JC-1 fluorescence in the stated concentration of Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Fluorescence, Concentration Assay, Plasmid Preparation, shRNA, Transfection, Functional Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Matrix metalloproteinase production by COOH-terminal heparin-binding fibronectin fragment in rheumatoid synovial cells.

doi: 10.1097/01.lab.0000056999.08437.b2

Figure Lengend Snippet: Figure 3. Activation of extracellular signal-regulated kinase (ERK), p38 mitogen- activated protein kinase (MAPK), and c-Jun NH2-terminal kinase (JNK) in COOH-HBFN-f–stimulated RSF. After incubation with 100 nM COOH-HBFN-f (indicated as HBFN-f) or 2 ng/ml IL-1 for the time periods indicated, RSF were lysed as described. Cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-pospho-ERK1/2 (ph-ERK1/2), anti-ERK1/2, anti-phospho-p38 MAPK (ph-p38), anti-p38, anti-phospho-JNK (ph-JNK), or anti-JNK antibody. RSF derived from four patients with RA were used for experiments with similar results.

Article Snippet: Anti-ERK (K-23; sc-94) and -p38 MAPK (C-20; sc-535) were purchased from Santa Cruz Biotechnology, Inc. Anti-phospho-ERK, -phospho-p38 kinase, -JNK, and -phospho-JNK were obtained from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: Activation Assay, Incubation, SDS Page, Derivative Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Matrix metalloproteinase production by COOH-terminal heparin-binding fibronectin fragment in rheumatoid synovial cells.

doi: 10.1097/01.lab.0000056999.08437.b2

Figure Lengend Snippet: Figure 4. Effects of MAPK inhibitors on MMP production by RSF stimulated with COOH-HBFN-f. After preincubation with PD98059 or SB203580 for 1 hour at the concentrations indicated, RSF were incubated for 48 hours with 100 nM COOH-HBFN-f (indicated as HBFN-f). Conditioned media were analyzed by immunoblotting using specific antibodies for MMP-1, MMP-3, and MMP-13. The amount of sample applied was determined on the basis of DNA content of RSF in the well. RSF derived from four patients with RA were used for experiments with similar results.

Article Snippet: Anti-ERK (K-23; sc-94) and -p38 MAPK (C-20; sc-535) were purchased from Santa Cruz Biotechnology, Inc. Anti-phospho-ERK, -phospho-p38 kinase, -JNK, and -phospho-JNK were obtained from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: Incubation, Western Blot, Derivative Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Matrix metalloproteinase production by COOH-terminal heparin-binding fibronectin fragment in rheumatoid synovial cells.

doi: 10.1097/01.lab.0000056999.08437.b2

Figure Lengend Snippet: Figure 7. Activation of ERK, p38, and JNK in RSF with 41 integrin ligation with CS-1. After incubation with CS-1 at 10 M for the time periods indicated, RSF were lysed as described. Cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-ph-ERK1/2, anti-ERK1/2, anti-ph- p38, anti-p38, anti-ph-JNK, or anti-JNK antibody. RSF derived from four patients with RA were used for experiments with similar results.

Article Snippet: Anti-ERK (K-23; sc-94) and -p38 MAPK (C-20; sc-535) were purchased from Santa Cruz Biotechnology, Inc. Anti-phospho-ERK, -phospho-p38 kinase, -JNK, and -phospho-JNK were obtained from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: Activation Assay, Ligation, Incubation, SDS Page, Derivative Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Matrix metalloproteinase production by COOH-terminal heparin-binding fibronectin fragment in rheumatoid synovial cells.

doi: 10.1097/01.lab.0000056999.08437.b2

Figure Lengend Snippet: Figure 8. Effects of MAPK inhibitors on MMP induction by 41 integrin stimulation with CS-1. RSF were pretreated for 1 hour with the indicated concentrations of PD98059 or SB203580 and thereafter stimulated for 48 hours with 10 M CS-1. Conditioned media were analyzed by SDS-PAGE and immunoblotting using specific antibodies for MMP-1, MMP-3, and MMP-13. The amount of sample applied was determined on the basis of DNA content of RSF in the well. RSF derived from four patients with RA were used for experiments with similar results.

Article Snippet: Anti-ERK (K-23; sc-94) and -p38 MAPK (C-20; sc-535) were purchased from Santa Cruz Biotechnology, Inc. Anti-phospho-ERK, -phospho-p38 kinase, -JNK, and -phospho-JNK were obtained from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: SDS Page, Western Blot, Derivative Assay

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Schematic representing the initial events of the metastatic cascade that can be measured using the CRC-OOC. Tumor cells in the top channel (1) can be visualized and analyzed separately from tumor cells that have invaded and adhered in the endothelial compartment (2). Additionally, tumor cells that are found in the endothelial effluent (3; circulating tumor cells (“CTC-like” cells)) can also be collected and analyzed. (B.) Circulating tumor cells were collected from the endothelial effluent of stretched and not stretched HCT116 CRC-Chips and RNAseq was performed. GO Pathway analysis was performed on a subset of genes with either a 2-fold difference between stretched and not stretched CTCs or an FDR-adjusted p-value <0.1. N=2 biological replicates with 3 pooled chips in each replicate. (C.) Gene expression of neurotransmitter-related genes were measured by a neurotransmitter-specific PCR array. HCT116 tumor cells of stretched and not stretched chips were harvested on day 6 from the top epithelial channel and isolated via FACs. Data is displayed as the gene expression fold change of stretch versus not stretch conditions Expression was normalized to the average of 5 housekeeping genes. Genes that had a 1.5-fold increase or decrease in the stretched condition are displayed. N=3 biological replicates with 3 chips pooled per biological replicate. (D.) Schematic of the production of GABA from glutamate by the enzyme GAD1. In this figure, experiments related to GABA are indicated in purple and experiments related to GAD1 are indicated in light blue. (E.) Representative confocal immunofluorescent images of the epithelial (top; 1) or endothelial (bottom; 2) channel of the CRC-Chips stained for GABA (purple) on day 6. Invaded HCT116 H2B-GFP stain positive for GABA, while HCT116 H2B-GFP tumor cells that are in the top channel stain weakly for GABA. DAPI stains the nuclei of Caco2 C2BBe1 cells in the top channel and endothelial cells in the bottom channel. Scale bars represent 200 μm in the top channel image and 100 μm in the bottom channel images. Top channel images are maximum projections that span a 35 μm Z-height with a 5 μm step size. Bottom channel images are maximum projections that span a 10 μm Z-height with a 5 μm step size. (F.) RNAseq analysis was performed on CRC organoids and normalized GAD1 expression is shown. N=5 independent donors with 2-3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test; ***p<0.001 ( G.) CRC organoids were isolated from stretched chips and qPCR analysis of GAD1 gene expression was performed. N=5 independent doners with 3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test p<0.05. ( H.) GAD1 mRNA expression from TCGA in KRAS, NRAS, or BRAF mutant primary colon cancer tumors. N=196 patients with KRAS, NRAS, or BRAF mutant tumors; N=201 patients with KRAS, NRAS, or BRAF wildtype tumors. Individual data points are shown and median with interquartile range is represented. Data was analyzed with an unpaired t-test; ****p<0.0001. ( I.) Kaplan-Meier curve with univariate analysis of the survival of patients with KRAS, NRAS, or BRAF mutated CRC tumors based on high versus low expression of GAD1 (defined as above or below the median GAD1 mRNA expression z-score of 0.3). Data was extracted from the TCGA. N=254 patients. Data was analyzed using a log-rank (Mantel-Cox test). ( J.) Effluent from the epithelial channel of the patient-derived organoids was collected on day 0 (D0) and day 6 (D6). GABA intensity was analyzed from extracted metabolites N=6 chips per timepoint per patient; n=4 on D0 and 2 on D6 for UK. Data was analyzed using a two-way ANOVA; ***p<0.001; ****p<0.0001. ( K .) US-H2B-GFP (top) and UP-H2B-GFP (bottom) stretched tumor-chips were stained for GABA (purple). Scale bars represent 200 μm. L. Representative 10x immunofluorescence images of the 5 tumors stained for EpCAM (green), CK20 (red), and GABA (purple). Scale bars represent 500 μm and 200 μm for UK. All schematics were made in or are from BioRender.

Article Snippet: ABAT human shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-93288-V) and control shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-108080) were used to produce HCT116 H2B-GFP ABAT knock down cells at a multiplicity of infection (MOI) of 5 and 2 mg/mL of puromycin (Santa Cruz Biotechnology, #sc-108071) was used to select for knocked down cells.

Techniques: Gene Expression, Isolation, Expressing, Staining, Mutagenesis, Derivative Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Invasion of HCT116 tumor-chips in the presence or absence of exogenous GABA (flowed through the epithelial channel) was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=6 chips. Individual data are shown, with mean ± SEM represented and analyzed using a one-way ANOVA; ****p<0.0001. (B.) Intracellular [ 13 C 4 ]GABA or unlabeled GABA was measured via mass spectrometry-based metabolomics in the HCT116 tumor-chips after the addition of exogenous GABA for six days. N=3 chips. (C.) Schematic of GABA catabolism by ABAT, subsequent entry into the TCA cycle, and inhibition of ABAT activity by vigabatrin. In this figure, experiments related to GABA are indicated as purple, and experiments related to ABAT are indicated as teal. (D.) Western blot analysis of ABAT in shRNA control or ABAT shRNA HCT116 tumor cells. Cropped western blot (left) and quantification (right) confirm knockdown of ABAT. (E.) Growth rate of ABAT-knockdown or control HCT116 tumor cells when grown in traditional cell culture methods. N=3. Individual data are shown and mean ± SEM are represented. Data was analyzed using a t-test; **p<0.01. (F.) Numbers of ABAT-knockdown or control HCT116 tumor cells in the top channel on-chip as measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (D6). N=5-6 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; *p<0.05;***p<0.001. (G.) Invasion of ABAT knockdown (KD) or control shRNA HCT116 tumor-chips in the presence or absence of stretching was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=5-6 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; *p<0.05. (H.) Numbers of HCT116 tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). Individual data are shown and mean ± SEM are represented. N=4 chips. Data was analyzed using a two-way ANOVA; **p<0.01. (I.) Invasion of HCT116 tumor-chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; ***p<0.001. (J.) Numbers of US-H2B-GFP (red) or UP-H2B-GFP (blue) tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; ns=p>0.05. (K.) Invasion of US-H2B-GFP or UP-H2B-GFP organoid-tumor chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; **p<0.01. All schematics were made in or are from BioRender.

Article Snippet: ABAT human shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-93288-V) and control shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-108080) were used to produce HCT116 H2B-GFP ABAT knock down cells at a multiplicity of infection (MOI) of 5 and 2 mg/mL of puromycin (Santa Cruz Biotechnology, #sc-108071) was used to select for knocked down cells.

Techniques: Mass Spectrometry, Inhibition, Activity Assay, Western Blot, shRNA, Control, Knockdown, Cell Culture, Fluorescence, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Death Receptor 6 Induces Apoptosis Not through Type I or Type II Pathways, but via a Unique Mitochondria-dependent Pathway by Interacting with Bax Protein *

doi: 10.1074/jbc.M112.362038

Figure Lengend Snippet: Time course of DR6-induced apoptosis. After induction of DR6 expression by the addition of tetracycline (Tet), cells were harvested at various time points as indicated and divided into two portions. Half of the harvested cells were directly lysed and separated by SDS-PAGE, followed by Western blot analysis. The other half of the cells were used for preparation of cytosolic extracts and mitochondria-containing membrane fraction as described under “Experimental Procedures.” A, top panel shows induction of DR6 expression. Second panel shows PARP cleavage. Third, fourth, fifth, and sixth panels show activation of caspase-3, caspase-9, caspase-8, and caspase-7, respectively. The activation of caspases was determined by the formation of active forms of these caspases. This membrane was also reprobed with anti-actin antibody to indicate relative loading of samples (bottom panel). B, top panels show release of cytochrome c from mitochondria (right column) to the cytosol (left column). Second panels show translocation of Bax from cytosol (left panel) to mitochondria (right panel). Third panel show tBid formation and translocation to mitochondria. Fourth panel shows the reprobe of the second panels with anti-COX I antibody confirming the mitochondria remained intact during preparation.

Article Snippet: The plasmid pcDNA4/TO/LacZ-Myc-His, which expresses LacZ protein with a Myc tag after the addition of tetracycline, and the conventional plasmid pcDNA3.1/LacZ-Myc-His, which expresses Myc-tagged LacZ protein, were provided in the vector packages by the vendor (Invitrogen).

Techniques: Expressing, SDS Page, Western Blot, Membrane, Activation Assay, Translocation Assay

Journal: The Journal of Biological Chemistry

Article Title: Death Receptor 6 Induces Apoptosis Not through Type I or Type II Pathways, but via a Unique Mitochondria-dependent Pathway by Interacting with Bax Protein *

doi: 10.1074/jbc.M112.362038

Figure Lengend Snippet: Overexpression of antiapoptotic protein Bcl-2 and Bcl-xL inhibited DR6-induced apoptosis. Cell lysis and fractionation were performed as described previously. A and B, after 24-h transfection, a significant amount of Bcl-2 and Bcl-xL was detected (lanes 3 and 4, top panel). Because the recombinant Bcl-2 and Bcl-xL were expressed as a FLAG-tagged protein, the recombinant Bcl-2 and Bcl-xL were detected with a slower migration rate than endogenous Bcl-2 and Bcl-xL (compare lanes 3 and 4 with lanes 1 and 2). This membrane was also reprobed with anti-actin antibody to indicate relative loading of samples (fourth panel). Second and third panels show tetracycline (Tet)-induced DR6 expression, respectively; fifth and sixth panels, cytochrome c release from mitochondria; seventh and eighth panels, Bax translocation from cytosol to mitochondria. C, top panel shows Western blot for PARP; second to sixth panels, Western blots for caspases; seventh panel, Western blot for actin to indicate relative loading of lysate samples; eighth and ninth panels, Western blots for Bid; tenth and eleventh panels were stained for mitochondrial protein COX I.

Article Snippet: The plasmid pcDNA4/TO/LacZ-Myc-His, which expresses LacZ protein with a Myc tag after the addition of tetracycline, and the conventional plasmid pcDNA3.1/LacZ-Myc-His, which expresses Myc-tagged LacZ protein, were provided in the vector packages by the vendor (Invitrogen).

Techniques: Over Expression, Lysis, Fractionation, Transfection, Recombinant, Migration, Membrane, Expressing, Translocation Assay, Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: miR-132-3p Modulates DUSP9-Dependent p38/JNK Signaling Pathways to Enhance Inflammation in the Amnion Leading to Labor

doi: 10.3390/ijms23031864

Figure Lengend Snippet: The role of p38 and JNK in DUSP9 siRNA-induced expression of proinflammatory cytokines and COX2 as well as PGE2 in WISH cells. WISH cells were transfected with si- DUSP9 or NC for 24 h, followed by 24 h of treatment with DMSO, p38 inhibitor SB203580 (10 μM), or JNK inhibitor SP600125 (20 μM). RT-qPCR analysis of the expression of IL-1β , TNF-α , COX2 ( A , G ), IL-6 , and IL-8 ( B , H ). Magnetic Luminex Assays of the secretion of IL-1β, TNF-α ( C , I ), IL-6, and IL-8 ( D , J ). Western blot analysis of COX2 expression ( E , K ). ELISA analysis of PGE2 level ( F , L ). Data were shown as mean ± SEM, n = 3. * p < 0.05, ** p < 0.01 vs. control (0), # p < 0.05, ## p < 0.01 vs. si- DUSP9 .

Article Snippet: To verify the involvement of p38 and JNK signaling pathways in the induction of IL-1β, IL-6, IL-8, TNF-α, and COX2 as well as PGE2 secretion by DUSP9 siRNA, the cells were transfected with NC or si- DUSP9 for 24 h, followed by 24 h of treatment with dimethyl sulfoxide (DMSO), p38 inhibitor SB203580 (10 μM, Selleck, Houston, TX, USA), or JNK inhibitor SP600125 (20 μM, Selleck, Houston, TX, USA).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Luminex, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Cells

Article Title: 2,3-Bisphosphoglycerate Mutase (BPGM), a Metabolic Player Shaping Stress-Adaptive Transcriptional States in Clear Cell Renal Cell Carcinoma

doi: 10.3390/cells15070633

Figure Lengend Snippet: BPGM expression is elevated in human clear cell renal cell carcinoma. ( a ) Representative immunoblot showing BPGM protein levels in ccRCC tumor tissue of four representative patients (P1–4) and matched adjacent normal kidney samples. ( b ) Paired analysis of BPGM protein expression in matched control and tumor samples from individual patients ( n = 61 paired samples). Each data point represents one biological replicate (individual patient sample). ( c ) Violin plot depicting the distribution of BPGM expression levels in normal kidney tissue and ccRCC samples, illustrating increased expression and interindividual variability in tumor tissue. The dashed line indicates median and dotted lines the interquartile range. Statistical analysis was performed using a paired Student’s t -test. Exact p -values are indicated in the figure.

Article Snippet: BPGM protein was detected using a polyclonal rabbit anti-BPGM antibody (catalog #NBP1-86064, Novus Biologicals, Centennial, CO, USA).

Techniques: Expressing, Western Blot, Control

Journal: Cells

Article Title: 2,3-Bisphosphoglycerate Mutase (BPGM), a Metabolic Player Shaping Stress-Adaptive Transcriptional States in Clear Cell Renal Cell Carcinoma

doi: 10.3390/cells15070633

Figure Lengend Snippet: ccRCC cells show limited sensitivity to epigenetic stress and elevated BPGM expression. ( a , b ) Crystal violet assays of ( a ) 2102EP and ( b ) A498 cells after 24 h treatment with increasing concentrations of Vorinostat (VS; 2–8 µM). ( a ) n = 10 per condition. ( b ) n = 10 (control, 2 µM, 8 µM) and n = 9 (3 µM, 4 µM; one outlier removed). ( c , d ) Basal BPGM protein expression in 2102EP and A498 cells shown as representative immunoblot ( c ) and quantification ( d ) ( n = 6 per condition). ( e ) BPGM mRNA expression after 24 h VS treatment in 2102EP and A498 cells. Expression is shown relative to control. n = 6 (2102EP); n = 5 (A498 control; one outlier removed) and n = 6 (A498 VS). Data are presented as box plots (median with interquartile range; whiskers indicate min–max). Each data point represents one biological replicate. Statistical analysis was performed using Brown–Forsythe ANOVA with Dunnett’s T3 multiple comparisons test ( a , b ), Welch’s t -test ( d ), and unpaired Student’s t -test (2102EP) or Welch’s t -test (A498) for ( e ). Details on test selection and normality assessment are provided in Methods. For clarity, only step-wise comparisons along the concentration series are displayed in panels ( a , b ). Complete multiple comparisons are provided in . Color code: white: 2102EP control; beige: A498 control; red: 2102EP + vorinostat; blue: A498 + vorinostat. Exact p -values are indicated in the figure.

Article Snippet: BPGM protein was detected using a polyclonal rabbit anti-BPGM antibody (catalog #NBP1-86064, Novus Biologicals, Centennial, CO, USA).

Techniques: Expressing, Control, Western Blot, Selection, Concentration Assay

Journal: Molecular and Cellular Biology

Article Title: A Multifunctional Domain in Human CRM1 (Exportin 1) Mediates RanBP3 Binding and Multimerization of Human T-Cell Leukemia Virus Type 1 Rex Protein

doi: 10.1128/mcb.23.23.8751-8761.2003

Figure Lengend Snippet: FIG. 1. The amino acid residues of hCRM1 required for Rex ac- tivity. (A) The ability of CRM1s to support Rex activity. HeLa cells were transfected with the indicated plasmids. After cell lysis, the amount of CAT and the activity of -Gal were measured and CAT/ -Gal ratios were calculated. The ratio for the control sample without pSRTAgRexM64 and pSRCRM1s was arbitrarily set at 1. The amount of CAT and the -Gal activity in control samples were over 300 pg and 3.0 103 U, respectively. Error bars represent standard deviations. (B) Restoration of Rex activity by overexpression of CRM1s in HeLa cells. The experimental procedure was the same as that described for panel A. (C) Effect of overexpressing CRM1s on Rex activity in REF52 cells. REF52 cells were transfected with the indicated plasmids. At 24 h of posttransfection, CAT/-Gal ratios were calculated. The ratio for the sample without pSRCRM1s was arbi- trarily set at 1. (D) Western blot analysis of various CRM1s. A fraction of each sample used in the experiments described for panel C was subjected to Western blot analysis using the anti-hCRM1 antibody to examine CRM1 protein synthesis. This antibody was raised with the peptide, which represents the carboxy-terminal region of hCRM1 and has a sequence different from that of rCRM1, so it does not recognize endogenous rCRM1. (E) Effect of CRM1s on Rex-mediated Gag expression from HTLV-1 molecular clone. At 48 h posttransfection, Gag/-Gal ratios were calculated. The ratio for the sample without pSRCRM1s was arbitrarily set at 1. (F) A schematic representation of hCRM1 functional domains. The amino acid sequence of hCRM1 is shown. Experiments characterizing the RanGTP binding domain (bro- ken line) (33), LMB binding residue (arrowhead) (24), Rev-interacting amino acids (asterisks) (2), and the domain binding to NES (under- line) (33) have been previously reported. The residues 411, 414, 474, and 481 in hCRM1 are indicated in bold characters, and the corre- sponding residues of rCRM1 are indicated under the hCRM1 se- quence in single-letter amino acid code.

Article Snippet: Mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and affinity-purified chicken antihCRM1 antibody (18) were used as primary antibodies to detect GAL-fused proteins and CRM1s, respectively.

Techniques: Activity Assay, Transfection, Lysis, Control, Over Expression, Western Blot, Sequencing, Expressing, Functional Assay, Binding Assay, Residue

Journal: Molecular and Cellular Biology

Article Title: A Multifunctional Domain in Human CRM1 (Exportin 1) Mediates RanBP3 Binding and Multimerization of Human T-Cell Leukemia Virus Type 1 Rex Protein

doi: 10.1128/mcb.23.23.8751-8761.2003

Figure Lengend Snippet: FIG. 2. In vivo interaction of Rex with CRM1 mutants in which one amino acid is replaced. REF52 cells were transfected with the plasmid expressing GAL-CRM1s in combination with pRexVP, pG5BCAT, and pCDM-gal. The cells were harvested and subjected to CAT and -Gal assays, and CAT/-Gal ratios were calculated. The ratio for the control sample, which detected the interaction between GAL-hCRM1 and Rex-VP, was arbitrarily set at 1. The amount of CAT and -Gal activity in control samples were over 400 pg and 3.0 103 U, respec- tively. GAL4 nonfusion protein, expressing only a GAL4 region, was used as a negative control. A fraction of each sample was subjected to Western blot analysis using the anti-GAL4 monoclonal antibody to examine GAL-CRM1 expression.

Article Snippet: Mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and affinity-purified chicken antihCRM1 antibody (18) were used as primary antibodies to detect GAL-fused proteins and CRM1s, respectively.

Techniques: In Vivo, Transfection, Plasmid Preparation, Expressing, Control, Activity Assay, Negative Control, Western Blot

Journal: Molecular and Cellular Biology

Article Title: A Multifunctional Domain in Human CRM1 (Exportin 1) Mediates RanBP3 Binding and Multimerization of Human T-Cell Leukemia Virus Type 1 Rex Protein

doi: 10.1128/mcb.23.23.8751-8761.2003

Figure Lengend Snippet: FIG. 6. Binding characterization of two amino acid-substituted CRM1s. (A) In vivo interaction of CRM1s with Rex. REF52 cells were treated as described for Fig. 2 except for the plasmids that expressed two amino acid-substituted CRM1s as a GAL4 fusion protein. A portion of each sample was subjected to Western blot analysis to confirm GAL-CRM1 expression. (B) In vitro binding of h411/414 to RanBP3. As shown in Fig. 5A, in vitro-translated CRM1s were incubated with GST or GST-RanBP3 immobilized on glutathione-Sepharose 4B.

Article Snippet: Mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and affinity-purified chicken antihCRM1 antibody (18) were used as primary antibodies to detect GAL-fused proteins and CRM1s, respectively.

Techniques: Binding Assay, In Vivo, Western Blot, Expressing, In Vitro, Incubation