Journal: International Journal of Molecular Sciences

Article Title: Identification of Actionable Gene Variants in Pulmonary Large-Cell Neuroendocrine Carcinoma: A Real-World Analysis of a Polish Cohort

doi: 10.3390/ijms27072939

Figure Lengend Snippet: The graphic representation of TMEM79::NTRK1 fusion, including exonic (TMEM79 NM_032323.3 exon 3; NTRK1 NM_002529.3 exon 2) and chromosomal (chr1:156256264, chr1:156834146) breakpoint positions based on the output data from the Archer Analysis software version 7.2 (ArcherDX, Inc., Boulder, CO, USA; ( A )) and on the variant visualization by the Alamut Visual Plus v1.12 software (SOPHiA GENETICS, Inc., Boston, MA, USA; ( B )).

Article Snippet: Gene fusion variants were further evaluated using Alamut Visual Plus (v1.12; SOPHiA GENETICS, Inc., Boston, MA, USA).

Techniques: Software, Variant Assay

Journal: F1000Research

Article Title: An unexpected effect of TNF-α on F508del-CFTR maturation and function

doi: 10.12688/f1000research.6683.2

Figure Lengend Snippet: Differentiated primary HBE cell cultures grown at an air-liquid interface were incubated with 50ng/ml TNFα for 10–30min, 3–6h and 24h. CFTR immunodetection was performed with 24.1 anti-CFTR antibody and analyzed with confocal microscopy. Green staining represents CFTR (Alexa Fluor 488), red color staining represents ZO-1 protein of tight junctions (Alexa Fluor 594) and blue DAPI staining visualizes nuclei. Independent TNFα treatments and CFTR immunodetection were performed on HBE cells from three different F508del/F508del CF patients. Representative images of one experiment are demonstrated (Scale bars = 20µm).

Article Snippet: Anti-CFTR antibodies (abs): MM13-4 mouse monoclonal ab against N-terminus of CFTR, (Millipore, France, 05-581); 24-1 mouse monoclonal ab against C-terminus of CFTR (R&D Systems, MAB, 25031).

Techniques: Incubation, Immunodetection, Confocal Microscopy, Staining

Journal: Journal of Biological Chemistry

Article Title: Probing Conformational Rescue Induced by a Chemical Corrector of F508del-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mutant

doi: 10.1074/jbc.m111.239699

Figure Lengend Snippet: FIGURE 5. The effect of chemical correction and ablation of the R553XR555 sequence on conformational correction of F508del-CFTR probed using limited proteolysis. Limited proteolysis of control or chemically corrected F508del-CFTR, F508del-KXK-CFTR, or WT-CFTR with increasing levels of trypsin (0, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 g/ml) was performed. Digests were analyzed by SDS-PAGE, and immunoreactive bands were detected using the Odyssey infrared imaging system from LI-COR Biosciences. The colored signal was converted to grayscale for the preparation of this figure. A, samples were immuno- blotted with anti-CFTR monoclonal antibody, L12B4 (NBD1-specific). Subsequent analyses focused on the larger NBD1 fragments (34–36 kDa) and the smaller NBD1 fragments (lower than 28 kDa). Both sets of fragments were indicated using brackets on the right-hand side of each image. B, samples were immuno- blotted with anti-CFTR monoclonal antibody, M3A7 (NBD2-specific). NBD2 fragments of 28 kDa were analyzed subsequently (indicated using brackets on the right-hand side of each image).

Article Snippet: L12B4 anti-CFTR antibody (1:1000) was used for immunodetection conjugated to IRDye800 (1:10 000) (Rockland Immunochemical Inc).

Techniques: Sequencing, Control, SDS Page, Imaging

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Activation of constitutive androstane receptor inhibits intestinal CFTR-mediated chloride transport.

doi: 10.1016/j.biopha.2019.01.015

Figure Lengend Snippet: Fig. 4. Effects of CAR agonists on CFTR-mediated Cl−

Article Snippet: After centrifugation of the cell and tissue homogenates at 12,000 rpm for 20min at 4 °C, equal amounts of protein (50 μg) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK).The membrane was then incubated for 1 h with 5% non-fat milk and probed overnight at 4 °C with anti-human CFTR, anti-β actin (Cell signaling), anti-mouse CFTR (Abcam) and anti-CAR (R&D systems) (all at 1000-fold dilution) followed by a further incubation with HRP-conjugated secondary antibody for 1 h at room temperature.

Techniques:

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Activation of constitutive androstane receptor inhibits intestinal CFTR-mediated chloride transport.

doi: 10.1016/j.biopha.2019.01.015

Figure Lengend Snippet: Fig. 5. Effects of CAR agonist on mRNA and protein expres- sion of CFTR in T84 cells (A): Quantitative real time PCR ana- lysis of CFTR mRNA expression in T84 cells after treatment with vehicle (0.01% DMSO), CITCO, or phenytoin at the indicated concentrations for 24 h. The results were normalized to GAPDH. (B) Representative blot and densitometric quantification of CFTR protein expression were normalized to β-actin levels in T84 cells treated with vehicle (0.01% DMSO), CITCO, or phenytoin at the indicated concentrations for 24 h. The data are expressed as means ± S.E. (n = 5–7). *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to control.

Article Snippet: After centrifugation of the cell and tissue homogenates at 12,000 rpm for 20min at 4 °C, equal amounts of protein (50 μg) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK).The membrane was then incubated for 1 h with 5% non-fat milk and probed overnight at 4 °C with anti-human CFTR, anti-β actin (Cell signaling), anti-mouse CFTR (Abcam) and anti-CAR (R&D systems) (all at 1000-fold dilution) followed by a further incubation with HRP-conjugated secondary antibody for 1 h at room temperature.

Techniques: Real-time Polymerase Chain Reaction, Lysis, Expressing, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Activation of constitutive androstane receptor inhibits intestinal CFTR-mediated chloride transport.

doi: 10.1016/j.biopha.2019.01.015

Figure Lengend Snippet: Fig. 6. Effects of CAR antagonist on CITCO- induced inhibition of Cl−secretion and CFTR protein expression in T84 cells (A). Effects of CAR antagonist on CITCO-induced inhibition of Cl−secretion in T84 cells. T84 cells were treated with vehicle (0.1% DMSO), CITCO (1 μM), CAR antagonist [CINPA1 (1 μM)], or CITCO plus CINPA1 for 24 h, then the forskolin-stimulated Isc was measured. (left) Representative Isc tracings and (right) summary of the data are shown. Data are ex- pressed as means ± S.E. of percentages of forskolin-stimulated Isc (n = 5). (B) Representative blot and densitometric quanti- fication of CFTR protein expression in T84 cells treated with vehicle alone or together with CITCO (1 μM) and/or CINPA1 (1 μM) for 24 h. The data are expressed as means ± S.E. (n = 5). *, P < 0.05 compared to the vehicle- treated control. NS, not significantly different from control. ##, P < 0.05; ##, P < 0.01 compared to CITCO-treatment group.

Article Snippet: After centrifugation of the cell and tissue homogenates at 12,000 rpm for 20min at 4 °C, equal amounts of protein (50 μg) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK).The membrane was then incubated for 1 h with 5% non-fat milk and probed overnight at 4 °C with anti-human CFTR, anti-β actin (Cell signaling), anti-mouse CFTR (Abcam) and anti-CAR (R&D systems) (all at 1000-fold dilution) followed by a further incubation with HRP-conjugated secondary antibody for 1 h at room temperature.

Techniques: Inhibition, Expressing, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Activation of constitutive androstane receptor inhibits intestinal CFTR-mediated chloride transport.

doi: 10.1016/j.biopha.2019.01.015

Figure Lengend Snippet: Fig. 7. Effect of CAR activation on CFTR-mediated Cl−transport during inhibition of mRNA synthesis in T84 cells. T84 cells were treated with ve- hicle (0.1% DMSO), CITCO (1 μM), or actinomycin D (Act D) (5 μg/ml), or CITCO plus Act D for 24 h, after that CFTR expression was measured by real- time PCR analysis. The results were normalized to GAPDH. The data are ex- pressed as means ± S.E. (n = 5). *, P < 0.05; ***, P < 0.001 compared to the vehicle-treated control. NS, not significantly different.

Article Snippet: After centrifugation of the cell and tissue homogenates at 12,000 rpm for 20min at 4 °C, equal amounts of protein (50 μg) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK).The membrane was then incubated for 1 h with 5% non-fat milk and probed overnight at 4 °C with anti-human CFTR, anti-β actin (Cell signaling), anti-mouse CFTR (Abcam) and anti-CAR (R&D systems) (all at 1000-fold dilution) followed by a further incubation with HRP-conjugated secondary antibody for 1 h at room temperature.

Techniques: Activation Assay, Inhibition, Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Activation of constitutive androstane receptor inhibits intestinal CFTR-mediated chloride transport.

doi: 10.1016/j.biopha.2019.01.015

Figure Lengend Snippet: Fig. 9. Effect of CAR activation on CFTR expression and function in mouse intestine. (A): Activation of CAR target gene (MDR1) in mouse intestine. B: Quantitative PCR analysis of CFTR mRNA expression in mouse intestinal tissues after intraperitoneally injected the mice with 3 mg/kgBW TCPOBOP (CAR agonist) or vehicle (corn oil) for 7 days. The results were normalized to GAPDH. C: Representative blot and densitometric quantification of CFTR pro- tein expression in mouse intestinal tissues after treated the mice with TCPOBOP or vehicle (corn oil) for 7 days. The results were nor- malized to β-actin levels. The data are pre- sented as mean ± SE (n = 8). *, P < 0.05; **, P < 0.01 compared to control. D. Antidiarrheal application of CAR activation on cholera toxin-induced intestinal fluid accumu- lation in mouse closed-loop model. ICR mice were treated with 3 mg/kgBW TCPOBOP or vehicle (corn oil) for 7 days. After treatment, PBS or PBS containing cholera toxin (CT) (1 μg/loop) were instilled into ileal loops. Four hours later, ileal loops were removed for de- termination of loop weight/length ratio. Representative photograph of ileal loops and summary of the data are shown. Data are ex- pressed as means ± S.E. of loop weight/ length ratio (n = 8). **, P < 0.01 compared with CT-treated control. NS, not significantly different compared with PBS-treated control.

Article Snippet: After centrifugation of the cell and tissue homogenates at 12,000 rpm for 20min at 4 °C, equal amounts of protein (50 μg) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK).The membrane was then incubated for 1 h with 5% non-fat milk and probed overnight at 4 °C with anti-human CFTR, anti-β actin (Cell signaling), anti-mouse CFTR (Abcam) and anti-CAR (R&D systems) (all at 1000-fold dilution) followed by a further incubation with HRP-conjugated secondary antibody for 1 h at room temperature.

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Injection, Control

Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Differential regulation of cystic fibrosis transmembrane conductance regulator by interferon gamma in mast cells and epithelial cells.

doi: 10.1124/jpet.105.087528

Figure Lengend Snippet: Fig. 1. Western blot analysis of CFTR and STAT1 expression in un- treated (lane 1), PMA-treated (lane 2), TNF-treated (lane 3), and IFN- treated (lane 4) T84 (A) and RCMC (B). T84 cells were treated with PMA (10 ng/ml), human recombinant TNF (10 ng/ml), or IFN (10 ng/ml) for 24 h. RCMC were treated with PMA (10 ng/ml) or rat recombinant TNF (10 ng/ml) or IFN (10 ng/ml) for 24 h. Cell lysates were resolved on an 4 to 12% SDS-PAGE and blotted with anti-CFTR, anti-STAT1, or anti-actin antibody. Western blots shown represent three independent experiments. The effect of 24 h IFN treatment (80 ng/ml) on CFTR expression was characterized in T84 cells (C) and rat peritoneal mast cells (D), using confocal laser scanning microscopy (bar on the figure represents 10 m).

Article Snippet: The membranes were blocked with 3% milk in Tris-buffered saline-0.05% Tween for 1 h and then probed with primary antibodies against CFTR (clone H-182) and STAT1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), phosphoSTAT1 (BD Transduction Laboratories, Chicago, IL), phospho-stress-activated MAPK/c-Jun NH2-terminal kinase (JNK) (Thr183/Tyr185; Cell Signaling Technology Inc.), phospho-p38 MAPK (Thr180/Tyr182; Cell Signaling Technology Inc.), and phospho ERK1/2 (Thr202/Tyr204; Cell Signaling Technology Inc.), or anti-actin (Sigma-Aldrich) in 4% BSA/PBS for 1 h at room temperature.

Techniques: Western Blot, Expressing, Recombinant, SDS Page, Confocal Laser Scanning Microscopy

Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Differential regulation of cystic fibrosis transmembrane conductance regulator by interferon gamma in mast cells and epithelial cells.

doi: 10.1124/jpet.105.087528

Figure Lengend Snippet: Fig. 4. A, IFN-mediated up-regulation of mast cell CFTR protein requires activation of JAK2 and/or p38 and ERK. RCMC, LAD2, and T84 were treated with IFN (10 ng/ml) with or without JAK2 inhibitor (AG-490; 30 mg/ml), p38 inhibitor (SB202190; 30 mg/ml), or ERK MAPK inhibitor (U0126; 30 mg/ml) for 24 h. Representative of three inde- pendent experiments. B, densitometry summary of data in Fig. 4. RCMC were treated with IFN (10 ng/ml) with or without JAK2 inhibitor (AG-490; 30 mg/ml), p38 inhibitor (SP202190; 30 mg/ml), or ERK inhibitor (U0126; 30 mg/ml) for 24 h, and expression of CFTR (black columns) and STAT1 (gray columns) was analyzed by Western blot (n 3 independent experiments; error bars represent S.E.M.). As- terisks represent statistical significance as determined by Student’s t test (p 0.05).

Article Snippet: The membranes were blocked with 3% milk in Tris-buffered saline-0.05% Tween for 1 h and then probed with primary antibodies against CFTR (clone H-182) and STAT1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), phosphoSTAT1 (BD Transduction Laboratories, Chicago, IL), phospho-stress-activated MAPK/c-Jun NH2-terminal kinase (JNK) (Thr183/Tyr185; Cell Signaling Technology Inc.), phospho-p38 MAPK (Thr180/Tyr182; Cell Signaling Technology Inc.), and phospho ERK1/2 (Thr202/Tyr204; Cell Signaling Technology Inc.), or anti-actin (Sigma-Aldrich) in 4% BSA/PBS for 1 h at room temperature.

Techniques: Activation Assay, Expressing, Western Blot

Journal: Scientific Reports

Article Title: PGE 2 is a direct and robust mediator of anion/fluid secretion by human intestinal epithelial cells

doi: 10.1038/srep36795

Figure Lengend Snippet: ( a ) mRNA of membrane-bound ion channels and transporters are expressed in cultured human small intestinal organoids. Semi-quantitative RT-PCR analysis shows expression of CFTR, NKCC1, KCNQ1 and KCNN4 in small intestine-derived organoids. Results acquired from the whole small intestinal tissue sample are shown as a reference. dH 2 O served as a negative control. The number of bp indicates the length of the targeted amplification region for each gene. ( b ) Immunostaining of NKCC1 and KCNQ1 shows their expression at the basolateral membrane of cultured small intestinal organoids. Organoids were fixed and subjected to immunostaining using primary antibodies specific for NKCC1 or KCNQ1. The expression was visualized by FITC-labeled Tyramide (green). The stainings using non-immunized mouse IgG and rabbit IgG served as negative controls. Scale bar represents 100 μm. ( c,d ) Inhibitory effects of various compounds on the PGE 2 -induced swelling of jejunal organoids that were established from the uninflamed region of a CD patient ( c ) and from the healthy mucosa of a non-IBD patient ( d ). Inhibitors were added 1 hour prior to PGE 2 -induction at the following concentration: GlyH-101 (50 μM), PPQ-102 (10 μM), CFTR Inhibitor-172 (CFTRinh-172) (50 μM), glyburide (500 μM), 4-AP (1 mM), XE 991 (10 μM), HMR 1556 (10 μM), TRAM 34 (10 μM) and bumetanide (100 μM). PGE 2 -induced swelling was quantified 30 min after the PGE 2 -induction (10 −9 M). Data are shown as the mean ± SEM of three independent wells. *indicates P < 0.05, **indicates P < 0.005, ***indicates P < 0.0005, ****indicates P < 0.0001 as determined by two-sided Student’s t-test compared to the data of Inhibitor (−). All results are representative of at least three independent experiments.

Article Snippet: The chemicals and reagents that were used in the present study were as follows: PGA 2 , PGD 2 , PGE 1 , PGE 2, PGF 2 α, PGI 2 and bumetanide (Cayman Chemical, Michigan, USA); VIP, ACh, histamine, bradykinin and serotonin hydrochloride (Sigma-Aldrich Japan, Tokyo, Japan); glyburide, GlyH-101, CFTRinh172 and PPQ-102 (Santa Cruz, Texas, USA); XE 991 dihydrochloride, 4-AP, HMR 1556 and TRAM 34 (Tocris Bioscience, Bristol, UK); recombinant human IL-10, IL-12, IL-17, IL-33 and TL1A/TNFSF15 (R&D Systems, Minneapolis, USA); and recombinant human TNF-α, IFN-α, IFN-γ, IL-1β, IL-4, IL-6, IL-22 and IL-24 (PeproTech, Rocky Hill, USA).

Techniques: Membrane, Cell Culture, Quantitative RT-PCR, Expressing, Derivative Assay, Negative Control, Amplification, Immunostaining, Labeling, Concentration Assay