Journal: Pharmaceutics

Article Title: The Influence of Short Motifs on the Anticancer Activity of HB43 Peptide

doi: 10.3390/pharmaceutics14051089

Figure Lengend Snippet: Effect of HB43 and mutants on cell viability of ( A ) SW480, ( B ) SW620, and ( C ) HT29 cells. Cells were treated with peptides for 24 h ( left ) and 48 h ( right ). Cell viability was measured using the AlamarBlue assay. Results were calculated as percent of control and represented as means ± S.D. of three independent experiments.

Article Snippet: Human colorectal cancer cell lines SW620 (ATCC, #CCL-227, isolated from the large intestine of a 51-year-old male Dukes C colorectal cancer patient), SW480 (ATCC, #CCL-228, isolated from the large intestine of a male Dukes B colorectal cancer patient), and HT29 (ATCC, #HTB-38, isolated from a white, female colorectal adenocarcinoma patient) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Alamar Blue Assay, Control

Journal: Pharmaceutics

Article Title: The Influence of Short Motifs on the Anticancer Activity of HB43 Peptide

doi: 10.3390/pharmaceutics14051089

Figure Lengend Snippet: Half-maximal inhibitory concentration (IC 50 ) of HB43, mut2 and mut3 peptides (μM) in colon cancer cell lines SW480, SW620, and HT29 after 24-h and 48-h treatment.

Article Snippet: Human colorectal cancer cell lines SW620 (ATCC, #CCL-227, isolated from the large intestine of a 51-year-old male Dukes C colorectal cancer patient), SW480 (ATCC, #CCL-228, isolated from the large intestine of a male Dukes B colorectal cancer patient), and HT29 (ATCC, #HTB-38, isolated from a white, female colorectal adenocarcinoma patient) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Concentration Assay

Journal: Pharmaceutics

Article Title: The Influence of Short Motifs on the Anticancer Activity of HB43 Peptide

doi: 10.3390/pharmaceutics14051089

Figure Lengend Snippet: Effect of HB43 and mutants on cell viability of ( A ) SW480, ( B ) SW620, and ( C ) HT29 cells. Cells were treated with peptides for 24 h ( left ) and 48 h ( right ). Cell viability was measured using the AlamarBlue assay. Results were calculated as percent of control and represented as means ± S.D. of three independent experiments.

Article Snippet: Human colorectal cancer cell lines SW620 (ATCC, #CCL-227, isolated from the large intestine of a 51-year-old male Dukes C colorectal cancer patient), SW480 (ATCC, #CCL-228, isolated from the large intestine of a male Dukes B colorectal cancer patient), and HT29 (ATCC, #HTB-38, isolated from a white, female colorectal adenocarcinoma patient) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Alamar Blue Assay, Control

Journal: Pharmaceutics

Article Title: The Influence of Short Motifs on the Anticancer Activity of HB43 Peptide

doi: 10.3390/pharmaceutics14051089

Figure Lengend Snippet: Half-maximal inhibitory concentration (IC 50 ) of HB43, mut2 and mut3 peptides (μM) in colon cancer cell lines SW480, SW620, and HT29 after 24-h and 48-h treatment.

Article Snippet: Human colorectal cancer cell lines SW620 (ATCC, #CCL-227, isolated from the large intestine of a 51-year-old male Dukes C colorectal cancer patient), SW480 (ATCC, #CCL-228, isolated from the large intestine of a male Dukes B colorectal cancer patient), and HT29 (ATCC, #HTB-38, isolated from a white, female colorectal adenocarcinoma patient) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Concentration Assay

Journal: BMC Microbiology

Article Title: Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-κB and proinflammatory gene program activation in intestinal epithelial cells

doi: 10.1186/1471-2180-4-33

Figure Lengend Snippet: Salmonella infection leads to NF-κB nuclear localization even in non-infected cells. HT29 cells were grown on glass coverslips and either mock-infected, left untreated, infected with Salmonella typhimurium , or treated with TNFα (10 ng/ml). Cells fixed after 30 min (TNF) and 1 h ( Salmonella ) as described in Experimental Procedures and Salmonella that had invaded HT29 cells were detected by direct fluorescence microscopy of GFP expression, p65(RelA) localization was monitored by indirect immunoflourescence of rabbit anti-p65 antibody detected with FITC-conjugated donkey anti-rabbit antibody. DAPI was used to stain nuclei. A, HT29 cells were mock-infected or infected at an MOI of 50 with Salmonella typhimurium strain SJW1103G which expresses GFP from the ssaH promoter that is only active inside infected host cells [10,34]. Cells were photographed using bright field microscopy (BF), and immunoflourescence to detect GFP or DAPI staining as indicated. Images were merged (overlay) to reveal cells that were infected. B, HT29 cells were left untreated, infected with Salmonella typhimurium strain 1103 or treated with TNFα. NF-κB p65(RelA) localization under various conditions as indicated was monitored by indirect immunofluorescence. Cells were visualized by bright field microscopy (BF), cell nuclei were stained with DAPI and p65(RelA) was visualized with FITC. DAPI staining was falsely colored red to make visualization of the merge (overlay) easier to distinguish.

Article Snippet: HT29 human intestinal (colorectal adenocarcinoma) epithelial cells (ATCC HTB-38), HeLa cervical epithelial adenocarcoma cells (ATCC CCL-2), 293T kidney cells (CRL-11268), A549 lung carcinoma cells (ATCC-185), and T98G glioblastoma cells (ATCC CRL-1690) were cultured in DMEM with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: Infection, Fluorescence, Microscopy, Expressing, Staining, Immunofluorescence

Journal: BMC Microbiology

Article Title: Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-κB and proinflammatory gene program activation in intestinal epithelial cells

doi: 10.1186/1471-2180-4-33

Figure Lengend Snippet: Protein factor in Salmonella culture broth leads to NF-κB activation. A, Salmonella dublin culture broth concentrated 100-fold was treated as indicated or infectious bacteria, as indicated was used to challenge HT29 cells. NF-κB DNA binding activity was assayed by EMSA from whole cell extracts prepared 45 min after treatment. Authenticity of the NF-κB DNA:protein complex was determined using p65(RelA)-specific and p50-specific antibody supershifts. B, Concentrated Salmonella dublin culture broth (IN) was chromatographed by gel permeation on a Superose 12 column. Eluted protein fractions were analyzed by fractionation on 10% SDS-PAGE and visualized by Coomassie blue (CB) staining. Molecular weight markers for chromatography and on the gels are indicated. Aliquots of each fraction as indicated was used to stimulate HT29 cells and resultant WCEs were analyzed by EMSA for NF-κB DNA binding activity. C, Concentrated Salmonella dublin culture broth (IN) was chromatographed by anion exchange chromatography on POROS HQ matrix. Proteins were eluted with an increasing NaCl gradient as indicated and analyzed on 10% SDS-PAGE and visualized by Coomassie blue (CB) staining. Input and aliquots of each fraction as indicated was used to stimulate HT29 cells and resultant WCEs were analyzed by EMSA for NF-κB DNA binding activity. Eluted material corresponding to protein bands B1-B6, a blank portion of the gel was isolated from a duplicate 10% SDS-PAGE gel as described in Experimental Procedures along with buffer samples from the beginning and end NaCl buffer gradient and used to stimulate HT29 cells and resultant WCEs were analyzed by EMSA for NF-κB DNA binding activity.

Article Snippet: HT29 human intestinal (colorectal adenocarcinoma) epithelial cells (ATCC HTB-38), HeLa cervical epithelial adenocarcoma cells (ATCC CCL-2), 293T kidney cells (CRL-11268), A549 lung carcinoma cells (ATCC-185), and T98G glioblastoma cells (ATCC CRL-1690) were cultured in DMEM with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: Activation Assay, Bacteria, Binding Assay, Activity Assay, Fractionation, SDS Page, Staining, Molecular Weight, Chromatography, Isolation

Journal: BMC Microbiology

Article Title: Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-κB and proinflammatory gene program activation in intestinal epithelial cells

doi: 10.1186/1471-2180-4-33

Figure Lengend Snippet: Flagellin mutants fail to activate NF-κB. EMSAs assaying for NF-κB DNA binding activity in WCEs prepared 45 min from non-infected cells (UN) and after direct infection of HT29 cells with wild-type E. coli DH5α, wild-type Salmonella dublin or SopE - mutant, SopB - mutant, the SopE - /SopB - double mutant, wild-type Salmonella typhimurium strain 1103, the fliC - mutant (fliC::Tn 10 ), the fliC - /fljB - double mutant as indicated at an MOI of 50. B, EMSAs assaying for NF-κB DNA binding activity in WCEs prepared 45 min after challenge of HT29 cells from non-infected cells (UN) or with sterile-filtered concentrated culture broths from wild-type and mutant bacteria as indicated.

Article Snippet: HT29 human intestinal (colorectal adenocarcinoma) epithelial cells (ATCC HTB-38), HeLa cervical epithelial adenocarcoma cells (ATCC CCL-2), 293T kidney cells (CRL-11268), A549 lung carcinoma cells (ATCC-185), and T98G glioblastoma cells (ATCC CRL-1690) were cultured in DMEM with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: Binding Assay, Activity Assay, Infection, Mutagenesis, Sterility, Bacteria

Journal: BMC Microbiology

Article Title: Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-κB and proinflammatory gene program activation in intestinal epithelial cells

doi: 10.1186/1471-2180-4-33

Figure Lengend Snippet: Flagellin is required for activating multiple signaling pathways during Salmonella infection and leads to nuclear localization of NF-κB. HT29 cells were left untreated, stimulated with TNFα (10 ng/ml) or a cocktail of anisomycin [An] (20 μg/ml)/PMA (12.5 ng/ml) for 15 min, or infected with either wild-type (WT) Salmonella typhimurium strain 1103 or the Salmonella typhimurium double fliC - /fljB - mutant strain 134 as indicated. WCE were prepared at the indicated times or at 10 min for TNF-treated cells or 15 min for anisomycin/PMA treated cells and used in EMSAs to analyze NF-κB DNA binding activity, or in immuno-kinase assays (KA) using anti-IKK or anti-JNK antibodies to measure IKK and JNK kinase activity on their respective substrates GST-IκBα 1–54 and GST-cJun 1–79 (as indicated). Immunoblot (IB) analysis of equivalent amounts (40 μg) of protein from each extract was fractionated on SDS-PAGE gels and transferred to PVDF membranes and probed with the indicated antibodies to detect bulk IKK, JNK, ERK and p38 as indicated. Immunoblot analysis using phospho-specific antibodies for ERK and p38 to detect activated ERK and p38 are indicated. B, Immunofluorescence demonstrating that flagellin mutant Salmonella fail to infect HT29 cells and that purified flagellin stimulation of HT29 cells leads to NF-κB nuclear p65 (RelA) localization as determined by indirect immunofluorescence. Imaging of the treatment indicated HT29 cells grown on coverslips was essentially the same as in Fig. 1A & 1B. False coloring of the DAPI stain was used to enhance the visualization of both DAPI stained nuclei and p65 nuclear localization.

Article Snippet: HT29 human intestinal (colorectal adenocarcinoma) epithelial cells (ATCC HTB-38), HeLa cervical epithelial adenocarcoma cells (ATCC CCL-2), 293T kidney cells (CRL-11268), A549 lung carcinoma cells (ATCC-185), and T98G glioblastoma cells (ATCC CRL-1690) were cultured in DMEM with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: Protein-Protein interactions, Infection, Mutagenesis, Binding Assay, Activity Assay, Western Blot, SDS Page, Immunofluorescence, Purification, Imaging, Staining

Journal: BMC Microbiology

Article Title: Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-κB and proinflammatory gene program activation in intestinal epithelial cells

doi: 10.1186/1471-2180-4-33

Figure Lengend Snippet: Purified flagellin activates signaling pathways and proinflammatory gene expression in intestinal epithelial cells mimicking that of wildtype a wild-type Salmonella infection. HT29 cells were left untreated or treated with TNFα (10 ng/ml) or a cocktail of anisomycin [An] (20 μg/ml)/PMA (12.5 ng/ml) for 10 min, or with flagellin (1 μg/ml) for the indicated times. WCE were prepared and analyzed by EMSA for NF-κB DNA binding activity, immuno-kinase assays (KA) or immunoblot analysis using phospho-specific antibodies for ERK or p38 to detect activation and with kinase-specific antibodies as described in Fig. 5A to detect bulk kinase abundance as indicated. A, EMSA to detect NF-κB DNA binding activity. Authenticity of the NF-κB bandshift was tested with supershift of the complex with p65(RelA)-specific antibody (α p65), normal rabbit serum (NRS) served as an irrelevant antibody control. B, immunoblot and kinase assays to detect IKK, JNK, ERK and p38 kinase activities and protein abundance as in Fig. 5A. C, semi-quantitative RT-PCR of proinflammatory gene expression of non-treated, wild-type and flagellin double mutant Salmonella typhimurium infected, TNFα (10 ng/ml) or flagellin (1 μg/ml) stimulated cells. HT29 cells were harvested at the indicated times after the indicated treatments and isolated RNA was used to make first strand cDNA that subsequently used in RT-PCR reactions (as described in Experimental Procedures) using gene-specific primers for IL1α, IL1β, IL-8, TNFα, MCP1 and β-actin. β-actin was used as a standard for normalizing expression patterns. Resulting PCR products were fractionated on 2% agarose gels and visualized by eithidium bromide staining.

Article Snippet: HT29 human intestinal (colorectal adenocarcinoma) epithelial cells (ATCC HTB-38), HeLa cervical epithelial adenocarcoma cells (ATCC CCL-2), 293T kidney cells (CRL-11268), A549 lung carcinoma cells (ATCC-185), and T98G glioblastoma cells (ATCC CRL-1690) were cultured in DMEM with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: Purification, Protein-Protein interactions, Gene Expression, Infection, Binding Assay, Activity Assay, Western Blot, Activation Assay, Control, Quantitative Proteomics, Quantitative RT-PCR, Mutagenesis, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Staining

Journal: BMC Microbiology

Article Title: Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-κB and proinflammatory gene program activation in intestinal epithelial cells

doi: 10.1186/1471-2180-4-33

Figure Lengend Snippet: TLR5 is expressed in numerous cell types and has variable responses to flagellin. A, whole cell extracts were prepared from non-stimulated T84, HT29, A549, HeLa, 293T and T98G cells and fractionated on a 8% SDS-PAGE gel, proteins were transferred to PVDF membrane and probed with anti-TLR5 antibody for immunoblot analysis (IB). Protein loading was examined by probing with anti-actin antibody. B, HT29, A549, HeLa, 293T and T98G cells were left untreated (--), treated with flagellin (F) or TNFα (T) and WCEs were prepared after 45 min and used in EMSA to monitor NF-κB DNA binding activity. Authenticity of the NF-κB bandshift was tested with supershift of the complex with p65(RelA)-specific antibody (αp65), normal rabbit serum (NRS) served as an irrelevant antibody control. C, HT29, A549, HeLa, 293T and T98G cells WCEs (50 μg) were fractionated on a 8% SDS-PAGE gel, proteins transferred to Immobilon P and immunoprobed with anti-muc1 (1:450, Santa Cruz). Size markers are listed and muc1 position is indicated with an arrow.

Article Snippet: HT29 human intestinal (colorectal adenocarcinoma) epithelial cells (ATCC HTB-38), HeLa cervical epithelial adenocarcoma cells (ATCC CCL-2), 293T kidney cells (CRL-11268), A549 lung carcinoma cells (ATCC-185), and T98G glioblastoma cells (ATCC CRL-1690) were cultured in DMEM with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: SDS Page, Membrane, Western Blot, Binding Assay, Activity Assay, Control

Journal: BMC Microbiology

Article Title: Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-κB and proinflammatory gene program activation in intestinal epithelial cells

doi: 10.1186/1471-2180-4-33

Figure Lengend Snippet: TLR5 inhibits flagellin-mediated NF-κB reporter gene activity. HT29 cells were transfected in triplicate in 6-well dishes using the indicated DN-TLR mammalian expression vectors or antisense TLR5 (AS TLR5) (2 μg/well), 2× NF-κB Luc reporter gene (100 ng/well), pRL-TK Renilla luciferase for normalization (50 ng/well) adjusted to 4 μg total DNA/well with empty vector pCDNA3.1 DNA. A, Fold-induction of 2× NF-κB Luc reporter gene in non-stimulated cells (light shading) and in TNFα (10 ng/ml) treated cells (dark shading). Lysates were prepared 12 h after stimulation. Results of a representative experiment are shown. B, HT29 cells transfected as in A were treated with flagellin (1 μg/ml) and cell lysates were prepared and analyzed as in Fig. 8A. Results of a representative experiment are shown.

Article Snippet: HT29 human intestinal (colorectal adenocarcinoma) epithelial cells (ATCC HTB-38), HeLa cervical epithelial adenocarcoma cells (ATCC CCL-2), 293T kidney cells (CRL-11268), A549 lung carcinoma cells (ATCC-185), and T98G glioblastoma cells (ATCC CRL-1690) were cultured in DMEM with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: Activity Assay, Transfection, Expressing, Luciferase, Plasmid Preparation

Journal: Biochemical pharmacology

Article Title: Thiazide-like diuretic drug metolazone activates human pregnane X receptor to induce cytochrome 3A4 and multidrug-resistance protein 1

doi: 10.1016/j.bcp.2014.08.025

Figure Lengend Snippet: (A) HepG2, (B) LS180, and (C) LS174T cells transiently transfected with hPXR, CYP3A4-luc, and CMV-Renilla were treated for 24 h with indicated concentrations of rifampicin or MET prior to luciferase assay.

Article Snippet: 2.1 Materials and chemicals HepG2 liver carcinoma cells, HEK293T cells, LS180 and LS174T human intestinal epithelial cells (derived from human colorectal adenocarcinoma), were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Transfection, Luciferase

Journal: Biochemical pharmacology

Article Title: Thiazide-like diuretic drug metolazone activates human pregnane X receptor to induce cytochrome 3A4 and multidrug-resistance protein 1

doi: 10.1016/j.bcp.2014.08.025

Figure Lengend Snippet: Rifampicin and MET activities in CYP3A4 promoter luciferase reporter assays

Article Snippet: 2.1 Materials and chemicals HepG2 liver carcinoma cells, HEK293T cells, LS180 and LS174T human intestinal epithelial cells (derived from human colorectal adenocarcinoma), were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Luciferase

Journal: Biochemical pharmacology

Article Title: Thiazide-like diuretic drug metolazone activates human pregnane X receptor to induce cytochrome 3A4 and multidrug-resistance protein 1

doi: 10.1016/j.bcp.2014.08.025

Figure Lengend Snippet: (A) HepG2, (B) LS180, and (C) LS174T cells transiently transfected with hPXR, CYP3A4-luc, and CMV-Renilla were treated for 24 h with indicated concentrations of rifampicin or indicated diuretics prior to luciferase assay.

Article Snippet: 2.1 Materials and chemicals HepG2 liver carcinoma cells, HEK293T cells, LS180 and LS174T human intestinal epithelial cells (derived from human colorectal adenocarcinoma), were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Transfection, Luciferase

Journal: Biochemical pharmacology

Article Title: Thiazide-like diuretic drug metolazone activates human pregnane X receptor to induce cytochrome 3A4 and multidrug-resistance protein 1

doi: 10.1016/j.bcp.2014.08.025

Figure Lengend Snippet: Real-time PCR analysis of (A) CYP3A4 and (B) MDR1 mRNA expression in LS180 cells and (C) CYP3A4 and (D) MDR1 mRNA expression in LS174T cells after 48-h treatment with different compounds as indicated (*p < 0.05; in the t-test comparisons were made between compound-treated and DMSO-treated samples). Western blot showing CYP3A4 and MDR1 protein levels in (E) LS180 and (F) LS174T cells upon MET treatment. The experiments were repeated two times and the data show one representative experiment. The numbers below the protein bands indicate the relative intensity of the protein bands, with the DMSO treated sample set as “1”. In both (E) and (F), anti-CYP3A4 and anti-MDR1 were used to detect CYP3A4 and MDR1 sequentially. Anti-β-actin was finally used to detect β-actin.

Article Snippet: 2.1 Materials and chemicals HepG2 liver carcinoma cells, HEK293T cells, LS180 and LS174T human intestinal epithelial cells (derived from human colorectal adenocarcinoma), were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot