Journal: International journal of molecular medicine

Article Title: Crosstalk between TLR5 and Notch1 signaling in epithelial cells during intestinal inflammation.

doi: 10.3892/ijmm.2013.1501

Figure Lengend Snippet: Figure 3. ������������������������������������������������������������������������������������������������������ ����������������������������������������� Flagellin-induced expression of Notch in colon epithelial cells. (A and B) Mice were anesthetized for 90-120 min with an intra‑peritoneal pento barbital injection and then given an intrarectal administration of TNBS (100 µl, 125 mg/kg) dissolved in 50% ethanol, with or without flagellin (40 µg/kg) or LPS (80 µg/kg). Colonic epithelial cells were then harvested for Notch1 and Jagged1 expression from the extracted total RNA by real‑time PCR at different time‑points. β‑actin served as the internal control intrarectal flagellin injection induces the Notch1 and Jagged1 expression in a time‑dependent manner. *p<0.05 vs. Flagellin (-) or LPS (-). Error bars indicate the SEM for values obtained from three independent experiments. (C) Colon‑26 cells (1x105 cells) were cultured in 6‑well plates in the presence or absence of flagellin at various time points, and Hes1 expression was assessed using real‑time PCR. Flagellin sig nificantly induced the Hes1 expression in Colon‑26 cells. *p<0.05 vs. Flagellin(-)/Jagged1‑Fc(-), respectively. Error bars indicate the SEM for values obtained from three independent experiments. (D) Effects of flagellin on RBP‑Jκ‑mediated reporter gene expression. Colon‑26 cells (2.5x104 cells/well) were plated in 24‑well plates. After 12‑16 h, the cells were transfected with an RBP‑Jκ reporter construct with an internal control vector, as described in the manufacturer's instructions. Similarly, co‑transfection with mock and DN‑TLR5 vectors was also performed using Lipofectamine 2000 in corresponding wells. At 12 h after transfection, the cells were treated with DAPT (10 µM), Jagged1-Fc (5 µg/ml) and flagellin (10 ng/ml) for 12 h. A dual luciferase assay was then performed using total cell extracts. A significant induction of N1ICD‑RBP‑Jk‑mediated reporter gene expression following flagellin stimulation was observed. RBP-Jκ activity was increased in the presence of Notch ligand Jagged1, whereas it was reduced in the presence of DAPT. **p<0.01, *p<0.05 vs. Flagellin(-) Control; #p<0.05 vs. Flagellin(+) Control. Error bars indicate the SEM for values obtained from three independent experiments. (E) In order to block the endogenous expression of Jagged1, commercially available mouse Jagged1‑specific siRNA was used. For siRNA transfection, Colon‑26 cells (2.5x104cells/well) were plated in 24‑well culture plates. After 12-16 h, the cells were transfected with 20 pmols each of duplex siRNAs for Jagged1 or non‑target‑specific negative control, using the siRNA‑trasfection reagent. To determine inhibition of the target gene, the Jagged1 expression was assessed by real‑time PCR at ~24‑36 h after transfection. *p<0.05 vs. negative control siRNA. Error bars indicate the SEM for values obtained from three independent experiments. (F) Effects of Jagged1 siRNA on flagellin‑induced RBP‑Jκ reporter gene expression. Colon‑26 cells (2.5x104 cells/well) were plated in 24‑well plates. After 12‑16 h, the cells were co‑transfected with 20 pmols each of duplex siRNAs for Jagged1 or negative control and RBP‑Jκ reporter constructs with internal control using trasfec tion reagent, as described in the manufacturer's instructions. At 24-36 h after transfection, the cells were treated with flagellin (100 ng/ml) for 12 h, after which a dual luciferase assay was carried out using total cell extracts. Knockdown of Jagged1 significantly reduced the flaggelin effects on RBP-Jκ activity. *p<0.05 vs. Flagellin(-) siRNA(-)/NC‑si; #p<0.05 vs. Flagellin(+) siRNA(-)/NC‑si. Error bars indicate the SEM for values obtained from three independent experiments.

Article Snippet: Trinitrobenzene sulfonic acid (TNBS; Sigma, St. Louis, MO, USA), dextran sodium sulphate (DSS, 5 kDa; Wako Pure Chemicals), flagellin (Salmonella typhimurium; InvivoGen, San Diego, CA, USA), recombinant rat Jagged1-Fc (R&D Systems, Minneapolis, MS, USA), γ-secretase inhibitor DAPT (Tocris Bioscience, Bristol, UK), NEMO-binding domain (NBD) inhibitory peptide (Imgenex, San Diego, CA, USA), Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), pNF-κB-Luc (Stratagene, Santa Clara, CA, USA), pRL-TK (Promega, Madison, WI, USA), mock (pZERO-mcs), a mouse dominant-negative TLR5 (pZERO-mTLR5) vector set (InvivoGen), RBP-Jκ Cignal Reporter assay (SA Biosciences, Valencia, CA, USA), mouse RBP-Jκ siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were obtained from respective sources.

Techniques: Expressing, Injection, Real-time Polymerase Chain Reaction, Control, Cell Culture, Gene Expression, Transfection, Construct, Plasmid Preparation, Luciferase, Activity Assay, Blocking Assay, Negative Control, Inhibition, Knockdown

Journal: International journal of molecular medicine

Article Title: Crosstalk between TLR5 and Notch1 signaling in epithelial cells during intestinal inflammation.

doi: 10.3892/ijmm.2013.1501

Figure Lengend Snippet: Figure 4. (A) In vitro effects of Notch on TLR5‑mediated NF‑κB signaling with or without treatment of NEMO‑binding domain (NBD) inhibitor peptide. Colon‑26 cells (2.5x104 cells/well) were plated in 24‑well plates for 12‑16 h, then transfected with pNF‑κB‑luc and pRL‑TK‑luc vectors using Lipofectamine 2000. At 12 h after transfection, the cells were treated with or without NBD inhibitor peptide (100 µM), DAPT (10 µM), recombinant Jagged1 (5 µg/ml) and flagellin (100 ng/ml). N.S., not statistically significant. **p<0.01, *p<0.05 vs. Flagellin(-) DMSO; #p<0.05 vs. Flagellin(+) DMSO. Error bars indicate the SEM for values obtained from three independent experiments. (B-E) In vitro effects of Notch on TLR5‑mediated IL‑6 activation. (B) Colon‑26 cells (2.5x104cells/well) were plated in 24‑well culture plates in the presence or absence of flagellin (10 ng/ml) at various time‑points. IL‑6, IL‑1β and TNF‑α expression was assessed by real‑time PCR from the extracted RNA samples at different time‑points. Error bars indicate the SEM for values obtained from three independent experiments. (C) Mouse IL‑6 promoter construct, pIL‑6/1278‑luc (200 ng/well) and pRL‑TK‑luc (20 ng/well) were transfected using Lipofectamine 2000 (2.5 µl/well) into Colon‑26 cells (2.5x104 cells/well), plated in 24‑well cell culture plates. Following treatment for 12 h with or without DMSO, DAPT (10 µM), Jagged1-Fc (5 µg/ml) and flagellin (10 ng/ml) for 12 h, dual luciferase assays were performed with extracted proteins. **p<0.01, *p<0.05 vs. Flagellin(-) DMSO; #p < 0.05 vs. Flagellin(+) DMSO. Error bars indicate the SEM for values obtained from three independent experiments. (D) For RBP‑Jκ‑siRNA transfection, Colon‑26 cells (2.5x104cells/well) were plated in 24‑well culture plates. After 12‑16 h, the cells were transfected with 20 pmols each of duplex siRNAs for RBP‑Jκ- or non‑target‑specific negative control, using the siRNA‑trasfection reagent. Inhibition of RBP‑Jκ expression was assessed by real‑time PCR at ~24‑36 h after transfection. *p<0.05 vs. negative control siRNA. Error bars indicate the SEM for values obtained from three independent experiments. (E) At 24‑36 h after siRNA tansfection, Colon‑26 cells were stimulated with flagellin (10 ng/ml) and the IL‑6 expression was assessed at different time‑points. *p<0.05 vs. Flagellin(-) siRNA(-)/NC-si; #p<0.05 vs. Flagellin(+) siRNA(-)/NC‑si. Error bars indicate the SEM for values obtained from three independent experiments.

Article Snippet: Trinitrobenzene sulfonic acid (TNBS; Sigma, St. Louis, MO, USA), dextran sodium sulphate (DSS, 5 kDa; Wako Pure Chemicals), flagellin (Salmonella typhimurium; InvivoGen, San Diego, CA, USA), recombinant rat Jagged1-Fc (R&D Systems, Minneapolis, MS, USA), γ-secretase inhibitor DAPT (Tocris Bioscience, Bristol, UK), NEMO-binding domain (NBD) inhibitory peptide (Imgenex, San Diego, CA, USA), Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), pNF-κB-Luc (Stratagene, Santa Clara, CA, USA), pRL-TK (Promega, Madison, WI, USA), mock (pZERO-mcs), a mouse dominant-negative TLR5 (pZERO-mTLR5) vector set (InvivoGen), RBP-Jκ Cignal Reporter assay (SA Biosciences, Valencia, CA, USA), mouse RBP-Jκ siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were obtained from respective sources.

Techniques: In Vitro, Transfection, Recombinant, Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Construct, Cell Culture, Luciferase, Negative Control, Inhibition

Journal: International journal of molecular medicine

Article Title: Crosstalk between TLR5 and Notch1 signaling in epithelial cells during intestinal inflammation.

doi: 10.3892/ijmm.2013.1501

Figure Lengend Snippet: Figure 5. In vivo effects of DAPT during TNBS‑mediated acute colitis. (A) DAPT was intraperitoneally injected into mice (5 mice/group) at the time of intrarectal TNBS administration. Five days after TNBS treatment, the distal colonic part was dissected from mice treated with or without DAPT or TNBS, then N1ICD status was checked by western blotting using anti‑N1ICD antibody. β-actin served as the loading control. The image shown is representative of results obtained from three independent experiments. (B) IL‑6 contents in dissected colon tissues treated with or without DAPT or TNBS were assessed by ELISA. (C) Effects of DAPT on body weight changes in TNBS‑treated mice. Data are expressed as serial changes in percentage of weight change until eutha nasia. *p<0.05 vs. TNBS(-) DMSO; #p<0.05 vs. TNBS(+) DMSO. Error bars indicate the SEM for values from three independent experiments (5 mice/group). (D) Representative figure of in vivo effects of DAPT on colon length in mice with TNBS‑induced colitis. (E) Representative image of histological changes in distal colonic lesions in mice with TNBS‑induced colitis with or without DAPT (original magnification, x20). (F) Colonic histological scores. During each histological examination, three different parameters were determined: severity of inflammation (based on polymorphonuclear neutrophil infiltration; 0‑3: none, slight, moderate, severe), depth of injury (0-3: none, mucosal, mucosal and submucosal, transmural) and crypt damage (0-4: none, basal one-third damaged, basal two-thirds damaged, only surface epithelium intact, entire crypt and epithelium lost). The score for each parameter was multiplied by a factor reflecting the percentage of tissue involvement (x1, 0-25%; x2, 26-50%; x3, 51-75%; x4, 76-100%) and all values were added to a sum, with a maximum possible score of 40. N.D., not detected. *p<0.05 vs. TNBS(-) DMSO; #p<0.05 vs. TNBS(+) DMSO.

Article Snippet: Trinitrobenzene sulfonic acid (TNBS; Sigma, St. Louis, MO, USA), dextran sodium sulphate (DSS, 5 kDa; Wako Pure Chemicals), flagellin (Salmonella typhimurium; InvivoGen, San Diego, CA, USA), recombinant rat Jagged1-Fc (R&D Systems, Minneapolis, MS, USA), γ-secretase inhibitor DAPT (Tocris Bioscience, Bristol, UK), NEMO-binding domain (NBD) inhibitory peptide (Imgenex, San Diego, CA, USA), Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), pNF-κB-Luc (Stratagene, Santa Clara, CA, USA), pRL-TK (Promega, Madison, WI, USA), mock (pZERO-mcs), a mouse dominant-negative TLR5 (pZERO-mTLR5) vector set (InvivoGen), RBP-Jκ Cignal Reporter assay (SA Biosciences, Valencia, CA, USA), mouse RBP-Jκ siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were obtained from respective sources.

Techniques: In Vivo, Injection, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: International journal of molecular medicine

Article Title: Crosstalk between TLR5 and Notch1 signaling in epithelial cells during intestinal inflammation.

doi: 10.3892/ijmm.2013.1501

Figure Lengend Snippet: Figure 6. Crosstalk between TLR5 and Notch1 signaling in adjacent epithelial cells during intestinal inflammation. Flagellin‑induced TLR5 signaling leads to an increased expression of Notch1 and Jagged1, which facilitates receptor‑ligand‑induced cleavage of N1ICD by γ‑secretase for RBP‑Jκ‑mediated IL‑6, as well as the canonical Notch target gene, Hes1 expression. Using DAPT, a γ‑secretase inhibitor, we found evidence that TLR5 and Notch1 cumulative signaling exhibit control over target gene (IL‑6) expression in colonic epithelial cells. DAPT not only inhibited RBP‑Jκ functions by blocking production of N1ICD, but also reduced TLR5 canonical signaling by modulating NEMO. Since DAPT inhibits N1ICD generation, we speculated that the effects of N1ICD‑mediated crosstalk on TLR5‑NEMO‑NF‑κB indicate the involvement of RBP‑Jκ in this process. Dotted arrows indicate crosstalk projections.

Article Snippet: Trinitrobenzene sulfonic acid (TNBS; Sigma, St. Louis, MO, USA), dextran sodium sulphate (DSS, 5 kDa; Wako Pure Chemicals), flagellin (Salmonella typhimurium; InvivoGen, San Diego, CA, USA), recombinant rat Jagged1-Fc (R&D Systems, Minneapolis, MS, USA), γ-secretase inhibitor DAPT (Tocris Bioscience, Bristol, UK), NEMO-binding domain (NBD) inhibitory peptide (Imgenex, San Diego, CA, USA), Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), pNF-κB-Luc (Stratagene, Santa Clara, CA, USA), pRL-TK (Promega, Madison, WI, USA), mock (pZERO-mcs), a mouse dominant-negative TLR5 (pZERO-mTLR5) vector set (InvivoGen), RBP-Jκ Cignal Reporter assay (SA Biosciences, Valencia, CA, USA), mouse RBP-Jκ siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were obtained from respective sources.

Techniques: Expressing, Control, Blocking Assay

Journal: The Journal of Biological Chemistry

Article Title: Human SMC2 Protein, a Core Subunit of Human Condensin Complex, Is a Novel Transcriptional Target of the WNT Signaling Pathway and a New Therapeutic Target *

doi: 10.1074/jbc.M112.428466

Figure Lengend Snippet: SMC2 is up-regulated in human CRC. A, WB analysis of SMC2 in human CRC. A representative subset of 29 cases studied is shown. Actin was used as loading control. B, quantitative real-time PCR for SMC2 in 16 pairs of colon adenocarcinoma tumors and matched adjacent normal colonic tissues. Data are representative of three independent experiments. The mean values of SMC2 levels were compared using Student's t test (upper boxplot). C and D, SMC2, SMC4, and β-catenin levels were evaluated by WB in both colorectal cancer cell lines (n = 14) and samples from CRC patients (n = 27, a representative subset is shown). Actin was used as loading control. E and F, SMC2, SMC4, and β-catenin protein levels on WB were determined by gel band quantification and normalized to the corresponding actin levels. Values were used to perform correlation studies following Spearman test. G, immunohistochemistry of SMC2 in paraffin-embedded tissue. A representative specimen is shown. Magnified regions of the normal and tumor mucosa are shown on the right. N, normal tissue; T: tumor tissue (adenocarcinoma).

Article Snippet: Blots were probed using antibodies against SMC2 (ab10412, Abcam; and 07-710, Upstate-Millipore, dilution factor of 1:1000), SMC4 (ab17958, Abcam, dilution factor of 1:1000), TCF4 (05-511, Upstate-Millipore, dilution factor, 1:500), NCAPH (HPA003008, Sigma Aldrich, dilution factor, 1:2000), β-catenin (610154, BD Transduction Laboratories, dilution factor, 1:1000) or c-Myc (monoclonal 9E10, sc-40, Santa Cruz Biotechnology, 1:100).

Techniques: Real-time Polymerase Chain Reaction, Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: Human SMC2 Protein, a Core Subunit of Human Condensin Complex, Is a Novel Transcriptional Target of the WNT Signaling Pathway and a New Therapeutic Target *

doi: 10.1074/jbc.M112.428466

Figure Lengend Snippet: SMC2 protein is down-regulated upon WNT signaling inhibition. Ls174T/dnTCF4 (A and B) and Ls174T/pTER-β-catenin (C and D) cell lines were cultured in absence or presence of 5 μg/μl doxycycline (Dox) during the indicated time points. Cells were lysed and analyzed by WB using the indicated antibodies. Representative data from three replicates/independent experiments are shown.

Article Snippet: Blots were probed using antibodies against SMC2 (ab10412, Abcam; and 07-710, Upstate-Millipore, dilution factor of 1:1000), SMC4 (ab17958, Abcam, dilution factor of 1:1000), TCF4 (05-511, Upstate-Millipore, dilution factor, 1:500), NCAPH (HPA003008, Sigma Aldrich, dilution factor, 1:2000), β-catenin (610154, BD Transduction Laboratories, dilution factor, 1:1000) or c-Myc (monoclonal 9E10, sc-40, Santa Cruz Biotechnology, 1:100).

Techniques: Inhibition, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Human SMC2 Protein, a Core Subunit of Human Condensin Complex, Is a Novel Transcriptional Target of the WNT Signaling Pathway and a New Therapeutic Target *

doi: 10.1074/jbc.M112.428466

Figure Lengend Snippet: Functional study of SMC2 promoter activity. A, schematic representation of human SMC2 promoter. Predicted TCF response elements are also indicated; arrows indicate target sequence for ChIP PCR amplification. B, Ls174T/dnTCF4 (left) and Ls174T/pTER-β-catenin (right) cell lines were transfected with SMC2 promoter-luciferase reporter construct together with control Renilla luciferase reporter pRL-TK for normalization (RLU, relative luciferase units). Where indicated, cells were doxycline (Doxy)-treated to induce the TCF4 dominant-negative form (left) or a siRNA targeting β-catenin (right). TOP-flash vector was used as positive control for WNT signaling activity/repression. A representative result out of at least three different experiments run in triplicates is shown. C, DLD-1 or HCT116 cell lines were co-transfected with SMC2 promoter luciferase construct and pcDNA (empty vector), β-catenin, or VP16-TCF4 expression vectors. D, PCR analyses of DNA pulled down by isotypic antibody (negative control) or anti-TCF-4 monoclonal antibody in ChIP assay. c-myc promoter sequence containing TBE1 element and APC promoter region 1B sequences were amplified as positive and negative controls, respectively. Error bars indicate S.D. (Student's t test; **, p < 0.01).

Article Snippet: Blots were probed using antibodies against SMC2 (ab10412, Abcam; and 07-710, Upstate-Millipore, dilution factor of 1:1000), SMC4 (ab17958, Abcam, dilution factor of 1:1000), TCF4 (05-511, Upstate-Millipore, dilution factor, 1:500), NCAPH (HPA003008, Sigma Aldrich, dilution factor, 1:2000), β-catenin (610154, BD Transduction Laboratories, dilution factor, 1:1000) or c-Myc (monoclonal 9E10, sc-40, Santa Cruz Biotechnology, 1:100).

Techniques: Functional Assay, Activity Assay, Sequencing, Amplification, Transfection, Luciferase, Construct, Dominant Negative Mutation, Plasmid Preparation, Positive Control, Expressing, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Human SMC2 Protein, a Core Subunit of Human Condensin Complex, Is a Novel Transcriptional Target of the WNT Signaling Pathway and a New Therapeutic Target *

doi: 10.1074/jbc.M112.428466

Figure Lengend Snippet: Determination of the minimal regulatory region of SMC2 promoter. A, relative position and sequences of the putative TBEs predicted in silico in the SMC2 promoter and deletion mutants for luciferase (luc) reporters performed. B, determination of fragment 3 as the minimal regulatory region of the SMC2 promoter. Luciferase activity of each deletion mutant was normalized to Renilla luciferase internal control (RLU, relative luciferase units) in DLD1 (left) or HCT116 (right) cell lines; a representative result is shown of at least three independent experiments. *, p < 0.05; **, p < 0.01; Student's t test (promoter activity versus full-length SMC2 promoter (SMC2 FL).

Article Snippet: Blots were probed using antibodies against SMC2 (ab10412, Abcam; and 07-710, Upstate-Millipore, dilution factor of 1:1000), SMC4 (ab17958, Abcam, dilution factor of 1:1000), TCF4 (05-511, Upstate-Millipore, dilution factor, 1:500), NCAPH (HPA003008, Sigma Aldrich, dilution factor, 1:2000), β-catenin (610154, BD Transduction Laboratories, dilution factor, 1:1000) or c-Myc (monoclonal 9E10, sc-40, Santa Cruz Biotechnology, 1:100).

Techniques: In Silico, Luciferase, Activity Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Human SMC2 Protein, a Core Subunit of Human Condensin Complex, Is a Novel Transcriptional Target of the WNT Signaling Pathway and a New Therapeutic Target *

doi: 10.1074/jbc.M112.428466

Figure Lengend Snippet: Elucidation of the TBE responsible for β-catenin·TCF4 transactivation in the SMC2 promoter. A, sequence alignment of SMC2 promoter in different species; Hs, Homo sapiens; Pt, Pan troglodytes; Mmt, Macaca mulatta; Rn, Rattus novergicus; Mms, Mus musculus. Conserved TBEs are highlighted in gray background. B, schematic representation of SMC2 promoter mutant variants. C, DLD1 (left) or HCT116 (right) cell lines were transfected with constructs above. Luciferase activity was normalized to Renilla activity (RLU, relative luciferase units); a representative result is shown out of at least three independent experiments. D, DLD-1 (left) or HCT116 (right) cell lines were co-transfected with Δ3 fragment mutational combinations and expression vectors for β-catenin, TCF4-VP16 (constitutively active form of TCF4), or the empty vector pcDNA3 (pcDNA); a representative result is shown out of at least three independent experiments (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Article Snippet: Blots were probed using antibodies against SMC2 (ab10412, Abcam; and 07-710, Upstate-Millipore, dilution factor of 1:1000), SMC4 (ab17958, Abcam, dilution factor of 1:1000), TCF4 (05-511, Upstate-Millipore, dilution factor, 1:500), NCAPH (HPA003008, Sigma Aldrich, dilution factor, 1:2000), β-catenin (610154, BD Transduction Laboratories, dilution factor, 1:1000) or c-Myc (monoclonal 9E10, sc-40, Santa Cruz Biotechnology, 1:100).

Techniques: Sequencing, Mutagenesis, Transfection, Construct, Luciferase, Activity Assay, Expressing, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Human SMC2 Protein, a Core Subunit of Human Condensin Complex, Is a Novel Transcriptional Target of the WNT Signaling Pathway and a New Therapeutic Target *

doi: 10.1074/jbc.M112.428466

Figure Lengend Snippet: siRNA knockdown of SMC2 impairs tumor growth in a xenograft mouse model. A, schematic representation of the experimental design. DLD1 cells were transiently transfected with an siRNA targeting SMC2 or a scrambled sequence. After 48 h, a second round of transfection was performed. 24 h later, 1.5 × 106 cells were injected subcutaneously in the dorsal flanks of athymic nude mice. B, SMC2 knockdown was assessed by WB using whole cell extracts from in vitro culture until 120 h post-tranfection (sc, scrambled siRNA). C, representative resected tumors from the same animal at day 40 post-injection. Scale bar, 1 cm. D, tumor growth curves. Tumor volume was measured every 2–3 days for 36 days. The graph is representative of two independent experiments. Error bars represent S.E. (n = 11). Differences were evaluated with paired Student's t test (p = 0.0201); (*, p < 0.05, t test in each time point).

Article Snippet: Blots were probed using antibodies against SMC2 (ab10412, Abcam; and 07-710, Upstate-Millipore, dilution factor of 1:1000), SMC4 (ab17958, Abcam, dilution factor of 1:1000), TCF4 (05-511, Upstate-Millipore, dilution factor, 1:500), NCAPH (HPA003008, Sigma Aldrich, dilution factor, 1:2000), β-catenin (610154, BD Transduction Laboratories, dilution factor, 1:1000) or c-Myc (monoclonal 9E10, sc-40, Santa Cruz Biotechnology, 1:100).

Techniques: Transfection, Sequencing, Injection, In Vitro

Journal: Microbiology Spectrum

Article Title: The LiaFSR-LiaX System Mediates Resistance of Enterococcus faecium to Peptide Antibiotics and to Aureocin A53- and Enterocin L50-Like Bacteriocins

doi: 10.1128/spectrum.00343-23

Figure Lengend Snippet: Fold change in resistance levels of adaptive and deletion mutants of E. faecium to selected bacteriocins and antibiotics. The fold change values shown for the indicated agents were calculated by dividing the MIC (μg/mL) value for a given mutant by that for WT strain LMGT 2783. A fold change of 1 indicates no change in the level of resistance between the WT and mutant, a fold change of >1 indicates a decrease in the mutant's sensitivity compared to that of the WT, and a fold change of <1 indicates an increase in the mutant's sensitivity compared to that of the WT. A logarithmic scale with a base of 2 was used on the y axis. *, statistically significant results ( P value < 0.05). NIS, nisin; DAP, daptomycin; BAC, bacitracin; RAM, ramoplanin; GEN, gentamicin; GRA, gramicidin; KAN, kanamycin. Shown are only those agents to which at least one mutant strain was significantly more or less sensitive than the WT.

Article Snippet: In contrast, the BHT-B-resistant mutants obtained exhibited slightly higher DAP MIC values, up to ~8 μg/mL; they were sufficiently high to exceed the susceptible dose-dependent or even resistant clinical MIC breakpoints set for DAP and E. faecium by the Clinical and Laboratory Standards Institute (CLSI) (≤4 and ≥8 μg/mL, respectively) ( ).

Techniques: Mutagenesis

Journal: Microbiology Spectrum

Article Title: The LiaFSR-LiaX System Mediates Resistance of Enterococcus faecium to Peptide Antibiotics and to Aureocin A53- and Enterocin L50-Like Bacteriocins

doi: 10.1128/spectrum.00343-23

Figure Lengend Snippet: Sensitivities of liaFSR and liaFSR-liaX deletion mutants of E. faecium overexpressing liaS and/or liaR gene to selected bacteriocins and antibiotics ^a

Article Snippet: In contrast, the BHT-B-resistant mutants obtained exhibited slightly higher DAP MIC values, up to ~8 μg/mL; they were sufficiently high to exceed the susceptible dose-dependent or even resistant clinical MIC breakpoints set for DAP and E. faecium by the Clinical and Laboratory Standards Institute (CLSI) (≤4 and ≥8 μg/mL, respectively) ( ).

Techniques: Plasmid Preparation