Journal: International journal of food microbiology

Article Title: Emerging of Shiga toxin-producing Escherichia coli O177:H11 and O177:H25 from cattle at slaughter in Italy.

doi: 10.1016/j.ijfoodmicro.2024.110846

Figure Lengend Snippet: Fig. 1. Heatmap showing the presence/absence of virulence genes (y-axis) within the STEC isolates identified in this study (x-axis). Presence of virulence genes shown in blue, while absence in light green.

Article Snippet: Following the ISO/TS 13136:2012 method (ISO, 2012) each sponge was put into a sterile bag containing 90 ml of modified Tryptone Soya Broth (mTSB; Oxoid) added with novobiocin (Oxoid), homogenised in a stomacher blender for 2 min and incubated at 37 ◦C ± 1 ◦C for 18 to 24 h. After enrichment, a Real-Time PCR was conducted as described in the ISO method using the iQ-Check STEC VirX kit (Biorad, Hercules, CA, USA).

Techniques:

Journal: International journal of food microbiology

Article Title: Emerging of Shiga toxin-producing Escherichia coli O177:H11 and O177:H25 from cattle at slaughter in Italy.

doi: 10.1016/j.ijfoodmicro.2024.110846

Figure Lengend Snippet: Fig. 2. a) SNPs distance matrix results of the STEC O177:H25 strains using A2 as reference. b) Phylogenetic tree based on the maximum likelihood method of the STEC O177:H25 strains. The A2 isolate was used as outgroup to root the tree.

Article Snippet: Following the ISO/TS 13136:2012 method (ISO, 2012) each sponge was put into a sterile bag containing 90 ml of modified Tryptone Soya Broth (mTSB; Oxoid) added with novobiocin (Oxoid), homogenised in a stomacher blender for 2 min and incubated at 37 ◦C ± 1 ◦C for 18 to 24 h. After enrichment, a Real-Time PCR was conducted as described in the ISO method using the iQ-Check STEC VirX kit (Biorad, Hercules, CA, USA).

Techniques:

Journal: Cancer biology & therapy

Article Title: A novel peroxisome proliferator-activated receptor delta antagonist, SR13904, has anti-proliferative activity in human cancer cells.

doi: 10.4161/cbt.8.13.8691

Figure Lengend Snippet: Figure 4. SR13904 exerts inhibitory effects on the cell cycle. (A) A representative experiment in which A549 cells were grown in phenol-red-free DMEM + 2% charcoal-treated FBS ± SR13904 (20 μM). Cells were treated in the exponential phase and assessed by flow cytometry at 24 and 48 h. (B) Statistical analysis of cell cycle distribution (G1, S and G2 phases) from three experiments. All differences (SR13904-treated vs. vehicle control-treated) were highly significant. p values for the G1 phase are shown. **p = 0.001–0.01, ***p = <0.001 (C) Western analysis of select cell cycle proteins. A549 cells were grown in phenol-red-free DMEM + 0.5% charcoal-treated FBS and exposed to the ligand. Relative protein levels were assessed at 24 h and 48 h following addition of SR13904 (20 μM) or control. (D) mRNA analysis of CDK2, CDK4 and cyclin D1. A549 cells were grown and treated as in (C). Real-time PCR for CDK2, CDK4 and cyclin D1 were performed as described in Materials and Methods. Each experimental measurement was normalized to the corresponding GAPDH mRNA levels. For CDK2 (24 and 48 h time points) and cyclin D1 (48 h) time point, the differences (SR13904- treated vs. vehicle control-treated) were significant. *p = 0.01–0.05, **p = 0.001–0.01.

Article Snippet: CDK4 (#2906), cyclin D1 (#2922), cyclin E (#4129), p21 (#2947) and p27 (#2552) antibodies were purchased from Cell Signaling Technology.

Techniques: Flow Cytometry, Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 1 NUAK1 co-immunoprecipitates with p53. Equal amount of cell extracts from A549 cells were immunoprecipitated with anti-NUAK1 antibody or normal rabbit IgG as negative control and were western blotted with anti-p53 antibody. Fifty percent of protein before immunoprecipitation was kept for input and was subjected to western blotting with anti-NUAK1 and anti- LKB1 antibodies. Vec, LKB1 and KDM indicate that A549 cells were stably transfected with vector control, wild-type (WT) LKB1 and kinase-deficient LKB1, respectively; L þ s and L þ c indicate that cells that stably expressed WT LKB1 were transiently transfected with NUAK1 siRNA pool or control siRNA as a control. Glucose: cells were incubated in medium without glucose for 2 h; glucose þ : no glucose starvation treatment.

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunoprecipitation, Negative Control, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Control, Incubation

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 2 NUAK1 directly phosphorylates p53. (a) In vitro phosphorylation of p53 by E. coli-produced and LKB1-activated NUAK1. In kinase buffer that contained (g-32P) ATP, His-p53 was incubated with His-NUAK1 (N), or His-NUAK1 incubated with active- LKB1 and then isolated (NL) or His-NUAK1 incubated with heat-inactivated active-LKB1 and then isolated (NiL). After separation by SDS–PAGE, proteins were transferred to PVDF membrane and detected by autoradiography. His-p53 and His-NUAK1 were also detected by anti–p53 and anti-NUAK1 antibodies. (b) In vitro phosphorylation of p53 by E. coli-produced mutants of NUAK1. His-p53 was incubated with His-NUAK1 (N), His-NUAK1 (T211E) (TE) or His-NUAK1 (T211D) (TD), and then detected by autoradiography. His-p53, His-NUAK1 and mutants were also examined by western blotting. (c) In vitro phosphorylation of p53 by NUAK1 and mutants produced in HEK293T cells. His-p53 was incubated with His-NUAK1 (N), T211A mutant (TA) or kinase-dead mutant K84A (KA) and then detected by autoradiography. Western blotting was the same as in (b). (d) Phosphorylation of p53 in Hep3B cells. Hep3B cells were stably transfected with vector control (Vec) or WT p53 (p53) or p53 and transiently expressed NUAK1 (p þ N) or p53 and NUAK1 with ATM siRNA pool (pNs) or p53 and NUAK1 with control siRNA (pNc). After incubated in glucose þ or glucose medium for 2 h, cells were lysed and subjected to western blotting with phospho-p53 antibody sampler kit (Cell Signaling Technology), anti-p53, anti-NUAK1, anti-ATM and b-actin antibodies.

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: In Vitro, Phospho-proteomics, Produced, Incubation, Isolation, SDS Page, Membrane, Autoradiography, Western Blot, Mutagenesis, Stable Transfection, Transfection, Plasmid Preparation, Control

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 3 LKB1 activation of NUAK1 stimulates phosphorylation of p53. (a) LKB1-dependent p53 phosphorylation requires NUAK1. A549 cells were stably transfected with vector control (Vec), kinase-deficient LKB1 (KDM) or WT LKB1 and transiently transfected with NUAK1 siRNA pool (L þ s) or WT LKB1 and transiently transfected with control siRNA (L þ c). Cells were incubated in glucose þ or glucose medium for 2 h. Western blotting was done using phospho-p53 antibody sampler kit (Cell Signaling Technology), anti-p53 antibody, anti-NUAK1 antibody, anti-LKB1 antibody and b-actin antibody. (b) Requirement of NUAK1 kinase activity in LKB1-dependent p53 phosphorylation. A549 cells stably transfected with LKB1 ( þ ) or vector control () were transiently transfected with NUAK1 ( þ ), T211A (TA), kinase-dead mutant K84A (KA) or vector control (Vec), and treated under glucose starvation for 2 h. Cells were lysed and western blotting was performed as in (a). (c) In vivo phosphorylation assay of NUAK1 by LKB1. A549 cells that stably expressed WT LKB1 , vector control (Vec) or kinase-deficient LKB1 (KDM) were transiently transfected with WT NUAK1 or NUAK1 T211A mutation. Transfection was the same in (d), (e) and (f). The cells were subjected to glucose starvation for 2 h and incubated for 3 h with [32P] Pi (300 cpm/pmol; Furi). Cells were then lysed and NUAK1 or NUAK1 (T211A) was immunoprecipitated with anti-NUAK1 antibody. The immunoprecipitates were separated by SDS–PAGE and subjected to autoradiography. (d) Cells were treated with 200 mM AMP. (e) In vitro kinase assay of NUAK1. After being subjected to glucose starvation for 2 h, cells were lysed and NUAK1 or NUAK1 (T211A) was immunoprecipitated with anti-NUAK1 antibody. The in vitro kinase activity of immunoprecipitates was assayed by measuring the 32P labeling of SAMS peptide. One unit of activity was defined as 1 nmol SAMS peptide phosphorylated per minute. (f) Cells were treated with 200 mM AMP.

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Phospho-proteomics, Stable Transfection, Transfection, Plasmid Preparation, Control, Incubation, Western Blot, Activity Assay, Mutagenesis, In Vivo, Immunoprecipitation, SDS Page, Autoradiography, In Vitro, Kinase Assay, Labeling

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 4 Cell cycle arrest induced by LKB1/NUAK1 requires p53. (a) A549 cells were stably transfected with vector control (Vec), kinase-deficient LKB1 (KDM) or WT LKB1 ( þ ). Cells stably expressed WT LKB1 were also transiently transfected with ( þ ) or without () WT NUAK1, NUAK1 siRNA pool (siRNA) or control siRNA (Ctl-si). After synchronization, cells were treated with glucose medium. Cells were then harvested, stained with propidium iodide and analyzed by flow cytometry. Each analysis was carried out in triplicate and also in (b). (b) A549 cells were stably transfected with vector control (Vec) or WT LKB1 ( þ ), and transiently transfected with p53 ( þ ), vector control (Vec), p53 S15A mutant (S15A), p53 S392A mutant (S392A), p53 siRNA pool (siRNA) or control siRNA (Ctl-si). Cells were treated as in (a) and subjected to flow cytometry analysis.

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Control, Staining, Cytometry, Mutagenesis

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 5 p21/WAF1 transcriptional activity induced by LKB1/ NUAK1. (a) Quantitative RT–PCR analysis of p21/WAF1 transcription. A549 cells were stably transfected with vector control (Vec), kinase-deficient LKB1 (KDM), WT LKB1 (LKB1) and transiently transfected with NUAK1 siRNA pool (L þ s), or WT LKB1 and transiently transfected with control siRNA (L þ c). RNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA and displayed as fold change relative to the value obtained from cells transfected with vector control, which was set at 1. (b) Western blotting of endogenous p21 protein. Transfection was the same as in (a).

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Quantitative RT-PCR, Stable Transfection, Transfection, Plasmid Preparation, Control, Western Blot

Journal: Oncogene

Article Title: A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.

doi: 10.1038/onc.2011.19

Figure Lengend Snippet: Figure 6 NUAK1 interacts with p53 in the nucleus and binds to p21/WAF1 promoter. (a) Endogenous NUAK1 was present in the p53RE region of p21/WAF1 promoter. A549 cells were stably transfected with vector control (Vec), wild-type LKB1 (LKB1), kinase- deficient LKB1 (KDM), or wild-type LKB1 and transiently transfected with NUAK1 siRNA pool (L þ s). Cells were subjected to glucose starvation for 2 h. ChIP was done using anti-NUAK1 antibody and normal rabbit IgG was used as a negative control for the specificity of the immunoprecipitation. Cells transiently transfected with NUAK1 siRNA pool were included as a negative control. Quantitative PCR was done with the specific primers for p53RE of p21/WAF1 promoter and the bars represent normalized quantitative PCR values expressed as percentage of input. (b) ChIP assay of NUAK1 at p21/WAF1 TATA-50UTR region. (c) ChIP assay of p53 at p21/WAF1 promoter p53RE region. (d) ChIP assay of LKB1 at p21/WAF1 promoter p53RE region. (e) The binding of NUAK1 to p53RE required wild-type p53. p53-null Hep3B cells were stably transfected with wild-type p53 (p53), p53 S15A mutant (S15A), p53 S392A mutant (S392A) or vector control (Vec). ChIP assay was done as described in A and p53-expressing cells transiently transfected with NUAK1 siRNA pool were included as a negative control (p þ s). (f) ChIP assay of LKB1 at p21/WAF1 promoter p53RE region in Hep3B cells. p53-expressing cells transiently transfected with LKB1 siRNA pool were included as a negative control (p þ s). (g) Co-immunoprecipitation analysis of endogenous NUAK1 and p53 from A549 cells that stably expressed vector control (Vec), wild-type LKB1 (LKB1), kinase-deficient LKB1 (KDM) or wild-type LKB1 and transiently transfected with NUAK1 siRNA pool (L þ s) as control. Cells were treated with glucose– medium and then fractionated (N: nuclear fraction; C: cytoplasmic fraction). Equal amounts of protein from each sample were immunoprecipitated using anti-NUAK1 antibody or using normal rabbit IgG as a negative control. Fifty percent of protein before immunoprecipitation was kept for input, and was subjected to western blotting with anti-NUAK1 and anti-p53 antibodies.

Article Snippet: Anti-NUAK1 polyclonal antibody, anti-LKB1 monoclonal antibody, b-actin polyclonal antibody, anti-GAPDH polyclonal antibody, normal mouse IgG, normal rabbit IgG, ATM siRNA pool, p53 siRNA pool, NUAK1 siRNA pool, LKB1 siRNA pool and control siRNA were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Control, Negative Control, Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Mutagenesis, Expressing, Western Blot