Journal: Infectious Agents and Cancer

Article Title: HPV-mediated down-regulation of NOD1 inhibits apoptosis in cervical cancer

doi: 10.1186/s13027-020-0272-3

Figure Lengend Snippet: Expression of NOD1 in CINs and ISCCs

Article Snippet: Immunohistochemistry (IHC) staining was conducted as described previously [ ] with antibodies against NOD1 (MAB7090, monoclonal mouse IgG2B clone, diluted at 1/60; R&D Systems, Minneapolis, USA), p16 INK4A (p16 INK4A (F-12): sc-1661, mouse monoclonal antibody, diluted at 1/100, Santa Cruz Biotechnology Inc., California, USA), and horseradish peroxidase-labelled secondary antibody (Maixin Biotechnology, Fuzhou, China) in accordance with manufacturer’s instructions.

Techniques: Expressing

Journal: Infectious Agents and Cancer

Article Title: HPV-mediated down-regulation of NOD1 inhibits apoptosis in cervical cancer

doi: 10.1186/s13027-020-0272-3

Figure Lengend Snippet: NOD1 expression in frozen fresh ISCC and normal cervical tissues. a. Expression of NOD1 protein in 3 paired tumor (T) and adjacent normal (N) tissues from 3 patients was detected by Western blot. b. Quantitative comparison of the NOD1 mRNA expression between ISCC and normal cervical tissues using RT-PCR with GAPDH as the control house keeping gene. The ratios of NOD1/GAPDH expression were calculated from triplicate. ( P = 0.22)

Article Snippet: Immunohistochemistry (IHC) staining was conducted as described previously [ ] with antibodies against NOD1 (MAB7090, monoclonal mouse IgG2B clone, diluted at 1/60; R&D Systems, Minneapolis, USA), p16 INK4A (p16 INK4A (F-12): sc-1661, mouse monoclonal antibody, diluted at 1/100, Santa Cruz Biotechnology Inc., California, USA), and horseradish peroxidase-labelled secondary antibody (Maixin Biotechnology, Fuzhou, China) in accordance with manufacturer’s instructions.

Techniques: Expressing, Western Blot, Comparison, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Infectious Agents and Cancer

Article Title: HPV-mediated down-regulation of NOD1 inhibits apoptosis in cervical cancer

doi: 10.1186/s13027-020-0272-3

Figure Lengend Snippet: Expression of NOD1 and P16 INK4A in normal cervix, CIN I, CIN II, CIN III and ISCC tissues detected by immunohistochemistry. Hematoxylin and eosin (HE) staining showed that in comparison to normal cervical squamous epithelium ( a ), atypical hyperplasia of squamous epithelium was restricted within lower third in CIN I ( e ), lower two-thirds in CIN II ( i ), or exceeded over lower two-thirds in CIN III ( m ), respectively. Carcinoma invaded into muscle tissue in ISCC ( q ). NOD1 showed the strongest immunoreactivity in normal cervical squamous epithelium ( b ), and the staining intensity of NOD1 gradually decreased from CIN I ( f ), CIN II ( j ) to CIN III ( n ). No NOD1 immunostaining was observed in cells within carcinoma nests ( r ). In contrast to NOD1, no p16 INK4A staining was detected in the normal cervical tissue ( c ). The expression of p16 INK4A gradually increased from CIN I ( g ), CIN II (K) to CIN III ( o ), and p16 INK4A positive cells were diffusely distributed within the atypical hyperplasia tissue. Diffuse and strong staining of p16 INK4A was observed in tumor epithelial cells of carcinoma nests in ISCC (S). NOD1 staining was only detected in normal cervical squamous epithelium ( d ) with double staining of NOD1 (red) and p16 INK4A (black), and NOD1 staining was not observed within atypical hyperplasia cells of CINs (H, L, P), where p16 INK4A was positive. Strong p16 INK4A staining but no NOD1 staining was observed in the tumor cells of carcinoma nests ( t )

Article Snippet: Immunohistochemistry (IHC) staining was conducted as described previously [ ] with antibodies against NOD1 (MAB7090, monoclonal mouse IgG2B clone, diluted at 1/60; R&D Systems, Minneapolis, USA), p16 INK4A (p16 INK4A (F-12): sc-1661, mouse monoclonal antibody, diluted at 1/100, Santa Cruz Biotechnology Inc., California, USA), and horseradish peroxidase-labelled secondary antibody (Maixin Biotechnology, Fuzhou, China) in accordance with manufacturer’s instructions.

Techniques: Expressing, Immunohistochemistry, Staining, Comparison, Immunostaining, Double Staining

Journal: Infectious Agents and Cancer

Article Title: HPV-mediated down-regulation of NOD1 inhibits apoptosis in cervical cancer

doi: 10.1186/s13027-020-0272-3

Figure Lengend Snippet: A cross tabulation of NOD1 and p16 INK4A expression

Article Snippet: Immunohistochemistry (IHC) staining was conducted as described previously [ ] with antibodies against NOD1 (MAB7090, monoclonal mouse IgG2B clone, diluted at 1/60; R&D Systems, Minneapolis, USA), p16 INK4A (p16 INK4A (F-12): sc-1661, mouse monoclonal antibody, diluted at 1/100, Santa Cruz Biotechnology Inc., California, USA), and horseradish peroxidase-labelled secondary antibody (Maixin Biotechnology, Fuzhou, China) in accordance with manufacturer’s instructions.

Techniques: Expressing

Journal: Infectious Agents and Cancer

Article Title: HPV-mediated down-regulation of NOD1 inhibits apoptosis in cervical cancer

doi: 10.1186/s13027-020-0272-3

Figure Lengend Snippet: Expression of NOD1 in 3 cervical squamous carcinoma cells was detected by immunocytochemistry ( a ), quantitative real-time PCR ( b ) and Western blot ( c ). NOD1 showed strongest expression in the HPV (−) C33A cells and was weakest in the HPV16 (+) SiHa cells. * P < 0.05, **** P < 0.0001

Article Snippet: Immunohistochemistry (IHC) staining was conducted as described previously [ ] with antibodies against NOD1 (MAB7090, monoclonal mouse IgG2B clone, diluted at 1/60; R&D Systems, Minneapolis, USA), p16 INK4A (p16 INK4A (F-12): sc-1661, mouse monoclonal antibody, diluted at 1/100, Santa Cruz Biotechnology Inc., California, USA), and horseradish peroxidase-labelled secondary antibody (Maixin Biotechnology, Fuzhou, China) in accordance with manufacturer’s instructions.

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

Journal: Infectious Agents and Cancer

Article Title: HPV-mediated down-regulation of NOD1 inhibits apoptosis in cervical cancer

doi: 10.1186/s13027-020-0272-3

Figure Lengend Snippet: HPV oncoproteins down-regulated the expression of NOD1 and RIP2 in C33A cells. NOD1 expression was down-regulated in the cancer cells transfected with E6- or E7-expressing plasmids detected by immunocytochemistry ( a ), Western blot ( b ), and RT-PCR ( c ). RIP2 mRNA expression was down-regulated in the cancer cells transfected with E6- or E7-expressing plasmids detected by RT-PCR ( d ). BLANK, without plasmid, VECT, empty vector, E6, E6-expressing plasmids, and E7, E7-expressing plasmids

Article Snippet: Immunohistochemistry (IHC) staining was conducted as described previously [ ] with antibodies against NOD1 (MAB7090, monoclonal mouse IgG2B clone, diluted at 1/60; R&D Systems, Minneapolis, USA), p16 INK4A (p16 INK4A (F-12): sc-1661, mouse monoclonal antibody, diluted at 1/100, Santa Cruz Biotechnology Inc., California, USA), and horseradish peroxidase-labelled secondary antibody (Maixin Biotechnology, Fuzhou, China) in accordance with manufacturer’s instructions.

Techniques: Expressing, Transfection, Immunocytochemistry, Western Blot, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

Journal: Infectious Agents and Cancer

Article Title: HPV-mediated down-regulation of NOD1 inhibits apoptosis in cervical cancer

doi: 10.1186/s13027-020-0272-3

Figure Lengend Snippet: The activation of NOD1 increased CHX-induced apoptosis. NOD1 expression increased in SiHa cells treated with iE-DAP (** P < 0.005, *** P < 0.0001) ( a ). The NOD1 activation enhanced CHX-induced apoptosis. HPV16-positive SiHa cells were treated with iE-DAP in the presence or absence of CHX. Cell viability was measured by flow cytometry. The apoptosis of SiHa cells increased significantly in the presence of CHX ( P < 0.005) ( b )

Article Snippet: Immunohistochemistry (IHC) staining was conducted as described previously [ ] with antibodies against NOD1 (MAB7090, monoclonal mouse IgG2B clone, diluted at 1/60; R&D Systems, Minneapolis, USA), p16 INK4A (p16 INK4A (F-12): sc-1661, mouse monoclonal antibody, diluted at 1/100, Santa Cruz Biotechnology Inc., California, USA), and horseradish peroxidase-labelled secondary antibody (Maixin Biotechnology, Fuzhou, China) in accordance with manufacturer’s instructions.

Techniques: Activation Assay, Expressing, Flow Cytometry

Journal:

Article Title: Bacterial Peptide Recognition and Immune Activation Facilitated by Human Peptide Transporter PEPT2

doi: 10.1165/rcmb.2008-0059OC

Figure Lengend Snippet: Lung epithelial expression of nucleotide-binding oligomerization domain (NOD)-related factors. Total RNA and protein were obtained from primary human upper airway epithelial cells (hLECs) after overnight culture with or without 250 units/ml of IFN-γ, or 100 ng/ml of TNF-α. (A) NOD1/CARD4 and (B) NOD2/CARD15 mRNA was quantified by real-time PCR and expressed as relative copy number (RCN). GAPDH and CAP were used as internal controls to standardize and quantify mRNA expression. (C) Whole cell lysates were immunoprecipitated with affinity-purified anti-Nod1/Card4 antibody (IMGENEX), and then immunoblotted with an anti-Nod1/Card4 antibody (provided by Dr. Dana Philpott). Pre-immune rabbit serum was used as a negative control. (D) Western analysis of RIP2. Samples were loaded with 40 μg/well protein from the same lysate, and then the membrane was blotted with an anti-RIP2 antibody. β-Actin served as control to determine equal loading. (E) An immunoblot demonstrating constitutive levels of NOD1 and RIP2 proteins in both BEAS-2B and hLEC lung epithelial cells; 80 μg/well protein was loaded for each sample.

Article Snippet: GAPDH and CAP were used as internal controls to standardize and quantify mRNA expression. ( C ) Whole cell lysates were immunoprecipitated with affinity-purified anti-Nod1/Card4 antibody (IMGENEX), and then immunoblotted with an anti-Nod1/Card4 antibody (provided by Dr. Dana Philpott).

Techniques: Expressing, Binding Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, Affinity Purification, Negative Control, Western Blot, Membrane

Journal:

Article Title: Bacterial Peptide Recognition and Immune Activation Facilitated by Human Peptide Transporter PEPT2

doi: 10.1165/rcmb.2008-0059OC

Figure Lengend Snippet: γ-iE-DAP stimulates interaction between NOD1 and RIP2, leading to NF-κB activation. (A) Immunoprecipitation of human NOD1 was performed in BEAS-2B whole cell extracts after transfection with flag-tagged NOD1 and treatment with γ-iE-DAP (5 μg/ml). (A) Samples were resolved and then immunoblotted with an anti-RIP2 antibody. Western analysis was also performed with a monoclonal anti-flag antibody (Sigma) and β-actin (lower panels), demonstrating equal amounts of NOD1, specifically in the NOD1-transfected cultures (lanes 3–6) and equal loading in all samples. Negative controls included immunoprecipitation with a mouse IgG isotype control antibody (lane 1) or empty vector transfected cells (lane 2). (B) siRNA Suppression of NOD1 expression: since constitutive levels of NOD1 are barely detectable, we evaluated siRNA efficacy in BEAS-2B exposed to IFN-γ. Compared with the scrambled control siRNA, siRNA targeted to NOD1 resulted in a significant reduction in IFN-γ–induced protein levels. (C) Next we evaluated the ability of α-iE-DAP and γ-iE-DAP to induce NF-κB–mediated transcription in baseline, siControl, and siNOD1-treated cultures. First, BEAS-2B cultures were transfected with siRNAs, followed by transfection with an NF-κB/luciferase promotor plasmid. At 4 hours after transfection, cells were administered γ-iE-DAP or α-iE-DAP (5 μg/ml each) for 2 hours. Lysates were than analyzed for firefly luciferase activity and normalized to renilla expression. We observed that only γ-iE-DAP treatment resulted in luciferase expression, and this was significantly decreased in cultures treated with siRNA against NOD1 (data are expressed as mean values ± σ of n = 3; P ≤ 0.01). (D) To further validate these findings, BEAS-2B cultures were transiently transfected with siRNA against NOD1 or a scrambled control and then exposed to γ-iE-DAP or α-iE-DAP. Muropeptides were removed after 4 hours and then cells were cultured in normal medium for an additional 20 hours. As shown, only γ-iE-DAP treatment resulted in a significant increase in IL-6 release that was inhibited back down to baseline levels in cells receiving siRNA directed against NOD1 (data are expressed as mean values ± σ of n = 3; P ≤ 0.05).

Article Snippet: GAPDH and CAP were used as internal controls to standardize and quantify mRNA expression. ( C ) Whole cell lysates were immunoprecipitated with affinity-purified anti-Nod1/Card4 antibody (IMGENEX), and then immunoblotted with an anti-Nod1/Card4 antibody (provided by Dr. Dana Philpott).

Techniques: Activation Assay, Immunoprecipitation, Transfection, Western Blot, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Cell Culture

Journal: Applied Sciences

Article Title: Toll-Like Receptors 1/2/4/6 and Nucleotide-Binding Oligomerization Domain-Like Receptor 2 Are Key Damage-Associated Molecular Patterns Sensors on Periodontal Resident Cells

doi: 10.3390/app11114724

Figure Lengend Snippet: Figure 1. TLR1/2/4/5/6, NOD1/2 or NLRP1/2 immunohistochemical detection in healthy gingival (rows 1–3) or peri- odontitis tissue (rows 4–5) biopsies. Specific damage-associated molecular patterns are detected as brown DAB stain. Scale bars = 100 µm. Column A-I showed micrographs of TLR1/2/4/5/6, NOD1/2 or NLRP1/2 detection, respectively. Rows 1–5 are: healthy oral epithelium (H-OE), oral sulcular epithelium (OSE), junctional epithelium (JE), periodontitis oral epithelium (P-OE) and pocket epithelium (PE), respectively. Shown are representative micrographs from healthy human gingival biopsies (n = 13) and periodontitis biopsies (n = 17) with staining from three independent experiments. TLR3/7/8/9/10 immuno-detection was attempted but had negative staining results (data not shown).

Article Snippet: The specimens were then incubated with anti-human TLR-1 to -8 and TLR10, NOD1/2 or NLRP1/2 primary antibodies, independently in 3% bovine serum albumin at 4 ◦C overnight (mouse anti-human: TLR1, TLR4 and NLRP2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), TLR2 (Imgenex, San Diego, CA, USA), TLR3 (eBioscience, San Diego, CA, USA), TLR5 (Abnova, Taipei, Taiwan), TLR8 (Enzo Life Science, Farmingdale, NY, USA); rabbit anti-human: TLR6, NLRP1 and NOD2 (Thermo Fischer Scientific, Waltham, MA, USA), TLR7 (LifeSpan BioSciences, Inc., Seattle, WA, USA), NOD1 (R&D systems, Inc., Minneapolis, MN, USA), TLR10 (Novus Biologicals, Littleton, CO, USA)).

Techniques: Immunohistochemical staining, Staining, Negative Staining

Journal: Applied Sciences

Article Title: Toll-Like Receptors 1/2/4/6 and Nucleotide-Binding Oligomerization Domain-Like Receptor 2 Are Key Damage-Associated Molecular Patterns Sensors on Periodontal Resident Cells

doi: 10.3390/app11114724

Figure Lengend Snippet: Figure 2. Selected DAMP sensors mRNA detection from HGT, or HGK/HGF primary cultures. By reverse transcription, PCR, TLR1-6, NOD1/2 and NLRP1/2 were detectable in HGT, HGK, and HGF with TLR4 or NLRP2 detectable in some but not all samples investigated (please refer to text and Table 4 for details). TLR7/8/10 mRNAs were only detectable from HGTs. THP-1 cell line was utilized as the positive control for TLR9 mRNA detection. HGF—human gingival fibroblasts, HGK—human gingival keratinocytes, HGT—human gingival tissue.

Article Snippet: The specimens were then incubated with anti-human TLR-1 to -8 and TLR10, NOD1/2 or NLRP1/2 primary antibodies, independently in 3% bovine serum albumin at 4 ◦C overnight (mouse anti-human: TLR1, TLR4 and NLRP2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), TLR2 (Imgenex, San Diego, CA, USA), TLR3 (eBioscience, San Diego, CA, USA), TLR5 (Abnova, Taipei, Taiwan), TLR8 (Enzo Life Science, Farmingdale, NY, USA); rabbit anti-human: TLR6, NLRP1 and NOD2 (Thermo Fischer Scientific, Waltham, MA, USA), TLR7 (LifeSpan BioSciences, Inc., Seattle, WA, USA), NOD1 (R&D systems, Inc., Minneapolis, MN, USA), TLR10 (Novus Biologicals, Littleton, CO, USA)).

Techniques: Reverse Transcription, Positive Control

Journal: Applied Sciences

Article Title: Toll-Like Receptors 1/2/4/6 and Nucleotide-Binding Oligomerization Domain-Like Receptor 2 Are Key Damage-Associated Molecular Patterns Sensors on Periodontal Resident Cells

doi: 10.3390/app11114724

Figure Lengend Snippet: Figure 3. Selected damage-associated molecular pattern (DAMP) sensors expression in human gingival keratinocytes (HGK) or human gingival fibroblasts (HGF) explants under 1% O2 and/or 2 µg/mL Escherichia coli LPS, 24 h. (A) Detection of TLR1, TLR2, TLR4-6, NOD1/2, NLRP1/2 from HGK/HGF cell lysates after various treatments. Shown are representative blots from three independent experiments; (B) relative HGK TLR1, TLR2, TLR4-6, NOD1/2, NLRP1/2 protein levels after various treatments; (C) relative HGF TLR1, TLR2, TLR4-6, NOD1/2, NLRP1/2 protein levels after various treatments. * p < 0.05, ** p < 0.01, one-way ANOVA with adjustments against multiple comparisons within same DAMP sensor group. Cells at passage three were used and the experiments were independently repeated three times.

Article Snippet: The specimens were then incubated with anti-human TLR-1 to -8 and TLR10, NOD1/2 or NLRP1/2 primary antibodies, independently in 3% bovine serum albumin at 4 ◦C overnight (mouse anti-human: TLR1, TLR4 and NLRP2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), TLR2 (Imgenex, San Diego, CA, USA), TLR3 (eBioscience, San Diego, CA, USA), TLR5 (Abnova, Taipei, Taiwan), TLR8 (Enzo Life Science, Farmingdale, NY, USA); rabbit anti-human: TLR6, NLRP1 and NOD2 (Thermo Fischer Scientific, Waltham, MA, USA), TLR7 (LifeSpan BioSciences, Inc., Seattle, WA, USA), NOD1 (R&D systems, Inc., Minneapolis, MN, USA), TLR10 (Novus Biologicals, Littleton, CO, USA)).

Techniques: Expressing

Journal: Journal of cellular and molecular medicine

Article Title: Role of nucleotide-binding oligomerization domain 1 (NOD1) in pericyte-mediated vascular inflammation.

doi: 10.1111/jcmm.12804

Figure Lengend Snippet: Fig. 1 NOD1 and NOD2 expression by human brain pericytes. (A) Pri- mary HBP were stimulated with TNFa (50 ng/ml) or IFNc (100 ng/ml) for 20 hrs, and NOD1 and NOD2 gene expression was analysed using qRT-PCR. Data shown are means standard deviation (S.D.) of tripli- cates of two independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). HL60 cells were used as a NOD2 positive control (insert). (B) FACS analysis of intracellular NOD1 expression in unstimulated HBP and Jurkat cells (positive control). Solid grey curves show negative controls in which the primary antibody was omitted. (C) Immunocytofluorescence staining of NOD1, detected in the cytoplasm of unstimulated HBP (top), compared to a negative control in which the same cells are incubated without primary antibody (bottom).

Article Snippet: Mouse anti-NOD1 monoclonal antibody (clone 626919) was from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Standard Deviation, Positive Control, Staining, Negative Control, Incubation

Journal: Journal of cellular and molecular medicine

Article Title: Role of nucleotide-binding oligomerization domain 1 (NOD1) in pericyte-mediated vascular inflammation.

doi: 10.1111/jcmm.12804

Figure Lengend Snippet: Fig. 2 Effect of NOD1 activation in human brain pericytes. HBP were stimulated with C12-iE-DAP (5 lg/ml) or ultrapure LPS (100 ng/ml) for 6 hrs and IL6 (A) and IL8 (B) gene expression was analysed using qRT-PCR. (C) In parallel, secretion of IL8 was assessed in cell culture supernatants by ELISA. (D) C12-iE-DAP (1 lg/ml) and LPS (5 ng/ml) synergistically enhanced IL8 gene expression by HBP. Results are expressed as means s- tandard deviation (S.D.) of triplicates of three independent experiments. (**P < 0.01, ***P < 0.001 -versus non-stimulated control; ###P < 0.001 - versus C12-iE-DAP or LPS alone).

Article Snippet: Mouse anti-NOD1 monoclonal antibody (clone 626919) was from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Gene Expression, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of cellular and molecular medicine

Article Title: Role of nucleotide-binding oligomerization domain 1 (NOD1) in pericyte-mediated vascular inflammation.

doi: 10.1111/jcmm.12804

Figure Lengend Snippet: Fig. 3 Knock-down of NOD1 abrogates C12-iE-DAP response. (A) The knock-down efficiency of HBP transduced with NOD1 shRNA or control shRNA lentiviral particles was assessed by qRT-PCR. (B) Western blot analysis was used to verify knock-down of NOD1. Alpha-tubulin was used as a load- ing control. Numbers indicate the expression fold change relative to the loading control. (C) NOD1 and control shRNA expressing HBP were treated with 1 lg/ml C12-iE-DAP for 6 hrs. Data are presented as fold change of IL8 expression compared to non-stimulated cells. Data represent mean standard deviation (S.D.) of three independent experiments, each performed in triplicates. (***P < 0.001).

Article Snippet: Mouse anti-NOD1 monoclonal antibody (clone 626919) was from R&D Systems (Minneapolis, MN, USA).

Techniques: Knockdown, Transduction, shRNA, Control, Quantitative RT-PCR, Western Blot, Expressing, Standard Deviation

Journal: Journal of cellular and molecular medicine

Article Title: Role of nucleotide-binding oligomerization domain 1 (NOD1) in pericyte-mediated vascular inflammation.

doi: 10.1111/jcmm.12804

Figure Lengend Snippet: Fig. 4 Role of RIPK2, NF-kB and MAP kinases on NOD1-mediated responses. (A) HBP were incubated with different concentrations of PP2 ranging from 0.01 lM to 10 lM for 30 min. prior to treatment with LPS (50 ng/ml) or with C12-iE-DAP (1 lg/ml) and analysed for IL8 gene expression. Results are expressed as a percentage of response to the agonist alone versus non-stimulated controls (###P < 0.001). (B) Effect of pre-treatment with the inhibitors SB203580 (p38 inhibitor), SC-514 (NF-jB inhibitor), SPS00125 (JNK inhibitor) and PD98059 (MEK-1 inhibitor) at 10 lM on response to C12-iE-DAP (1 lg/ml). (C) Dose-dependent effect of SB203580 and SC-514 (0.1–10 lM) on HBP activation by C12-iE-DAP. (D) Synergistic effects of C12-iE-DAP+LPS were inhibited by SB203580 and SC-514. HBP were pre-treated with 10 lM SB203580 or SC-514 prior to stimulation with C12-iE-DAP (1 lg/ml) and LPS (5 ng/ml). Data are expressed as mean standard deviation (S.D.) and represent three independent experiments, each performed in triplicates (**P < 0.01, ***P < 0.001).

Article Snippet: Mouse anti-NOD1 monoclonal antibody (clone 626919) was from R&D Systems (Minneapolis, MN, USA).

Techniques: Incubation, Gene Expression, Activation Assay, Standard Deviation

Journal: PLoS pathogens

Article Title: Cyclosporine A impairs nucleotide binding oligomerization domain (Nod1)-mediated innate antibacterial renal defenses in mice and human transplant recipients.

doi: 10.1371/journal.ppat.1003152

Figure Lengend Snippet: Figure 2. CsA impairs the migration of neutrophils and macrophages in UPEC-infected kidneys and neutrophil bacte- rial phagocytic killing capacity. (A and B) Flow cytometry analysis of the CD45+ leukocyte population infiltrating kidneys from CsA- and vehicle-treated mice, 24 h after UPEC inoculation. Representative dot- plots of CD45+ F4/802 renal cells (A) and percentages (A, upper right insets and B) of F4/802 CD11b+ Gr1HI neutrophils infiltrating the infected CsA- and vehicle-treated mice kidneys. The bars indicate the mean percentage of neutrophils over total CD45+ leukocytes (n = 4 mice per group). (C) Illustrations and quantification of Texas red-coupled E. coli internalized by peritoneal neutrophils from vehicle-treated (2) and CsA-treated mice. Values are represented as mean 6 SE of the mean count values (3–5 per condition) from three independent experiments. Cell membranes were stained with CD11b-FITC. Bars, 10 mm. (D) Killing of serum-opsonized E. coli by neutrophils from untreated or CsA-treated WT mice. Bacterial viability (n = 5–7 determinations from three experiments) was expressed relative to a control assay performed without neutrophils. (E) Variation in the levels of Tlr2, Tlr4, Tlr5, Tlr9, Nod1, and Nod2 mRNAs measured by quantitative real time PCR in kidneys from CsA-treated (+) and vehicle-treated mice 24 h after UPEC inoculation. (n = 6–8 determinations from 3–4 experiments). (F) Immunoblot analysis of the Tlr4, Nod1, and Nod2 proteins and the corresponding ß-actin in kidney homogenates from vehicle-treated and CsA-treated mice 24 h after the inoculation of UPEC. Values are presented as mean 6 SE. *, p,0.05 (B–D, Two-tailed, unpaired Student’s t test; E, Mann-Whitney test). doi:10.1371/journal.ppat.1003152.g002

Article Snippet: In parallel, aliquots of NH4Cl-treated blood samples were permeabilized with methanol, and then incubated with an antihuman NOD1 antibody (Imgenex Corp.).

Techniques: Migration, Infection, Flow Cytometry, Staining, Control, Real-time Polymerase Chain Reaction, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: PLoS pathogens

Article Title: Cyclosporine A impairs nucleotide binding oligomerization domain (Nod1)-mediated innate antibacterial renal defenses in mice and human transplant recipients.

doi: 10.1371/journal.ppat.1003152

Figure Lengend Snippet: Figure 4. CsA impairs Tlr4-mediated chemokine production in renal medullary collecting duct cells. (A) Illustration showing UPEC (in blue) adhering to the luminal surface of cells from collecting duct sections (arrows), identified by the AQP-2 positive staining of the collecting duct principal cells (in dark red). Bar = 20 mm. (B) Identification by reverse transcription PCR of amplified products of Tlr4 (311 bp), Nod1 (525 bp), and Nod2 (507 bp) mRNAs in a confluent culture of medullary collecting ducts (MCDs) dissected from the kidney of a WT mouse. (C) CXCL2 production in cultured WT MCD cells incubated with or without LPS, FK156, or MDP for 6 h at 37uC. (D) Relative levels of Tlr4, Nod1 and Nod2 mRNAs measured by quantitative real time PCR in confluent cultures of WT MCD cells incubated with various concentrations of CsA for 48 h and compared to untreated MCD cells. (E) Immunoblot analysis of Tlr4 and Nod1, and the corresponding b-actin in WT MCD cells incubated with or without various concentrations of CsA for 48 h. (F) CXCL2 production in cultured MCD cells dissected from WT, Tlr42/2, Nod12/2, or Nod22/2 kidneys incubated with or without CsA for 48 h, then with or without 104 UPEC (upper panel, n = 3–5 determinations from 3 independent experiments) or 10 ng/ml LPS (lower panel, values from 4 separate cultures of MCD cells dissected from a single mouse in each group) for 3 h or 6 h, respectively. Values are presented as mean 6 SE. *, p,0.05 (C, E, Mann-Whitney test; D, Two-tailed, unpaired Student’s t test). doi:10.1371/journal.ppat.1003152.g004

Article Snippet: In parallel, aliquots of NH4Cl-treated blood samples were permeabilized with methanol, and then incubated with an antihuman NOD1 antibody (Imgenex Corp.).

Techniques: Staining, Reverse Transcription, Amplification, Cell Culture, Incubation, Real-time Polymerase Chain Reaction, Western Blot, MANN-WHITNEY, Two Tailed Test

Journal: PLoS pathogens

Article Title: Cyclosporine A impairs nucleotide binding oligomerization domain (Nod1)-mediated innate antibacterial renal defenses in mice and human transplant recipients.

doi: 10.1371/journal.ppat.1003152

Figure Lengend Snippet: Figure 5. CsA preferentially impairs Nod1 expression in neutrophils, macrophages, and renal dendritic cells. (A, D, G) Fold variations of Tlr4, Nod1 and Nod2 mRNAs expression measured by quantitative real-time PCR in WT bone marrow neutrophils (A), macrophages (D), and renal CD11c+ DCs (G) incubated with CsA for 8 h (neutrophils) or 36–48 h (macrophages, DCs) at 37uC, and compared to corresponding untreated cells. (B and E). Immunodetection of the Nod1 protein in untreated (2 CsA) and CsA-treated (+ CsA) neutrophils and BMMs. (C, F, H) Effects of 100 nM CsA on CCL5 and CXCL2 production [mean values of mean individual values (n = 3–4) from 3 to 4 separate experiments] in supernatants from neutrophils (C) and macrophages (F) (106 cells/well, or renal DCs (105 cells/well) sequentially incubated without or with CsA for 8 h (neutrophils) or 36–48 h (macrophages, renal DCs), then with CsA and 10 ng/ml LPS, or 1 mM FK156 or MDP for a further 6 h at 37uC. Values are presented as mean 6 SE. *, p,0.05 between groups or versus untreated or CsA-treated cells (Two-tailed, unpaired Student’s t test). doi:10.1371/journal.ppat.1003152.g005

Article Snippet: In parallel, aliquots of NH4Cl-treated blood samples were permeabilized with methanol, and then incubated with an antihuman NOD1 antibody (Imgenex Corp.).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation, Immunodetection, Two Tailed Test

Journal: PLoS pathogens

Article Title: Cyclosporine A impairs nucleotide binding oligomerization domain (Nod1)-mediated innate antibacterial renal defenses in mice and human transplant recipients.

doi: 10.1371/journal.ppat.1003152

Figure Lengend Snippet: Figure 6. NFATc1 controls Nod1 mRNA expression in macrophages. (A) Representative reverse transcription-PCR of the inhibition of NFATc1 in BMMs transfected with a multiple set (n = 4) of NFATc1a–d siRNAs compared to non-transfected BMMS or with cells transfected with a negative control (Control) siRNA. GAPDH mRNA expression was used as internal control. (B and C) Fold variation of Nod1 and Nod2 mRNAs measured by quantitative real time PCR in BMMs transfected with the set of NFATc1a–d siRNAs, or with a negative control (Control) siRNA and compared to non- transfected BMMs (B), and in BMMs incubated with or without 1 mM 11R-VIVIT for 48 h (C) (n = 3 independent experiments in each condition tested). (D) Nucleo-cytoplasmic distribution of the NFATc1 immunostaining (shown in red) (left panel) and percentage of NFATc1 immunostaining co- localized with Sytox green nuclear acid staining (n = 7–13 nuclei analyzed for each condition from two separate experiments) (right panel) in WT BMMs sequentially incubated without or with 11R-VIVIT or 1027 M CsA for 48 h, then without or with 2 mM ionomycin for additional 60 min. Bar = 10 mm. (E) Fold variation of Nod1 and Nod2 mRNAs expression over corresponding b-actin mRNA measured by quantitative real-time PCR in WT BMMs incubated or not with 11R-VIVIT or CsA for 48 h, then with or without 2 mM ionomycin. (n = 3 independent experiments). *, p,0.05 between groups (Two-tailed, unpaired Student’s t test). doi:10.1371/journal.ppat.1003152.g006

Article Snippet: In parallel, aliquots of NH4Cl-treated blood samples were permeabilized with methanol, and then incubated with an antihuman NOD1 antibody (Imgenex Corp.).

Techniques: Expressing, Reverse Transcription, Inhibition, Transfection, Negative Control, Control, Real-time Polymerase Chain Reaction, Incubation, Immunostaining, Staining, Two Tailed Test

Journal: PLoS pathogens

Article Title: Cyclosporine A impairs nucleotide binding oligomerization domain (Nod1)-mediated innate antibacterial renal defenses in mice and human transplant recipients.

doi: 10.1371/journal.ppat.1003152

Figure Lengend Snippet: Figure 7. The 11R-VIVIT peptide inhibitor of NFATs decreases bacterial phagocytic killing capacity by neutrophils and increases renal susceptibility to UPEC. WT mice were given daily intraperitoneal injections of 10 mg/kg 11R-VIVIT for 48 h before the transurethral inoculation of UPEC. (A) Illustrations and quantification of internalized Texas red-coupled E. coli by peritoneal neutrophils from untreated (2) and 11R-VIVIT-treated (+) mice. Values are represented as mean 6 SE of the mean count values (2–4 per condition) from three independent experiments. Cell membranes were stained with CD11b- FITC. Bars = 10 mm. (B) Killing of serum-opsonized E. coli by peritoneal neutrophils from untreated and 11R-VIVIT-treated mice. Bacterial viability was expressed relative to control assay performed without neutrophils (n = 3–5 determinations from 3 experiments). (C to E) MPO activity (C), representative immunoblot analysis of Nod1 and corre- sponding ß-actin (D), and bacterial counts (E) in kidneys from untreated and 11R-VIVIT-treated WT mice 24 h after UPEC inoculation. The numbers indicate the ratio of Nod1 over ß-actin densitometric values from 4 experiments and the horizontal bars show the mean of bacterial counts of each group (n = 6 determinations in each group). Values are presented as mean 6 SE. *, p,0.05 between groups (Two-tailed, unpaired Student’s t test). doi:10.1371/journal.ppat.1003152.g007

Article Snippet: In parallel, aliquots of NH4Cl-treated blood samples were permeabilized with methanol, and then incubated with an antihuman NOD1 antibody (Imgenex Corp.).

Techniques: Staining, Control, Activity Assay, Western Blot, Two Tailed Test

Journal: PLoS pathogens

Article Title: Cyclosporine A impairs nucleotide binding oligomerization domain (Nod1)-mediated innate antibacterial renal defenses in mice and human transplant recipients.

doi: 10.1371/journal.ppat.1003152

Figure Lengend Snippet: Figure 8. The Nod1 stimulating agonists C12-iEDAP and FK156 stimulate neutrophil bacterial phagocytosis impaired by CsA and increase renal defenses against UPEC. (A) Illustrations and quantification of Texas red-coupled E. coli internalized by circulating neutrophils collected from WT mice treated with CsA for 5 days then with CsA plus C12-iEDAP (80 mg per mouse) for a further 5 h. Neutrophils were isolated from the blood of 3 mice under each of the conditions tested. Representative values (mean 6 SE) of the mean counts (9–15 per condition) from 3 independent experiments. Cell membranes were stained with WGA Alexa Fluor 647 conjugate. Bars, 10 mm. (B and C) Bacterial counts (B) and MPO activity (C) in kidneys of CsA-treated WT mice pre-treated or not with C12-iEDAP (n = 6), FK156 (n = 9), or MDP (n = 7) (80 mg per mouse), and then challenged 24 h later with E. coli HT7. (D) Immunoblot analysis of Nod1 and the corresponding ß-actin in the post-infected kidney homogenates from vehicle- and CsA-treated mice which had received FK156 12 h before UPEC inoculation. (E) Production of CXCL2 and CXCL1 in the 24 h post-infected kidneys from vehicle- or CsA-treated WT mice pre-treated or not with C12-iEDAP or FK156 (n = 6–9 per group). *, p,0.05 (A, C, E, Two-tailed, unpaired Student’s t test; B, Mann-Whitney test). doi:10.1371/journal.ppat.1003152.g008

Article Snippet: In parallel, aliquots of NH4Cl-treated blood samples were permeabilized with methanol, and then incubated with an antihuman NOD1 antibody (Imgenex Corp.).

Techniques: Isolation, Staining, Activity Assay, Western Blot, Infection, Two Tailed Test, MANN-WHITNEY

Journal: PLoS pathogens

Article Title: Cyclosporine A impairs nucleotide binding oligomerization domain (Nod1)-mediated innate antibacterial renal defenses in mice and human transplant recipients.

doi: 10.1371/journal.ppat.1003152

Figure Lengend Snippet: Figure 9. Functional downexpression of NOD1 in leukocytes from CsA-treated renal transplant recipients. (A) IL-8 production in blood samples from healthy volunteers (Controls, n = 10) and renal transplant recipients treated with CsA (n = 25) incubated without (None) or with 1 ng/ml Pam3CSK4 or LPS, 1 mg/ml flagellin, 50 mg/ml CpG-DNA, 1 mM M-TriDAP or MDP. (B) IL-8 production in blood samples from Controls and renal transplant recipients incubated with increasing concentrations of M-TriDAP. (C) Relative fold variation of TLR2, TLR4, NOD1 and NOD2 mRNAs in blood samples from renal transplant recipients (n = 25) compared to that found in Controls (n = 10). (D) Flow cytometry analyses of phagocytosed E. coli (expressed as fluorescence intensity of internalized E. coli) and mean fluorescence intensity (MFI) of NOD1 expression in neutrophils from healthy volunteers (n = 10) and renal transplant recipients treated with CsA (n = 25). * Transplant recipients who experienced UTI/APN. Values are presented as mean 6 SE from n determinations per condition tested. *, p,0.05 (A, B, Two-tailed, unpaired t test; C, Mann-Whitney test). doi:10.1371/journal.ppat.1003152.g009

Article Snippet: In parallel, aliquots of NH4Cl-treated blood samples were permeabilized with methanol, and then incubated with an antihuman NOD1 antibody (Imgenex Corp.).

Techniques: Functional Assay, Incubation, Flow Cytometry, Fluorescence, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Reproductive Sciences

Article Title: A Role for the Inflammasome in Spontaneous Labor at Term with Acute Histologic Chorioamnionitis

doi: 10.1177/1933719116675058

Figure Lengend Snippet: NLRC4 and NOD1 in the chorioamniotic membranes. A and D, Messenger RNA abundance of NLRC4 and NOD1 in the chorioamniotic membranes from women in spontaneous labor at term with (TIL-ACA, n = 32) or without (TIL, n = 28) acute histologic chorioamnionitis. Relative gene expressions are presented as −ΔCt values. B and E, Protein quantity of NLRC4 and NOD1 in chorioamniotic membrane tissue lysates (n = 5 each). C and F, Intensity of the immunostainings for NLRC4 and NOD1 in the chorioamniotic membranes (n = 10 each) and representative immunostainings, 200× magnifications. NOD indicates nucleotide-binding oligomerization domain.

Article Snippet: Next, membranes were submerged in blocking solution (5% nonfat dry milk in tris-buffered saline containing 0.1% Tween-20 [Bio-Rad] or StartingBlock T20 Block Buffer [ThermoFisher Scientific, Inc, Rockford, Illinois]) and probed overnight at 4°C with the following human antibodies—mouse anti-NLRC4 antibody (BioLegend, San Diego, California), rabbit anti-NOD1 polyclonal antibody (Enzo Life Sciences, Farmingdale, New York), 130 mouse anti-CASP-1 monoclonal antibody (R&D Systems), rabbit anti-CASP-4 polyclonal antibody (Abcam, Cambridge, Massachusetts), or rabbit anti-IL-18 polyclonal antibody (Santa Cruz Biotechnology, Dallas, Texas).

Techniques: Binding Assay