Journal: Infection and Immunity

Article Title: NALP1 Influences Susceptibility to Human Congenital Toxoplasmosis, Proinflammatory Cytokine Response, and Fate of Toxoplasma gondii -Infected Monocytic Cells

doi: 10.1128/iai.00898-10

Figure Lengend Snippet: FIG. 2. Analysis of the effect of NALP1 and TetR shRNAs on expression of NALP1. (A) Illustration of the promoter features of the pLenti4/Block-iT-DEST shRNA expression vector. In the absence of a Tet repressor protein, there is constitutive expression of the cloned NALP1 or TetR shRNA fragment. (B) Quantitative real-time PCR analysis of NALP1 transcript levels in wild-type MonoMac6 cells (MC) and MonoMac6 cells engineered to express either NALP1 gene shRNA (N-KO) or tetracycline repressor gene shRNA (TetRep) after 72 h of culture. The relative transcript levels of NALP1 were derived by dividing the obtained NALP1 value by the GAPDH value for each respective sample. The data shown are means for three independent experiments with standard error bars. The reduction in NALP1 transcript levels in N-KO cells was found to be significant (**, P 0.001) compared to both the wild-type and TetR shRNA-expressing cells. (C) Western blotting of NALP1 protein expression in N-KO, TetRep, and MC cells was performed on cell lysates after 72 h of culture, using anti-NALP1 antibody. A similar blot was hybridized with anti-GAPDH primary antibody to check for loading. The NALP1 protein was detectable as an 155-kDa band.

Article Snippet: Short hairpin RNA (shRNA) sequences for the human NALP1 gene coding sequence (GenBank accession no. NM_001033053) and the Salmonella enterica tetracycline repressor (TetR) gene coding sequence (GenBank accession no. NC_006856) were designed for cloning into a Gatewayadapted entry vector (Invitrogen).

Techniques: Expressing, Blocking Assay, shRNA, Plasmid Preparation, Clone Assay, Real-time Polymerase Chain Reaction, Derivative Assay, Western Blot

Journal: Infection and Immunity

Article Title: NALP1 Influences Susceptibility to Human Congenital Toxoplasmosis, Proinflammatory Cytokine Response, and Fate of Toxoplasma gondii -Infected Monocytic Cells

doi: 10.1128/iai.00898-10

Figure Lengend Snippet: FIG. 5. Analysis of effects of NALP1 gene knockdown on expression of proinflammatory cytokines (IL-1, IL-18, TNF-, and IL-12) in a human monocytic cell line (MonoMac6). Quantitative real-time PCR was performed on cDNAs synthesized using equal amounts of total RNA from wild-type MonoMac6 cells (MC) and MonoMac6 cells engineered to express either NALP1 gene shRNA (NALP1-KO) or tetracycline repressor gene shRNA (TetR) that had been cultured with (gray columns) or without (white columns) parasites for 36 h. The transcript values for IL-1 (A), IL-18 (B), TNF- (C), and IL-12 (D) were normalized by dividing the observed value by the value for GAPDH for each respective sample to derive the relative gene transcript level. The significance of augmentation of cytokine expression attributed to infection is shown by asterisks (*, P 0.05; **, P 0.001). The data are shown as means for three independent experiments with standard error bars.

Article Snippet: Short hairpin RNA (shRNA) sequences for the human NALP1 gene coding sequence (GenBank accession no. NM_001033053) and the Salmonella enterica tetracycline repressor (TetR) gene coding sequence (GenBank accession no. NC_006856) were designed for cloning into a Gatewayadapted entry vector (Invitrogen).

Techniques: Knockdown, Expressing, Real-time Polymerase Chain Reaction, Synthesized, shRNA, Cell Culture, Infection

Journal: Infection and Immunity

Article Title: NALP1 Influences Susceptibility to Human Congenital Toxoplasmosis, Proinflammatory Cytokine Response, and Fate of Toxoplasma gondii -Infected Monocytic Cells

doi: 10.1128/iai.00898-10

Figure Lengend Snippet: FIG. 6. Analysis of effects of NALP1 gene knockdown on expression of caspase-1 and BCL2A1 in MonoMac6 cells infected with T. gondii. Quantitative real-time PCR was performed on cDNAs synthesized from equal amounts of total RNA from wild-type MonoMac6 cells (MC) and MonoMac6 cells engineered to express either NALP1 gene shRNA (NALP1-KO) or tetracycline repressor gene shRNA (TetR) that had been cultured with (gray columns) or without (white columns) parasites for 36 h. The obtained transcript values for caspase-1 (A) and BCL2A1 (B) were normalized by dividing the observed value by the value for GAPDH for each respective sample to derive the relative gene transcript level. The significance of the levels of augmentation of caspase-1 and BCL2A1 expression attributed to infection is shown by asterisks (**, P 0.001). The data are shown as means for three independent experiments with standard error bars.

Article Snippet: Short hairpin RNA (shRNA) sequences for the human NALP1 gene coding sequence (GenBank accession no. NM_001033053) and the Salmonella enterica tetracycline repressor (TetR) gene coding sequence (GenBank accession no. NC_006856) were designed for cloning into a Gatewayadapted entry vector (Invitrogen).

Techniques: Knockdown, Expressing, Infection, Real-time Polymerase Chain Reaction, Synthesized, shRNA, Cell Culture

Journal: Toxicological Sciences

Article Title: In vitro transcriptomic analyses reveal pathway perturbations, estrogenic activities, and potencies of data-poor BPA alternative chemicals

doi: 10.1093/toxsci/kfac127

Figure Lengend Snippet: Comparison of estrogen receptor alpha (ERα) bioactivity and transcriptomic points of departure (tPODs) derived from the whole transcriptome analysis in MCF-7 cells ( n = 3–4 per concentration) exposed to bisphenol A and 15 alternative chemicals at a range of concentrations (0.0005–100 µM) for 48 h. ERα prediction is based on at least one concentration yielding an agonist or antagonist call. For tPOD derivation, data were prefiltered using the Williams trend test ( p ≤ .05) and a fold-change of ≥1.5 or ≤1.5. Data were also postfiltered with the following settings in BMDExpress v2.3: Best BMD/BMDL <20, Best BMDU/BMD <20, Best BMDU/BMDL <40, and Best fitPvalue >.1. tPODs representing the 25th rank ordered gene benchmark concentration (BMC) (shown in µM), the median gene BMC for the lowest pathway (at least 3 genes and 5% of pathway) as well as the median gene BMC for the ERα biomarker gene set are shown in the table and in the top panel. The chemicals are shown in decreasing order of potency based on tPODS from the 25th gene BMC.

Article Snippet: Data were processed as described below and reads were aligned to the BioSpyder TempO-Seq Human Whole Transcriptome probe set (22 537 probes over 19 687 genes) using their purpose-built pipeline.

Techniques: Comparison, Derivative Assay, Concentration Assay, Biomarker Discovery

Journal: Toxicological Sciences

Article Title: In vitro transcriptomic analyses reveal pathway perturbations, estrogenic activities, and potencies of data-poor BPA alternative chemicals

doi: 10.1093/toxsci/kfac127

Figure Lengend Snippet: Comparison of ERα bioactivity and tPODs derived from the whole transcriptome analysis in MCF-7 cells ( n = 3–4 per concentration) exposed to BPA and 15 alternative chemicals at a range of concentrations (0.0005–100 μM) for 48 h. ERα prediction is based on at least one concentration yielding an agonist or antagonist call. For tPOD derivation, data were prefiltered using the Williams trend test ( p ≤ .05) and a fold-change of ≥1.5 or ≤1.5. Data were also post-filtered with the following settings in BMDExpress v2.3: Best BMD/BMDL <20, Best BMDU/BMD <20, Best BMDU/BMDL <40, and Best fitPvalue >.1. tPODs representing the 25th rank ordered gene BMC (shown in μM), the median gene BMC for the lowest pathway (at least 3 genes and 5% of pathway) as well as the median gene BMC for the ERα biomarker gene set are shown in the table and in the top panel. The chemicals are shown in decreasing order of potency based on tPODS from the 25th gene BMC. 25th gene, ERα biomarker, lowest median pathway.

Article Snippet: Data were processed as described below and reads were aligned to the BioSpyder TempO-Seq Human Whole Transcriptome probe set (22 537 probes over 19 687 genes) using their purpose-built pipeline.

Techniques: Comparison, Derivative Assay, Concentration Assay, Biomarker Discovery