cas9 t2a puromycin resistance cassette  (Integrated DNA Technologies)

Journal: Science signaling

Article Title: A small sustained increase in NOD1 abundance promotes ligand-independent inflammatory and oncogene transcriptional responses

doi: 10.1126/scisignal.aba3244

Figure Lengend Snippet: (A-C) qPCR (A), immunoblot (B) and flow cytometry plots (C) showing oncogene expression in THP-1 NOD1 cells in absence and presence of DOX. (D) Immunoblot showing the abundance of the oncogene C-MYC and the phosphorylation (p) of AKT in THP-1 NOD1 cells in presence or absence of DOX. (E) Kinetics of C-JUN and C-MYC expression following ligand treatment as measured by qPCR. Grey arrows indicate times of recurring ligand addition (0, 24 and 48 h). Expression at each time point is represented relative to the untreated control at that timepoint (dashed line set to 1). (F-H) qPCR (F) and immunoblots (G, H) for the indicated genes in THP-1 NOD1 cells following CRISPR/Cas9 mediated ablation of FLAG-NOD1 in three independent single cell clones (NF1, NF2 and NF3). (I) qPCR for the indicated genes in THP-1 NOD1 cells following CRISPR/Cas9 mediated ablation of RIPK2 in three independent single cell clones. NT: non-targeting gRNA. NF: gRNA targeting FLAG-NOD1. RIPK2: gRNA targeting RIPK2. Data in (A) to (I) are representative of three independent experiments. Error bars on graphs are mean±SEM of at least three biological replicates and where not visible in (E) are shorter than the height of the symbol. p values from an unpaired t test (two-tailed) without assuming a consistent standard deviation are shown. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: CRISPR mediated ablation of RIPK2 and FLAG NOD1 For CRISPR/Cas9 targeting of RIPK2 or FLAG NOD1 , the guide RNA targeting RIPK2 or FLAG NOD1 (IDT) was cloned into a single self-inactivating lentivirus plasmid pRRL-U6-empty-gRNA-MND-Cas9-t2A-Puro, that expresses a Cas9-T2A-puromycin resistance cassette controlled by an MND promoter ( 45 ).

Techniques: Western Blot, Flow Cytometry, Expressing, CRISPR, Clone Assay, Two Tailed Test, Standard Deviation

Journal: Science signaling

Article Title: A small sustained increase in NOD1 abundance promotes ligand-independent inflammatory and oncogene transcriptional responses

doi: 10.1126/scisignal.aba3244

Figure Lengend Snippet: (A) Histograms (left) and quantification (right) of NOD1 protein in THP-1 cells treated with scramble or miR-191 LNA or miR-15b/16 TSB. (B) qPCR for the indicated oncogenes in cells treated with miR-15b/16 TSB. Gene expression at each timepoint is represented relative to the scramble LNA-treated control at that timepoint (dashed line set to 1). (C) qPCR for miR-15b, miR-16, NOD1 and oncogenes in ex vivo human monocytes following inhibition of miR-15b and miR-16 with LNA. Gene expression is represented relative to that in the scramble LNA control (dashed line set to 1). (D) qPCR for miR-15b, miR-16 and NOD1 (left) and the indicated oncogenes (right) in THP-1 cells following CRISPR/Cas9 mediated reduction of miR15b/16 in two independent clones analyzed 4–6 weeks after miR-15b/16 targeting and single cell cloning. Gene expression is represented relative to that in the non-targeting (NT) gRNA control (dashed line set to 1). (E) qPCR for the indicated inflammatory genes in THP-1 cells following CRISPR/Cas9 reduction of miR15b/16. Data in A, B and D are representative of three independent experiments, and in E are representative of two independent experiments with three biological replicates per condition. In C, one representative of two independent donors is shown with three replicates per condition. Error bars on graphs are mean±SEM and p values from an unpaired t test (two-tailed) without assuming a consistent standard deviation are shown. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns: not significant.

Article Snippet: CRISPR mediated ablation of RIPK2 and FLAG NOD1 For CRISPR/Cas9 targeting of RIPK2 or FLAG NOD1 , the guide RNA targeting RIPK2 or FLAG NOD1 (IDT) was cloned into a single self-inactivating lentivirus plasmid pRRL-U6-empty-gRNA-MND-Cas9-t2A-Puro, that expresses a Cas9-T2A-puromycin resistance cassette controlled by an MND promoter ( 45 ).

Techniques: Expressing, Ex Vivo, Inhibition, CRISPR, Clone Assay, Two Tailed Test, Standard Deviation