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: 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: 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

Journal: bioRxiv

Article Title: Wnt-inducible Lrp6-APEX2 Interacting Proteins Identify ESCRT Machinery and Trk-Fused Gene as Components of the Wnt Signaling Pathway

doi: 10.1101/2020.05.03.072579

Figure Lengend Snippet: CRISPR-Cas9 mediated TFG knock-out inhibits Wnt-dependent β-catenin stabilization and Wnt-induced reporter activity. ( A ) Schematic diagram of Cas9 cleaving the TFG genomic DNA target sequence. The target sequence (which is on the reverse strand) is shown in 5’ to 3’ orientation (left to right); the PAM (protospacer adjacent motif) sequence is in bold. ( B ) Western blot of HEK293T cells confirming complete elimination of TFG protein in Cas9 knock-out cells. β-actin was used as loading control. ( C-F ) Immunostaining for β-catenin on WT or TFG KO cells. Cells were treated with control conditioned medium or with Wnt3a conditioned medium for 3 hours before immunostaining. Note that TFG knock-out decreases Wnt3a-induced β-catenin accumulation; DAPI was used for nuclear counter-staining. Scale bars represent 20 µm. ( G ) Cas9-mediated TFG knock-out reduces response to Wnt3a by 50% in HEK293T BAR/Renilla β-catenin reporter cells. Cells were treated with Wnt3a or control medium for 16 hours before luciferase analysis. ( H ) Luciferase assay of HEK293T BAR reporter cells treated with the GSK3 inhibitor CHIR99021 at 5 µM concentration for 16 hours before being processed for luciferase assay. Note that TFG KO causes an 80% reduction of β-catenin luciferase activity in response to the GSK3 inhibition. Error bars represent standard deviation from triplicate experiments. Statistical significance was calculated with a paired 2-tailed t-Student test. * = P < 0.05; ** = P < 0.01.

Article Snippet: A plasmid based on the original PX459 from Zhang lab, containing Streptococcus pyogenes Cas9 (spCas9)-2A-blasticidin resistance was obtained from Addgene (#118055, from Ken-Ichi Takemaru lab).

Techniques: CRISPR, Knock-Out, Activity Assay, Sequencing, Western Blot, Control, Immunostaining, Staining, Luciferase, Concentration Assay, Inhibition, Standard Deviation

Journal: bioRxiv

Article Title: Wnt-inducible Lrp6-APEX2 Interacting Proteins Identify ESCRT Machinery and Trk-Fused Gene as Components of the Wnt Signaling Pathway

doi: 10.1101/2020.05.03.072579

Figure Lengend Snippet: Genomic DNA was extracted from untransfected WT HEK293T cells, and from a cell clone stably transfected with a Cas9 expression vector containing an sgRNA-encoding sequence specific for human TFG. A 600 nucleotide region spanning the target sequence was amplified by PCR, cloned into sequencing vectors, and analyzed by Sanger sequencing. The reference chromatogram from sequencing results is shown below the DNA sequence. ( A ) Nucleotide sequence of TFG from wild-type (WT) HEK293 cells; the coding frame is indicated by black dots between each codon. The boxed area marks the target sequence recognized by the spacer region in the sgRNA in the WT TFG sequence. The predicted amino acid sequence is shown above the DNA sequence. ( B and C ) Sequences from a TFG knock-out clonal cell line showing the two types of mutant sequences resulting after PCR amplification of mutated genomic DNA. Each TFG allele harbored independent insertion-deletion (indel) events induced by Cas9 as a result of non-homologous end joining (NHEJ) repair. In Mutation 1 (Mut1) Cas9 induced a deletion of a short nucleotide sequence (8 nucleotides, AAGACCCC). Mutation 2 (Mut 2) resulted in the insertion of an extra adenine between the original CCA and AGA codons. Both indel events induced a frameshift mutation, introducing an early STOP codon indicated by an asterisk, generating truncated TFG mutant proteins of approximately 90 amino acids.

Article Snippet: A plasmid based on the original PX459 from Zhang lab, containing Streptococcus pyogenes Cas9 (spCas9)-2A-blasticidin resistance was obtained from Addgene (#118055, from Ken-Ichi Takemaru lab).

Techniques: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Sequencing, Amplification, Clone Assay, Knock-Out, Mutagenesis, Non-Homologous End Joining

Journal: Frontiers in Immunology

Article Title: Human ILC3 Exert TRAIL-Mediated Cytotoxicity Towards Cancer Cells

doi: 10.3389/fimmu.2022.742571

Figure Lengend Snippet: ILC3s kill tumor cells via TRAIL-TRAILR2 pathway. (A) Representative histograms of TRAILR1 and TRAILR2 expression by parental HuH6, HepG2 and SK-Mel-37 cells, and TRAILR2 expression by TRAILR2 KO and control cell lines expressing Cas9. (B) Specific target cell lysis of HuH6 TRAILR2 KO, HepG2 TRAILR2 KO, SK-Mel-37 TRAILR2 KO and the control cell lines expressing Cas9 after 24h co-culture with ILC3s (n=3 blood donors, E:T ratios 4-10:1). *p <0.05, by paired Student’s t-test (HepG2 and HuH6); p=0.0625 by Wilcoxon matched-pairs signed rank test (SK-Mel-37).

Article Snippet: HepG2, HuH6 and SK-Mel-37 cells were first transduced with the lentiCas9-Blast (Addgene #52962) vector, encoding Cas9 protein and Blasticidin-resistance gene, used to select Cas9-expressing cells.

Techniques: Expressing, Lysis, Co-Culture Assay