paper n a plko 1 puro addgene  (Addgene inc)

Journal: Cell reports

Article Title: Depletion of Mettl3 in cholinergic neurons causes adult-onset neuromuscular degeneration

doi: 10.1016/j.celrep.2024.113999

Figure Lengend Snippet: Key Resources Table

Article Snippet: PT 58.5553804, exon location 2-3 IDT Cat#447472065 For all guide RNA and HDR oligo sequences for CRISPR/Cas9 deletion of Mettl3 check Table S2 This study N/A siRNA 1-ON-TARGET plus siRNA METTL3 (Human) SMARTPool Horizon 22 Discovery, Dharmacon N/A Nontargeting siRNA #1 Horizon 22 Discovery, Dharmacon N/A Recombinant DNA lentiCRISPR v2 plasmid Addgene RRID:Addgene_52961 pLKO.1-puro vector Sigma MISSION Cat#SHC001 pLKO.1-puro Non-Mammalian shRNA control transduction particles Sigma MISSION Cat#SHC002H pLKO.1-Ythdf1 Sigma MISSION TRCN0000348429 pLKO.1-Ythdf2 Sigma MISSION TRCN0000197932 pLKO.1-Ythdf3 Sigma MISSION TRCN0000329409 pLKO.1-Mettl3 Sigma MISSION TRCN0000039111 pcDNA3-flag-YTHDF2 Addgene RRID:Addgene_52300 pcDNA-FLAG-METTL3 Addgene RRID:Addgene_53739 pcDNA-FLAG-METTL3-APPA Addgene RRID:Addgene_160251 pRRL-CMV-flag-YTHDF2 This study N/A pRRL-CMV-FLAG-METTL3 This study N/A pRRL-CMV-FLAG-METTL3-APPA This study N/A Software and algorithms GraphPad Prism 9.2.0 GraphPad https://www.graphpad.com/features Fiji (Image J) NIH https://imagej.nih.gov/ij/ Adobe Photoshop Adobe https://adobe.com Nanocompore (Leger et al.) 56 https://github.com/tleonardi/nanocompore_pipeline biomaRt, v2.42.0 (Durinck et al.) 76 (Durinck et al.) 77 https://dec2021.archive.ensembl.org:443/biomart/martservice DESeq2, v1.26.0 (Love et al.) 78 DOI: 10.18129/B9.bioc.DESeq2 MinKNOW (v19.12.6) Oxford Nanopore N/A Guitar package (v2.2.0) (Cui et al.) 79 DOI: 10.18129/B9.bioc.Guitar MEME suite (Bailey et al.) 80 https://meme-suite.org/meme/ clusterProfiler, v3.14.3 (Wu et al.) 81 (Yu et al.) 82 DOI: 10.18129/B9.bioc.clusterProfiler compEpiTools, v1.20.0 (Kishore et al,) 83 DOI: 10.18129/B9.bioc.compEpiTools Nanopolish, v0.13.2 (Loman et al.) 74 https://github.com/jts/nanopolish NanopolishComp, v0.6.11 (Leger et al.) 56 https://github.com/a-slide/NanopolishComp Guppy v3.2.10 Oxford Nanopore N/A pycoQC (2.5.0.21) (Leger et al.) 56 https://github.com/a-slide/pycoQC Minimap2 (v2.10-r761) (Li et al.) 70 https://github.com/lh3/minimap2 GRCm38.p6 Ensembl Genome browser http://nov2020.archive.ensembl.org/Mus_musculus/Info/Index Other Open in a separate window Key Resources Table. . METTL3 downregulation and TDP-43 autoregulation The stable HEK293 TDP-43 cell line expressing human TDP-43 upon tetracycline induction was generated as described 59 .

Techniques: Virus, Recombinant, cDNA Synthesis, Purification, RNA Sequencing, CRISPR, Plasmid Preparation, shRNA, Control, Transduction, Software

Journal: Communications Biology

Article Title: Caspase-4 promotes metastasis and interferon-γ-induced pyroptosis in lung adenocarcinoma

doi: 10.1038/s42003-024-06402-3

Figure Lengend Snippet: a Comparison of CASP4 mRNA expression between early-stage and advanced-stage lung adenocarcinoma tissues (stages I–II vs. III–IV) based on RNA sequencing (RNA-seq) data (n = 56). b Representative images of immunoblotting analysis of CASP4 protein in mock and CASP4 -overexpressing (C4OE) NCI-H292 cells. Bar graphs indicate the band intensities of CASP4 normalized to beta-actin bands (n = 3). Data from mock was set at 1. c Flow chart of the RNA-seq experiment and data analysis. d The top eight upregulated gene ontology (GO) terms in NCI-H292 cells following CASP4 overexpression based on GO analysis of coding sequence expression data obtained from RNA sequencing. The horizontal axis shows accumulative hypergeometric p values. e Representative heatmap of cell migration- and angiogenesis-related genes upregulated by CASP4 . f RNA-seq analysis of antiapoptosis-related mRNAs, such as BCL2 , BCL2A1 , BCL2L1 , and MCL1 , between C4OE and mock cells. Each gene is presented as fragments per kilobase of exon million mapped reads (FPKM). N.S: nonspecific; n.s.: not significant. Data are expressed as mean ± standard deviation (n = 3). * P < 0.05, ** P < 0.01 compared with mock. Densitometry values are shown above each blot.

Article Snippet: These sequences were used to generate the lentiviral vector pLKO.1 puro (Addgene #8453)-based shRNAs targeting CASP4 or STAT1 .

Techniques: Comparison, Expressing, RNA Sequencing, Western Blot, Over Expression, Sequencing, Migration, Standard Deviation

Journal: Communications Biology

Article Title: Caspase-4 promotes metastasis and interferon-γ-induced pyroptosis in lung adenocarcinoma

doi: 10.1038/s42003-024-06402-3

Figure Lengend Snippet: a Representative images of immunoblotting analysis of CASP4 protein in mock and CASP4 -knockout (C4KO) NCI-H292 cells. b Representative images of fluorescent immunostaining of CASP4 protein in mock and CASP4 -knockout (C4KO) cells. Scale bar, 20 μm. c Sequence showing insertion and deletion in exon 2 of human CASP4 in C4KO NCI-H292 cells. d Flowchart of the RNA-seq experiment and data analysis. e The top eight downregulated GO terms in NCI-H292 cells following CASP4 KO based on GO analysis of CDS expression data obtained from RNA sequencing. The horizontal axis shows accumulative hypergeometric p values. f Representative heatmap of cell migration- and angiogenesis-related genes downregulated by CASP4 KO . g Differentially expressed genes that overlapped between C4OE upregulated and C4KO downregulated in “angiogenesis” and “positive cell migration. h Positive regulation of cell migration- and angiogenesis-related mRNAs, such as EPHA2 and PTGS2 , based on RT-qPCR in C4OE and mock cells. i Positive regulation of cell migration- and angiogenesis-related mRNAs, such as EPHA2 , and PTGS2 , based on RT-qPCR in C4KO and mock cells.” N.S: nonspecific. Data are expressed as mean ± standard deviation (n = 3). ** P < 0.01 compared with mock. Densitometry values are shown above each blot.

Article Snippet: These sequences were used to generate the lentiviral vector pLKO.1 puro (Addgene #8453)-based shRNAs targeting CASP4 or STAT1 .

Techniques: Western Blot, Knock-Out, Immunostaining, Sequencing, RNA Sequencing, Expressing, Migration, Quantitative RT-PCR, Standard Deviation

Journal: Communications Biology

Article Title: Caspase-4 promotes metastasis and interferon-γ-induced pyroptosis in lung adenocarcinoma

doi: 10.1038/s42003-024-06402-3

Figure Lengend Snippet: Ranking of the top ten upregulated transcription factors presumed to be involved in C4OE ( a ) and C4KO ( b ) by TRRUST database analysis of the CDS expression data obtained from RNA sequencing of NCI-H292 cells. The horizontal axis shows accumulative hypergeometric p values. c Comparison of relative luciferase activity between mock and C4KO NCI-H292 cells. d Immunoblotting analysis of phospho-p65, p65, and CASP4 in mock and C4KO cells. e Comparison of relative luciferase activity between mock and C4KO cells with rescued CASP4 gene (C4KO + C4) (n = 3). f Immunoblotting analysis of phospho-p65 and CASP4 protein in mock, C4KO, and C4KO + C4 cells. g mRNA levels of CASP4 , PTGS2, SERPINE1 , and EPHA2 in mock and C4OE cells with or without treatment with the NF-κB inhibitor BAY11-7082. h Protein levels of CASP4 in mock and C4OE cells with or without treatment with BAY11-7082. i Representative images of the wound healing assay with or without treatment with BAY11-7082 using mock or C4OE NCI-H292 cells at 0 and 24 h (scale bar, 500 μm). j The ratios of wound width after 24 h to initial wound width in ( i ) were graphed as percent wound closure (n = 3). In each experiment, treatment with BAY11-7082 (2.5 μM) lasted 8 h. n.s.: not significant. Data are expressed as mean ± standard deviation (n = 3). * P < 0.05, ** P < 0.01 compared with mock. Densitometry values are shown above each blot.

Article Snippet: These sequences were used to generate the lentiviral vector pLKO.1 puro (Addgene #8453)-based shRNAs targeting CASP4 or STAT1 .

Techniques: Expressing, RNA Sequencing, Comparison, Luciferase, Activity Assay, Western Blot, Wound Healing Assay, Standard Deviation

Journal: Communications Biology

Article Title: Caspase-4 promotes metastasis and interferon-γ-induced pyroptosis in lung adenocarcinoma

doi: 10.1038/s42003-024-06402-3

Figure Lengend Snippet: a Identification of the top 133 genes (Spearman’s correlation >0.4) coexpressed with CASP4 using the TGCA-LUAD dataset. b The top 133 genes coexpressed with CASP4 were analyzed via TRRUST analysis to determine the transcription factors involved. The horizontal axis shows accumulative hypergeometric p-values. c CASP4 mRNA induction by IFN-γ stimulation in NCI-H292 cells. Cells were treated for 24 h. d Immunoblotting analysis of CASP4 in NCI-H292 cells treated with PBS and 1 ng/mL of IFN-γ. Cells were treated for 48 h. e Evaluation of CASP4 mRNA expression in NCI-H292 cells by RT-qPCR and assessment of cell viability by CCK-8 assay after treatment with IFN-γ at different concentrations. The samples for RT-qPCR and CCK-8 assay were collected after 24 h and 72 h, respectively. f Comparison of CASP4 mRNA expression by RT-qPCR between mock and CASP4 OE after treatment with high-dose IFN-γ (100 ng/mL) for 24 h. g Downregulation of CASP4 mRNA expression by STAT1 knockdown (shSTAT1) in NCI-H292 cells. h Representative images of immunoblotting analysis of CASP4 and STAT1 protein in mock and shSTAT1 NCI-H292 cells. n.s.: not significant; N.S: nonspecific. Data are expressed as mean ± standard deviation (n = 3). * P < 0.05, ** P < 0.01 compared with PBS treatment. Densitometry values are shown above each blot.

Article Snippet: These sequences were used to generate the lentiviral vector pLKO.1 puro (Addgene #8453)-based shRNAs targeting CASP4 or STAT1 .

Techniques: Western Blot, Expressing, Quantitative RT-PCR, CCK-8 Assay, Comparison, Knockdown, Standard Deviation