sgrna targeting pkn1 and pkn2  (Addgene inc)

Journal: EMBO Molecular Medicine

Article Title: Pyrin dephosphorylation is sufficient to trigger inflammasome activation in familial Mediterranean fever patients

doi: 10.15252/emmm.201910547

Figure Lengend Snippet: A PKN1 KO clones expressing p.M694V or WT Pyrin were obtained and confirmed by Western blot analysis. Four clones are shown. Clones 3 and 6 were selected. B Absolute transcript level of the indicated genes was quantified by RNAseq in U937 cells (Benaoudia et al , ) and expressed as count per million (cpm). PYCARD encodes ASC and is shown as a reference. C PKN2 transcript levels were assessed in U937 cells at 24 h post‐electroporation with the indicated non‐targeting (NT) siRNA or three different siRNAs targeting PKN2 . D–F U937 cells with the indicated Pyrin variant were treated (+Dox, plain lines) or not (No Dox, dotted lines) with doxycycline (Dox) at 24 h post‐electroporation with the indicated siRNA. Cell death was monitored every 15 min. (F) SiRNA 14 did not reduce substantially PKN2 levels. Data information: (D, E, F) Cell death was normalized to the maximal propidium iodide (PI) incorporation determined using TX‐100‐treated cells. Each dot represents the mean ± SD of a biological triplicate from one experiment representative of three independent experiments. Source data are available online for this figure.

Article Snippet: The sgRNA targeting PKN1 and PKN2 ( ) were selected from the Brunello library (Addgene) and cloned into the PGKpuro2ABFP vector (from Kosuke Yusa; Addgene plasmid # 50946). sgRNA plasmids were transduced in a previously described Cas9‐expressing U937 clone (Lagrange et al , ) by spinoculation.

Techniques: Clone Assay, Expressing, Western Blot, Electroporation, Variant Assay

Journal: EMBO Molecular Medicine

Article Title: Pyrin dephosphorylation is sufficient to trigger inflammasome activation in familial Mediterranean fever patients

doi: 10.15252/emmm.201910547

Figure Lengend Snippet: A PKN1 KO clones expressing p.M694V or WT Pyrin were obtained and confirmed by Western blot analysis. Four clones are shown. Clones 3 and 6 were selected. B Absolute transcript level of the indicated genes was quantified by RNAseq in U937 cells (Benaoudia et al , ) and expressed as count per million (cpm). PYCARD encodes ASC and is shown as a reference. C PKN2 transcript levels were assessed in U937 cells at 24 h post‐electroporation with the indicated non‐targeting (NT) siRNA or three different siRNAs targeting PKN2 . D–F U937 cells with the indicated Pyrin variant were treated (+Dox, plain lines) or not (No Dox, dotted lines) with doxycycline (Dox) at 24 h post‐electroporation with the indicated siRNA. Cell death was monitored every 15 min. (F) SiRNA 14 did not reduce substantially PKN2 levels. Data information: (D, E, F) Cell death was normalized to the maximal propidium iodide (PI) incorporation determined using TX‐100‐treated cells. Each dot represents the mean ± SD of a biological triplicate from one experiment representative of three independent experiments. Source data are available online for this figure.

Article Snippet: The sgRNA targeting PKN1 and PKN2 ( ) were selected from the Brunello library (Addgene) and cloned into the PGKpuro2ABFP vector (from Kosuke Yusa; Addgene plasmid # 50946). sgRNA plasmids were transduced in a previously described Cas9‐expressing U937 clone (Lagrange et al , ) by spinoculation.

Techniques: Clone Assay, Expressing, Western Blot, Electroporation, Variant Assay