Article Title: Induction of innate immune memory via microRNA targeting of chromatin remodeling factors
Figure Lengend Snippet: Tnf is a direct target of miR-222, but suppression of Tnf does not account for miR-222-mediated transcriptional silencing of late LPS response genes a , Sequence and prediction scores of a miR-222 binding site in the Tnf UTR. b , Activity of a luciferase reporter construct in which the luciferase coding sequence is followed by either the complete Tnf UTR, or a UTR in which the predicted miR-222 binding site has been mutated to the sequence shown in (a) (n=6 independent experiments). c , CRISPR-Cas9 targeting strategy to delete predicted binding sites. d , RAW clones were screened for successful deletion of the miR-222 binding site by PCR across the targeted region of the UTR, using genomic DNA from the given clonal line as a template. Screening for Tnf UTR deletion is shown. Experiment was repeated twice with similar results. e , Successful deletion of the miR-222 binding site in RAW cell clones was confirmed by sequencing genomic DNA of the given cell line. miR-222 binding site in the TNF UTR is highlighted in yellow. f , LPS-induced Tnf expression in control and CRISPR-Cas9 targeted RAW cells (n=4 independent experiments). g , Average effect of miR-222 mimic transfection on LPS-induced Tnf mRNA levels in either control MEFs or MEFs which have undergone CRISPR targeting and clonal selection for deletion of the miR-222 binding site. Average of the effects from the 3 clonal lines (n=3 independent experiments). h , Wildtype BMDMs were transfected with a control or miR-222 mimic oligonucleotide. 24 hours later, cells were pre-treated with an isotype control (IgG) or TNF neutralizing (α-TNF) antibody for two hours, and stimulated with 10 ng/ml LPS. Expression of the given genes was measured by qPCR (n=4 biologically independent samples). i , Efficacy of TNF neutralization was confirmed by treating cells with IgG or α-TNF as above, followed by stimulation with 100 ng/ml recombinant mouse TNF (n=3 biologically independent samples). Gene upregulation was not detected (ND) in 2/3 samples treated with α-TNF. For all bar graphs, mean +/− SEM is plotted. ** p
Article Snippet: DNA was purified from fractions using the Qiagen PCR Purification Kit (28104).
Techniques: Sequencing, Binding Assay, Activity Assay, Luciferase, Construct, CRISPR, Clone Assay, Polymerase Chain Reaction, Expressing, Transfection, Selection, Real-time Polymerase Chain Reaction, Neutralization, Recombinant