Journal: Molecular cell

Article Title: ZC3H14 facilitates backsplicing by binding to exon-intron boundary and 3' UTR.

doi: 10.1016/j.molcel.2024.10.001

Figure Lengend Snippet: Figure 1. Genome-wide CRISPR screen for regulators of circRNA biogenesis with circReporter cells (A) Illustration of circReporter cell construction. The circGFP minigene included circVN1R1 flanking sequences, inverted split-GFP fragments, and IRES. (B) GFP and mCherry protein levels in circReporter cells treated with siRNA (sicircGFP) against circGFP backsplicing junction (BSJ). siNC, negative control siRNA with scrambled sequences. (C) Procedure of genome-wide CRISPR knockout screen. (D) Overlapped positive and negative regulators identified from GFPhigh and GFPlow cells. (E) GO analysis of 83 overlapped regulators identified from GFPhigh and GFPlow cells. (F) 20 regulators enriched in the RNA-binding GO term.

Article Snippet: Genome-wide CRISPR knockout screen and data analysis The Human CRISPR knockout pooled library (Brunello) was purchased from the Addgene (https://www.addgene.org/).

Techniques: Genome Wide, CRISPR, Negative Control, Knock-Out, RNA Binding Assay

Journal: Nature immunology

Article Title: Toll-like receptor-mediated induction of type I interferon in plasmacytoid dendritic cells requires the rapamycin-sensitive PI(3)K-mTOR-p70S6K pathway

doi: 10.1038/ni.1645

Figure Lengend Snippet: Expression of rapamycin-sensitive mTOR pathway components in pDCs. (a) Immunoblot analysis of phosphorylated (phospho-) and total mTOR in lysates of RAW cells (4 × 106) transiently transfected for 40 h with cyan fluorescent protein–tagged MyD88 and IRF7-YFP, treated with vehicle (−) or rapamycin (+) and then stimulated for 0–60 min with CpG-A–DOTAP. Results are representative of two independent experiments. (b) Immunoblot analysis of phosphorylated and total mTOR, p70S6K, 4E-BP1 and Akt in lysates of purified pDCs (1 × 106) stimulated for 15 min with CpG-A in the presence (Rap) or absence (Medium) of rapamycin. Results are representative of three independent experiments.

Article Snippet: The following primary antibodies were from Cell Signaling (all at a dilution of 1:1,000): anti-mTOR (2972); antibody to mTOR phosphorylated at Ser2448 (2971); anti-Akt (9272); antibody to Akt phosphorylated at Thr308 (9275); anti-4E-BP1 (9452); antibody to 4E-BP1 phosphorylated at Thr37 and Thr46 (9459), Ser65 (9451) or Thr70 (9455); anti-p70S6K (9202); and antibody to p70S6K phosphorylated at Thr389 (9205).

Techniques: Expressing, Western Blot, Transfection, Purification

Journal: Nature immunology

Article Title: Toll-like receptor-mediated induction of type I interferon in plasmacytoid dendritic cells requires the rapamycin-sensitive PI(3)K-mTOR-p70S6K pathway

doi: 10.1038/ni.1645

Figure Lengend Snippet: TLR-mediated induction of IFN-α in pDCs depends on the mTOR signaling pathway. (a) ELISA of IFN-α in supernatants (above) and immunoblot analysis of mTOR in lysates (below) of purified mouse pDCs (5 × 105) transfected for 5 h with control or mTOR-specific siRNA and then stimulated for 24 h with CpG-A. Below, β-actin serves as a loading control. (b) ELISA of IFN-α in supernatants of pDCs treated with rapamycin or the PI(3)K inhibitor LY294002 (1–25 μM), then stimulated with CpG-A and assessed 24 h later. (c) ELISA of IFN-α in supernatants (above) and immunoblot analysis of p70S6K in lysates (below) of mouse pDCs transfected for 5 h with siRNA pools specific for p70S6K1 (S6K1), p70S6K2 (S6K2) or both (S6K1,2), then stimulated for 24 h with CpG-A. *, P < 0.05. (d) ELISA of IFN-α in supernatants of pDCs isolated from wild-type (WT) and Rps6k1−/−Rps6k2−/− double-knockout (S6K1,2-KO) mouse spleens and stimulated in vitro with CpG-A. Data are representative of three independent experiments (a–c) or two independent experiments with at least two mice per group per experiment (d); error bars, s.e.m. of replicate wells.

Article Snippet: The following primary antibodies were from Cell Signaling (all at a dilution of 1:1,000): anti-mTOR (2972); antibody to mTOR phosphorylated at Ser2448 (2971); anti-Akt (9272); antibody to Akt phosphorylated at Thr308 (9275); anti-4E-BP1 (9452); antibody to 4E-BP1 phosphorylated at Thr37 and Thr46 (9459), Ser65 (9451) or Thr70 (9455); anti-p70S6K (9202); and antibody to p70S6K phosphorylated at Thr389 (9205).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Purification, Transfection, Isolation, Double Knockout, In Vitro