Journal: Cell & bioscience

Article Title: CRISPRa-mediated FOXP3 gene upregulation in mammalian cells.

doi: 10.1186/s13578-019-0357-0

Figure Lengend Snippet: Fig. 2 Targeted up and down-regulation of the FOXP3 transcript levels in HEK293 cells. a Schematic presentation of the transcriptional regulation with CRISPR–dCas9. b, c HEK293 cells were transfected with plasmids encoding sgRNAs targeting six distinct regulatory regions of the FOXP3 gene (see Fig. 1) and cotransfected with plasmids encoding dCas9-based activators (VP16 or VPR) or a repressor (KRAB). Fold expression of FOXP3 was analyzed in respect to a control sample transfected with a random sgRNA (rnd) and normalized to a house-keeping gene (GAPDH). A mixture of four sgRNAs was used for each region (sgRNA sequences are listed in Table 1). RNA was isolated from samples 48 h post transfection. Experiments were repeated 3 times. b Results using dCas9-VP16 or VPR activators show that regulatory target sites downstream (CNS3, CNS1) and upstream (Cage, Cage2) of core promoter can result in higher activation of FOXP3 transcription than sgRNA targeting core promoter. c Results using dCas9-KRAB demonstrate that FOXP3 basal transcription can be further downregulated using fusion of dCas9 with a repressor domain KRAB

Article Snippet: The following primary antibodies were used for western blotting: Mouse monoclonal to GAPDH diluted 1:2000, purchased from Proteintech (#60004-1-Ig), Mouse monoclonal [236A/E7] to FOXP3 from Abcam (1:1000) and Goat Anti-Mouse IgG (H+L)-HRP diluted 1:3000 (Jackson ImmunoResearch #115-035-003).

Techniques: CRISPR, Transfection, Expressing, Control, Isolation, Activation Assay

Journal: The Journal of Neuroscience

Article Title: Calpain Inhibition Is Protective in Machado–Joseph Disease Zebrafish Due to Induction of Autophagy

doi: 10.1523/JNEUROSCI.1142-17.2017

Figure Lengend Snippet: Human ataxin-3 proteolysis is calpain dependent. A, The effect of calpain or caspase inhibition on the presence of ataxin-3 cleavage fragments at 3 dpf was examined by immunoblotting protein lysates from control or EGFP-ataxin-3-84Q larva following 2 d of treatment with DMSO, calpain inhibitor compounds (ALLN or calpeptin), a pan-caspase inhibitor (zVAD-fmk), or a cathepsin inhibitor (CAT-1). The immunoblot was probed with an antibody against cleaved spectrin, ataxin-3, and a loading control (β-actin or GAPDH). The first lane of each immunoblot contained control samples (nontransgenic) and the subsequent lanes contained EGFP-ataxin-3-84Q samples treated with DMSO and increasing concentrations of the protease inhibitor. Calpeptin (50, 100, 200 μm) treatment produced a dose-dependent increase in the amount of full-length ataxin-3 and decrease in ataxin-3 cleavage product. Exposure to ALLN produced a similar result to calpeptin treatment. Treatment with zVAD-fmk or CAT-1 failed to preserve full-length ataxin-3 or decrease the amount of cleavage fragments. B, Quantification of levels of full-length ataxin-3 within separate immunoblots (n = 3–6) for treatment with each drug revealed that 25–50 μm calpeptin significantly increased the amount of full-length ataxin-3 compared with DMSO treatment, *p < 0.04. C, Quantification of the level of ataxin-3 cleavage fragments within samples of zebrafish treated with each protease inhibitor compound revealed a trend of decreased level of cleavage fragments following treatment with calpain inhibitor compounds, with 100 μm calpeptin significantly decreasing levels of the cleavage fragment (p = 0.031). FL, Full-length; CF, cleavage fragment; ZF, zebrafish. Error bars represent mean ± SEM.

Article Snippet: Antibodies used included rabbit anti-MJD (kind gift from H. Paulson), rabbit anti-beclin-1 (11306-1-AP, Proteintech), rabbit anti-p62 (SQSTM1, MBL PM045, MBL Life Science), rabbit anti-LC3B (ab51520, Abcam), mouse anti-spectrin (sc-48382, Santa Cruz Biotechnology), mouse anti-β-actin (A5441, Sigma-Aldrich) and mouse anti-GAPDH (60004-1, Proteintech) for loading controls.

Techniques: Inhibition, Western Blot, Protease Inhibitor, Produced

Journal: eLife

Article Title: ORMDL3 restrains type I interferon signaling and anti-tumor immunity by promoting RIG-I degradation

doi: 10.7554/eLife.101973

Figure Lengend Snippet: (A) HEK293T cells were transfected with an empty vector (EV) or ORMDL3 plasmid for 12 hr and were then infected with vesicular stomatitis virus (VSV) (MOI = 0.01) or transfected with poly(I:C). The transcription of IFNB1 mRNA was detected using quantitative real-time PCR (qRT-PCR). (B) HEK293T-EV and HEK293T-Flag-ORMDL3 stable cell lines were transfected with or without poly(I:C) and immunoblot analyses of phosphorylated IRF3 (p-IRF3), total IRF3, GAPDH, and ORMDL3 levels were performed. (C) Results of the qRT-PCR assays showing mRNA levels of IFNB1 in A549 cells transfected with EV or ORMDL3 followed by stimulating with poly(I:C) or poly(dG:dC). (D) Results of the qRT-PCR assays showing mRNA levels of Ifnb1 in bone marrow-derived primary macrophages (BMDM) infected with EV or psc-AAV-Ormdl3 virus followed by transfecting with poly(I:C) or poly(dG:dC). (E–F) Results of the luciferase assay showing IFNβ-Luc activity (E) and ISRE-Luc activity (F) in HEK293T cells transfected with EV or ORMDL3 plasmids together with individual EV, RIG-I, cGAS plus STING, or TRIF plasmids for 24 hr. (G) ORMDL3 stable knockdown or overexpression A549 cells, and the control cells were infected with VSV-GFP (MOI = 0.01) for 12 hr. The viral infection was observed using fluorescence microscopy, and the viral amount was detected using qRT-PCR. Scale bars, 200 μm. (H) The control and ORMDL3 stable knockdown A549 cells were infected with herpes simplex virus-1 (HSV-1) (MOI = 0.1) for 24 hr. The viral infection was observed using fluorescence microscopy, and the viral amount was detected using qRT-PCR. Scale bars, 200 μm. Data from three independent experiments are presented as mean ± SD and were analyzed by two-tailed Student’s t test (A, C, D-F, G-H, bottom) ,**p<0.01, ***p<0.001, ****p<0.0001, and ns = no significance. Figure 1—source data 1. Original files for western blots shown in , indicating relevant bands. Figure 1—source data 2. Original files for western blots shown in .

Article Snippet: The mouse antibody GAPDH (1:2000 for immunoblot, 60004-1-Ig) and rabbit antibody beta-Actin (1:2000 for immunoblot, #GB15003) were purchased from Servicebio Biotechnology (Wuhan, China).

Techniques: Transfection, Plasmid Preparation, Infection, Virus, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Stable Transfection, Western Blot, Derivative Assay, Luciferase, Activity Assay, Knockdown, Over Expression, Control, Fluorescence, Microscopy, Two Tailed Test

Journal: eLife

Article Title: ORMDL3 restrains type I interferon signaling and anti-tumor immunity by promoting RIG-I degradation

doi: 10.7554/eLife.101973

Figure Lengend Snippet:

Article Snippet: The mouse antibody GAPDH (1:2000 for immunoblot, 60004-1-Ig) and rabbit antibody beta-Actin (1:2000 for immunoblot, #GB15003) were purchased from Servicebio Biotechnology (Wuhan, China).

Techniques: Sequencing, shRNA, Software, Reporter Assay