Journal: Nature Communications

Article Title: Identification of DAXX as a restriction factor of SARS-CoV-2 through a CRISPR/Cas9 screen

doi: 10.1038/s41467-022-30134-9

Figure Lengend Snippet: a DAXX degradation after infection. 293T-ACE2 cells were infected with SARS-CoV-2 at MOI 0.1. After 24 h, cells were harvested and levels of DAXX, Lamin B, HSP90, Actin, GAPDH, Tubulin, TRIM22, RIG-I and of the viral protein spike were analyzed by Western Blot. A Western Blot representative of 3 independent experiments is shown. b GRL0617 and MG132 treatments restore DAXX expression. 293T-ACE2 cells were infected with SARS-CoV-2 at an MOI of 0.1. When indicated, cells were pretreated 2 h before infection with GRL0617 (at the indicated concentrations), or with MG132 (10 µM), a proteasome inhibitor, or Masitinib (10 µM) a 3CL inhibitor. After 24 h, cells were harvested and levels of DAXX, GAPDH and of the viral protein spike were analyzed by Western Blot. A Western Blot representative of 3 independent experiments is shown. c GRL0617 treatment restores DAXX localization. 293T-ACE2 cells were infected with SARS-CoV-2 at an MOI of 0.1. 24 h post-infection, cells were labelled with Hoescht and with antibodies against dsRNA (detecting viral RNA, in green) and HA (detecting DAXX, in red). When indicated, cells were treated with 50 µM of GRL0617 at the time of infection. Scale bars correspond to 10 µm. Images are representative of 3–6 different fields from 2 independent experiments. d – f : Nsp3 induces DAXX degradation. d 293T-ACE2 cells were transfected with 1 μg of the indicated viral proteins. After 24 h, the levels of DAXX and GAPDH were analyzed by Western Blot. e 293T-ACE2 cells were transfected with the indicated amounts of Nsp3. After 24 h, the levels of DAXX and GAPDH were analyzed by Western Blot. f 293T-ACE2 cells were transfected with 1 μg of Nsp3 or of pcDNA. 6 h post transfection, cells were also, when indicated, treated with 50 µM of GRL0617. Of, 24 h after transfection, the levels of DAXX and GAPDH were analyzed by Western Blot. Western Blots representative from 2 independent experiments are shown. The quantification of band intensity for Fig. 6d–f is shown in Fig. . Source data are provided as a Source Data file.

Article Snippet: The plasmids encoding mCherry-tagged viral proteins were a gift from Bruno Antonny and ordered through Addgene: Nsp3 -mCherry (#165131); Nsp4-mCherry (#165132); Nsp6-mCherry (#165133); Nsp7-mCherry (#165134); Nsp10-mCherry (#165135); Nsp13-mCherry (#165136); Nsp14-mCherry (#165137).

Techniques: Infection, Western Blot, Expressing, Transfection

Journal: Cell Death & Disease

Article Title: MDM2 facilitates adipocyte differentiation through CRTC-mediated activation of STAT3

doi: 10.1038/cddis.2016.188

Figure Lengend Snippet: CRTCs can bind STAT3, STAT5 and MDM2. ( a ) 293T cells were transfected with FLAG-tagged CRTCs and/or STATs. FLAG-immunoprecipitates (IPs) were analyzed for STAT3 and STAT5 by western blotting. ( b ) 293T cells were transfected with MYC-tagged MDM2 and/or STATs. MYC-IPs were analyzed for STAT3 and STAT5 by western blotting. ( c ) 293T cells were transfected with MYC-tagged MDM2 and/or JAK1. MYC-IPs were analyzed for JAK1 by western blotting. ( d ) 293T cells were transfected with FLAG-tagged CRTC2 and/or MDM2. FLAG-IPs were analyzed for MDM2 by western blotting. ( e ) 293T cells were transfected with FLAG-tagged CRTC3 and/or MDM2. FLAG-IPs were analyzed for MDM2 by western blotting

Article Snippet: pBABE-puro was a generous gift from Dr Ormond MacDougald. pBABE-puro C/EBPδ was described previously. pcDNA3 was kindly provided by Dr Jean-Christophe Marine. pCMV FLAG-MYC-CRTC2 was donated by Dr. Jeffrey Meier. pcDNA FLAG CRTC3 was a gift from Dr. Marc Montminy (Addgene, Cambridge, MA, USA; plasmid # 22976). pRK5 JAK1 was used previously. pcDNA3 STAT3 was a generous gift from Dr Jim Darnell (Addgene; plasmid #8706).

Techniques: Transfection, Western Blot

Journal: Genes & Development

Article Title: miRNAs cooperate in apoptosis regulation during C. elegans development

doi: 10.1101/gad.288555.116

Figure Lengend Snippet: The 3′ UTR of egl-1 is a target of miR-35 and miR-58 family miRNAs in vivo. ( A ) The 3′ UTR of egl-1 is illustrated in blue, with a reported C. elegans Argonaut ALG-1-binding site from a recent study indicated above . Sequences are given for predicted miR-35 family-binding sites and miR-58 family - binding sites, and conserved bases within each site are highlighted in gray across four Caenorhabditis species. The mutated sequences corresponding to egl-1 mut mir-35 (red) and egl-1 mut mir-58 (purple) are shown (for complete mutated sequences, see the Materials and Methods). ( B ) A schematic representation of the 3′ UTR reporters constructed for this study. ( C ) Analysis of single-copy 3′ UTR reporter expression during embryogenesis. The genetic background and transgene under investigation are indicated at the left of each image sequence, which shows representative embryos from three developmental stages. For each embryo, a DIC image ( left ) and GFP image ( right ) are shown. The following alleles were used: bcSi25 [P mai-2 gfp::h2b::mai-2 3′ UTR ], bcSi26 [P mai-2 gfp::h2b::egl-1 wt 3 ′ UTR], bcSi27 [P mai-2 gfp::h2b::egl-1 mut mir-35 3′ UTR ], bcSi46 [P mai-2 gfp::h2b::egl-1 mut mir-35 3′ UTR ], and bcSi47 [P mai-2 gfp::h2b::egl-1 mut mir-35 mir-58 3′ UTR ]. Asterisks indicate autofluorescence. Transgenic strains were homozygous for unc-119(ed3) and the cb-unc-119(+) selection marker. Bars, 10 µm.

Article Snippet: Second, this P mai-2 fragment was fused to the 1287-bp coding sequence of gfp::h2b as amplified from the plasmid pCM1.35 (a gift from G. Seydoux; Addgene plasmid no. 17248) using the primers 5′-CTAATTCACTCAATTTTCAGAATGAGTAAAGGAGAAGAACTTTT-3′ and 5′-GCTCGCGTTCTTGTACTGCAAATTACTTGCTGGAAGTGTACTTG-3′.

Techniques: In Vivo, Binding Assay, Construct, Expressing, Sequencing, Transgenic Assay, Selection, Marker