Journal: bioRxiv

Article Title: Clu1/Clu form mitochondria-associated granules upon metabolic transitions and regulate mitochondrial protein translation via ribosome interactions

doi: 10.1101/2024.08.17.608283

Figure Lengend Snippet: (A) Maximum intensity projection of confocal images of Clu1-GFP cells grown in glucose-containing media until mid-log phase, then subjected to different stresses: heat shock (HS; 46 °C, 30 min), without any carbon source (No glucose, 10 min), exposure to hyperosmotic stress (1 M KCl, 30 min), hypotonic stress (H 2 O, 10 min), oxidative stress (3 mM H2O2, 15 min), and mitochondrial stress sodium azide (0.5%, 15 min) and CCCP (30 μM, 15 min), or untreated (UT). The percentage of cells containing Clu1 foci is indicated in each image. (B) Confocal imaging (maximum intensity projection) of mid-log Clu1-GFP cells shifted from glucose to galactose-containing-media and incubated for 6 h, then subjected to stresses as in A, plus oligomycin (10 µM) and antimycin A (40 µM) (OA; 30 min). The percentage of cells containing Clu1 foci is indicated in each image. (C) Maximum intensity projection of Clu1-GFP cells grown in galactose-containing media for 6 h, treated with CCCP for 15 min (Gal+CCCP), spiked with glucose and imaged 20 min later (+ Glucose). (D) Confocal microscopy of Clu1-GFP strain expressing Ded1-mCherry, a marker of stress granules (SGs) in the log and PD phases, and after heat shock or sodium azide treatment, as described above. (E) Confocal microscopy images of Clu1-GFP strain expressing Dcp1-DsRed, a marker of P-bodies (PBs) in the log, late log and PD phases, and after sodium azide treatment. Scale bars = 5 µm; D, E inset = 2 µm.

Article Snippet: The mCherry cassette was amplified from pBS35 (Addgene plasmid; 83797), TEV-GFP from pFA6a-GFP(S65T) (Addgene plasmid; 41598) using a 5’ primer containing the TEV sequence, and the BirA* cassette from pYM28-BirA* (Addgene plasmid; 160290). pRS313-pTDH3-Su9-TagBFP was generated using a yeast recombination-based cloning approach, replacing the URA3 marker from pRS316-pTDH3-Su9-TagBFP (Addgene plasmid # 62383) by HIS3.

Techniques: Imaging, Incubation, Confocal Microscopy, Expressing, Marker

Journal: bioRxiv

Article Title: Clu1/Clu form mitochondria-associated granules upon metabolic transitions and regulate mitochondrial protein translation via ribosome interactions

doi: 10.1101/2024.08.17.608283

Figure Lengend Snippet: (A, B) Confocal microscopy of yeast Clu1-GFP cells co-expressing the mitochondrial marker mito-mCherry in the log (A) and PD phases (B). (C) 3D rendering of cells in PD phase. (D) Inset of panel B with an intensity profile plot showing the fluorescence distribution of Clu1-GFP and mito-mCherry. (E) Confocal image of Clu1-GFP cells expressing mito-mCherry after being shifted to galactose for 6 h and treated with CCCP. (F) Confocal imaging of Clu1-GFP cells co-expressing mitochondrial fission marker ffRFP-Mdv1 grown into PD phase. (G) Confocal imaging of a GFP-Clu egg chamber from 3-day-old flies (6 h of feeding after overnight fasting) immunostained for ATP5A to visualise mitochondria. (H) Correlative light and electron microscopy images of a GFP-Clu egg chamber nurse cell showing the juxtaposition of a GFP-Clu granule and mitochondria. The serial transmission electron microscopy (TEM) images, taken at 5000 × magnification, correspond to the confocal image shown. Scale bars: A, B, E, F = 5 µm; G = 20 µm, inset = 5 µm; H = 1 µm.

Article Snippet: The mCherry cassette was amplified from pBS35 (Addgene plasmid; 83797), TEV-GFP from pFA6a-GFP(S65T) (Addgene plasmid; 41598) using a 5’ primer containing the TEV sequence, and the BirA* cassette from pYM28-BirA* (Addgene plasmid; 160290). pRS313-pTDH3-Su9-TagBFP was generated using a yeast recombination-based cloning approach, replacing the URA3 marker from pRS316-pTDH3-Su9-TagBFP (Addgene plasmid # 62383) by HIS3.

Techniques: Confocal Microscopy, Expressing, Marker, Fluorescence, Imaging, Electron Microscopy, Transmission Assay

Journal: Viruses

Article Title: Schlafen14 Impairs HIV-1 Expression in a Codon Usage-Dependent Manner

doi: 10.3390/v16040502

Figure Lengend Snippet: Effect of SLFN14 on viral replication. ( A ) Role of the RNA Polymerase III-RIG-I-IFN signaling pathway in the SLFN14 activity. HEK293T cells co-transfected with HIVluc expression plasmid and an empty plasmid (control cells) or a plasmid expressing human SLFN14 were treated or not with inhibitors indicated. Luciferase activity measured in these cells was expressed as % of control cells. Data correspond to a triplicate experiment and are representative of three independent experiments. Although not indicated, statistically significant differences ( p ≤ 0.0001) were found between the control and each of the other groups, as calculated with two-way ANOVA and Dunnett post-hoc tests. ( B ) Effect of SLFN14 on HIV-1 replication. ( I ) CD4 and CXCR4 expression. HEK293T cells were co-transfected with plasmids expressing CD4 and a bicistronic plasmid encoding CXCR4 and mCherry, and either, an empty plasmid (control cells) or a human SLFN14 expression plasmid. CD4 was detected by immunostaining and MFI values of CD4 and mCherry (CXCR4) were quantified by flow cytometry. ( II ) These cells were infected with HIV-1 wild-type and viral replication was followed by measuring HIV-1 p24 in the cell supernatant. Data pertain to a triplicate experiment and they are representative of two independent experiments. Statistically significant differences were calculated with one-way ANOVA and Bonferroni post-hoc tests **** p ≤ 0.0001. Figure is created by Valenzuela et al.

Article Snippet: The CXCR4 expression plasmid contains an independent mCherry expression cassette (bicistronic plasmid). pCI Luc contains firefly luciferase cDNA cloned MluI/Xba I in pCI (Promega, Madison, WI, USA). pNLENG1-ES-IRES (a gift of D.N.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Control, Luciferase, Immunostaining, Flow Cytometry, Infection