EMBO/SourceData huggingface dataset:
Figure 3C Quantification of peripheral ER structures in WT and ∆ice2 cells harboring the inducible system (SSY1405, 1603) and treated with 800 nM estradiol for the times indicated. Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant.E Quantification of peripheral ER structures in untreated WT, Δice2, Δopi1 and Δice2 Δopi1 cells (SSY1404, 2356, 2595, 2811). Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant.
Figure 4C, D Fluorescence images of cortical sections of WT and Δice2 cells expressing Sec63-mNeon and Rtn1-mCherry (SSY1405, 1603) that were untreated (C) or treated with 8 mM DTT for 1 h (D).
Figure 5B Growth assays of untreated WT, Δice2, Δnem1, Δnem1 Δice2, Δspo7, Δspo7 Δice2, Δpah1 and Δpah1 Δice2 cells (SSY1404, 2356, 2482, 2484, 2481, 2483, 2807, 2808). Numbers represent areas under the curves and serve as growth indices. Mean + s.e.m., n = 3 biological replicates. Data for WT and ∆ice2 cells are the same in the left and middle panels.C, D Lipidomic analysis of WT, Δice2, Δnem1, Δice2 Δnem1, Δspo7 and Δice2 Δspo7 cells (SSY1404, 2356, 2482, 2484, 2481, 2483). Mean + s.e.m., n = 4 biological replicates. Asterisks indicate statistical significance compared with WT cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01. Data for WT and Δice2 cells are the same as in both panels.E Sec63-mNeon images of untreated WT, Δnem1, Δnem1Δice2, Δspo7 and Δspo7 Δice2 cells (SSY1404, 2482, 2484, 2481, 2483).
Figure 6D Western blot of HA from Pah1 dephosphorylation reactions that contained phosphorylated Pah1-HA from ∆nem1 mutants (SSY3065) and microsomes from cells of the indicated genotypes (SSY3053, 3074, 3075, 3095). Phosphorylated Pah1 and dephosphorylated Pah1 resulting from treatment with recombinant alkaline phosphatase (PPase) are shown for reference.E Western blot of HA, Sec61 and Pgk1 from cell lysates and microsomes prepared from WT and ∆ice2 cells expressing Nem1-HA (SSY3140, 3141). Nem1 is undetectable in cell lysates due to its low abundance.
Figure 7Western blots of FLAG, HA and Dpm1 from cell lysates or anti-FLAG immunoprecipitates of WT or ∆nem1 cells containing Spo7-FLAG, Nem1-FLAG or Ice2-HA as indicated (SSY122, 2421, 3183, 3184, 3195, 3196, 3197). The asterisk marks the position of the light chain from the antibody used for immunoprecipitation. The diamond marks a protein that is non-specifically precipitated by the anti-FLAG antibody. IP, immunoprecipitate.E Images of cells with endogenously tagged Ice2-mScarlet and Spo7-mNeon or Nem1-mNeon (SSY3244, 3245). Arrows indicate foci containg both Ice2 and either Spo7 or Nem1. F Quantification of Spo7 and Nem1 foci in WT and ∆ice2 cells (SSY2916, 3238, 2917, 3239). Mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with the respective WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01.
Figure 8A Lipidomic analysis of WT and ∆ice2 cells in which PAH1 was replaced with PAH1-HA or pah1(7A)-HA as indicated (SSY2841, SSY2842, SSY2970). Mean + s.e.m., n = 4 biological replicates. Asterisks indicate statistical significance compared with WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01.
Figure 9A Sec63-mNeon images of mid and cortical sections of untreated WT and ∆opi1 cells, overexpressing ICE2 where indicated (SSY1404, 2588, 2595, 2596).D Growth assays on solid media of WT, ∆hac1, Δice2, and Δhac1 Δice2 cells (SSY1404, 2356, 2805, 2806) in the absence or presence of 0.2 µg/ml tunicamycin. For each series, cells were diluted fivefold from one step to the next.
Figure 2(A-F) Immunocytochemistry of DRG neurons and the corresponding quantifications of the indicated genotypes for surface TrkA (A), surface TRPV1 (B), Rab3A (C), synaptotagmin (D), DENN/MADD (E) and α-tubulin (F). TrkA and TRPV1 were visualized without permeabilization. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; Welch's t test from at least three independent cultures. Error bars show SEM. Scale bars, 2 µm.
Figure 3Time-lapse fluorescence images of DRG neurons expressing KIF1A-EGFP and KIF1A(E239K)-EGFP (B) Arrowheads in (B) show the anterogradely moving particles. Scale bar in (B), 2 μm. See Movies EV1 and 2.(F) Movements of KIF1A-EGFP and KIF1A(E239K)-EGFP expressed in KIF1A knockdown DRG and the scrambled control. White scale bar, 1 μm. (G) Anterograde velocities of the indicated genotypes calculated from the kymograph slopes. Data are shown as mean and SD. KD+KIF1A-EGFP, n = 29; KD+KIF1A(E239K)-EGFP, n = 32; scrambled+KIF1A-EGFP, n = 44; scrambled+KIF1A(E239K)-EGFP, n = 42 from three independent cultures. ns, p = 0.3329; **, p < 0.01; ****, p < 0.0001; Unpaired t test.
Figure 4(A) Representative kymographs of tetramethylrhodamine-labelled microtubule movement driven by the WT and the E239K mutant. The arrowheads represent the end positions of the WT and E239K.(D) ATPase activity of WT (green) and E239K mutant (magenta) activated by microtubules. Data are represented as the mean ± SD. The data from at least three independent experiments were analysed. ****, p < 0.0001, two-way ANOVA.(F) Rate constants (kobs) determined for the reaction of mant-ATP with nucleotide-free KIF1A(WT) and KIF1A(E239K) were plotted against concentration of mant-ATP. Data are represented as the mean ± SD. The data from three independent measurements were analysed.
Figure 6(A) The positions of the β6-L10-β7 and neck-coil peptides are shown in brown and green. The positions of the neck-linker peripheral peptides (α1-β3-P loop, α4-L12 and L12-α5) are shown in light blue. (B-D) Quantitation of site-specific intramolecular interactions. Representative MS/MS spectra of the indicated peptides, including the mutated amino acid (β6-L10-β7) (B and C) and neck-coil (D). In the inserted graphs, the intensities in the ADP-BeFx and ADP-AlFx states were measured with respect to the nucleotide-free state as 1.0. Grey bar graphs, WT; black bar graphs, E239K; (-), nucleotide-free.
Figure 1.(e) Binding of monoclonal antibodies to 2019-nCoV RBD determined by ELISA.(f) Binding profiles of 2019-nCoV RBD to ACE2 and antibodies(g) competition of CR3022 and ACE2 with 2019-nCoV RBD measured by BLI in OctetRED96. Binding kinetics was evaluated using a 1:1 Langmuir binding model by ForteBio Data Analysis 7.0 software.
Figure 4C Comparison of meiotic chromosome segregation in cells lacking H3K9me3 depending on Swi6's affinity towards H3K9me2. Exemplary IF microscopy images of the indicated fission yeast strains are shown. Homothallic strains were incubated on SPAS plates. DNA and tubulin were stained with DAPI and anti-TAT-1, respectively. Missegregating chromosome is indicated with a yellow arrow; D Quantification of meiotic chromosome segregation phenotypes. The data for clr4+ and clr4F449Y is the same as in Fig 3B. P values were calculated using Fisher's exact test. Number of total cells counted is indicated in each bar.E Comparison of co-segregation of centromere I (CenI) during MI in cells lacking H3K9me3 depending on Swi6's affinity towards H3K9me2. Exemplary live cell microscopy images of the indicated fission yeast strains during MI and MII are shown. Heterothallic strains were crossed and dyads as well as tetrads were subjected to the analysis. Heterozygous CenI-GFP was used to follow the segregation pattern during meiosis. The GFP-signal is additionally displayed in greyscale for better visibility F Quantification of CenI-GFP segregation during meiosis. The data for clr4+ and clr4F449Y is the same as in Fig 3D. P values were calculated using Fisher's exact test. Number of total cells counted is indicated in each bar.G Tetrad dissection to asses spore viability in clr4F449Y swi6Chp1-like-CD cells. Exemplary image (left) and quantification of the spore viability (right) are shown. Meiosis was induced in homothallic cells and the resulting spores were dissected. The data for clr4+ and clr4F449Y is the same as in Fig 3F. P values were calculated using Fisher's exact test. Number of dissected tetrads is indicated in each bar.
Figure 6A Western blot of whole-cell lysates (WCL) using a PhosTag gel for anti-FLAG (top) and a Bis-Tris gel for anti-Act1 detection (bottom), respectively. WCL were prepared from mitotic and meiotic 3xFLAG-clr4+/3xFLAG-clr4+ cultures at the indicated time points.C Western blot of FLAG IPs using a PhosTag gel to asses phosphorylation levels of Clr4 in the indicated homothallic strains.D Western blot of FLAG IPs in 3xFLAG-clr4+ cdc2-as cells, treated with 2 µM 1-NM-PP1 for the indicated amount of time. The blot was first probed with the phospho-specific anti-pS458 antibody, stripped, and reprobed with an anti-FLAG antibody.
Figure 2RNA extracted from Ruxolitinib-treated or mock-treated, and SARS-CoV-, SARS-CoV-2- PBMCs was analyzed for (A) IFIT1, (B) IFNA1 and IFNB1 mRNA expression by Q-RT-PCR at indicated time points. Suspension and adherent cell fractions were analyzed separately, except at the four hours time point. Values were normalized to cellular RNASEP expression and are shown as fold change over mock-inoculated conditions. The dotted line indicates the expression level of mock-inoculated cell cultures and is set to 1. Data information: Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M (A-B Statistical significance between mock- and Ruxolitinib-treated samples was tested using paired Student's t-testing and comparing SARS-CoV to SARS-CoV-2-treated samples from the same donor and time points. P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant (not shown).(C) Supernatants from Ruxolitinib- or mock-treated and SARS-CoV-, SARS-CoV-2 or mock-inoculated PBMCs were collected 48 hours post exposure and cytokine expression of IFN-α2, IP-10/CXCL10, MCP-1/CCL2 and MCP-3/CCL7 were quantified using a Luminex-based immunoassay. PHA- or LPS-treated PBMCs were used as a positive control. Bars represent the results of a pool of four individual samples per condition. Data information: Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols, bars represent the mean, Statistical significance between mock- and Ruxolitinib-treated samples was tested using paired Student's t-testing and comparing SARS-CoV to SARS-CoV-2-treated samples from the same donor P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant (not shown). n.d. = not detectable.(D) Supernatants collected from SARS-CoV-, SARS-CoV-2- or mock-exposed PBMCs at indicated time points were analyzed for the release of bioactive IFN using the HL116 reporter cell assay. Data information: Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant (not shown).
Figure 4(A) Pseudotime cell trajectory analysis and GSEA analysis using genes differentially regulated between mock-, SARS-CoV- and SARS-CoV-2-challenged conditions for indicated cell types. Data information: Data shown in this figure are based on the analysis of two donors.(B) Representative UMAPs showing IFIT1 and ISG15 mRNA expression in the indicated conditions. Data information: Data shown in this figure are based on the analysis of two donors.(C) Volcano plot of all DEGs in SARS-CoV-2-exposed cells compared to mock-exposed cells in the indicated cell types. Known ISGs were colored in green based on their presence in the interferome database Data information: Data shown in this figure are based on the analysis of two donors.(D) Cell trajectory maps of indicated cell types with cells colored by expression of the genes in an IFN-module gene set. Data information: Data shown in this figure are based on the analysis of two donors.(E) Dot plot depicting expression of selected ISGs and cytokines. Expression levels are color-coded, the percentage of cells expressing the respective gene is coded by symbol size. Data information: Data shown in this figure are based on the analysis of two donors.
Figure 5(C) Bar graph showing the absolute number (bars) and relative percentage of cells that were identified as virus RNA-Positive in SARS-CoV- and SARS-CoV-2-inoculated cultures, respectively. Data information: Data shown in this figure are based on the analysis of two donors.(D) Plot of Log10 average expression of genes showing a Log2(fold change) >0.2 in viral RNA-Positive versus viral RNA-Negative monocytes from the SARS-CoV- (left panel) and SARS-CoV-2- (right panel) inoculated PBMCs with genes showing the highest expression fold change between both conditions. Data information: Data shown in this figure are based on the analysis of two donors.(E) IFN Module Score of viral-RNA-negative (grey; 715 and 958 cells in SARS-CoV and SARS-CoV-2 exposed cultures, respectively), SARS-CoV-RNA-Positive (blue; 56 cells) and SARS-CoV-2-RNA-Positive (magenta; 173 cells) monocytes. Statistical significance was tested using a Wilcoxon rank sum test with continuity correction. P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant Data information: Data shown in this figure are based on the analysis of two donors.
Figure 1Results of the ELISAs used to determine IFN-β (A) secretion in PMA-differentiated THP-1 cells transfected with siCtrl, siRNF34-1, siRNF34-2 or siRNF34-3 oligos and infected with VSV for the indicated times. ELISA data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.Results of the ELISAs used to determine IL6 (B) secretion in PMA-differentiated THP-1 cells transfected with siCtrl, siRNF34-1, siRNF34-2 or siRNF34-3 oligos and infected with VSV for the indicated times. ELISA data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.Luciferase activity driven by the IFN-β (C) promoter in HEK293T cells transfected with Flag-RNF34 or its E3 ligase-dead mutant H342A followed by VSV infection for the indicated times. Luciferase assays were performed 24 hrs after transfection, and luciferase activity was reported as the fold induction. The luciferase reporter data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.Luciferase activity driven by the NF-κB (D) promoter in HEK293T cells transfected with Flag-RNF34 or its E3 ligase-dead mutant H342A followed by VSV infection for the indicated times. Luciferase assays were performed 24 hrs after transfection, and luciferase activity was reported as the fold induction. The luciferase reporter data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.(E) Plaque assay of VSV loads in supernatants from cells transfected with shCtrl, shRNF34-1, shRNF34-2 or shRNF34-3 and subsequently infected with VSV. Cell-based studies were performed independently at least three times with comparable results.(F) Microscopy images of shCtrl-, shRNF34-1-, shRNF34-2- or shRNF34-3 cells infected with NDV-GFP (MOI = 1.0) for the indicated times. Gray represents cells; green represents NDV. Scale bar, 300 μm.(G) Immunoblot showing the levels of the phosphorylated (p) and total IRF3, TBK1, and VSV-G proteins in PMA-differentiated THP-1 cells transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times.RT-PCR analysis of the expression of the IFN-β (H) mRNAs in PBMCs transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times. The results were normalized to the values obtained prior to VSV infection.(I) Results of the ELISA used to determine IFN-β secretion in PBMCs transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times. ELISA data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.RT-PCR analysis of the expression of the IL8, ISG54 and ISG56 (J) mRNAs in PBMCs transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times. The results were normalized to the values obtained prior to VSV infection.
Figure 2(A) Luciferase activity driven by the ISRE promoter in HEK293T cells transfected with Myc-RNF34 and Flag-V, Flag-N-RIG-I, Flag-MAVS, Flag-STING or Flag-TBK1. Luciferase assays were performed 24 hrs after transfection. The luciferase reporter data are presented as means ± SEM. Two-tailed Student's t-tests were used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.(B) Y2H analysis in the AH109 yeast strain co-transformed with the indicated plasmids. A positive RNF34-MAVS interaction resulted in colony formation on synthetic medium lacking tryptophan, leucine, adenine, and histidine containing X-gal. pGBKT7-TP53+pGADT7-T and pGBKT7-lam+pGADT7-T were used as positive and negative controls, respectively. AH109 co-transfected with pGBKT7-RNF34+pACT-2 was used to exclude the self-activation of RNF34.(C) Immunoprecipitation analysis of HEK293T cells transfected with Myc-RNF34 and Flag-MAVS or Flag-V. Anti-Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody.(D) GST-tagged RNF34 was subjected to a pull-down assay with HEK293T cell lysates. Immunoblot with an anti-MAVS antibody are shown in the top panel. Loading of the GST proteins assessed using Coomassie blue staining is shown in the bottom panel. GST was used as a negative control.(E) Anti-Flag or IgG immunoprecipitates prepared from cells transfected with Flag-MAVS or Flag-vector-expressing plasmids were subjected to SDS-PAGE and blotted onto a nitrocellulose membrane. The nitrocellulose membrane was incubated with soluble GST-RNF34 (upper panel) or GST (middle panel) for 2 hrs and then analyzed with anti-Flag antibody.(F) Representative confocal images of immunofluorescence staining for Flag-RNF34 colocalization with endogenous MAVS in THP-1 cells infected with VSV for 12 hrs. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.(G) Immunoblot showing the levels of the RNF34 protein in THP-1 cells infected with VSV (MOI = 1.0) for the indicated times. α-Tubulin was used as a loading control.(H) In situ PLA assay of the RNF34-MAVS complex in HEK293T cells infected with VSV (MOI =1.0) for the indicated times using an anti-RNF34 or anti-MAVS antibody. RNF34-MAVS complex, red; nuclei, blue. Scale bar, 5 μm. Cell-based studies were performed independently at least three times with comparable results.(I) One hundred cells were counted, and the quantification of PLA signals per cell is shown. Cell-based studies were performed independently at least three times with comparable results.(J) Immunoprecipitation analysis of HEK293T cells transfected with Myc-RNF34 and Flag-MAVS or Flag-mMAVS. Anti-Flag immunoprecipitates were analyzed using immunoblotting with anti-Myc or anti-Flag antibody.
Figure 3(A) Immunoprecipitation analysis in HEK293T cells expressing Flag-MAVS and Myc-Ub together with HA-RNF34 or HA-H342A. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. The levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.(B) Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS and HA-RNF34 together with Myc-Ub (K6, K11, K27, K29, K33, K48, or K63 only) as indicated. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.(C) Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS and HA-RNF34 together with Myc-Ub (wild-type, WT; Lys-to-Arg mutants of all Ub lysines, KO; K27/29R). Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.(D) Immunoprecipitation analysis of K27 ubiquitination of MAVS in HEK293T cells infected with VSV for the indicated times. Anti-MAVS immunoprecipitates were analyzed using immunoblotting with an anti-MAVS or anti-Ub-K27 antibody. The WCL was immunoblotted with an anti-MAVS or anti-Ub-K27 antibody. Cell-based studies were performed independently at least three times with comparable results.(E) Immunoprecipitation analysis of siCtrl- or siRNF34-1-transfected cells infected with VSV for the indicated times. Anti-MAVS immunoprecipitates were analyzed using immunoblotting with an anti-MAVS or anti-Ub-K27 antibody. The WCL was immunoblotted with an anti-MAVS or anti-Ub-K27 antibody. Cell-based studies were performed independently at least three times with comparable results.
Figure 4Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or Lys to Arg mutants together with Myc-Ub and HA-RNF34. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or 4KR mutant together with Myc-Ub and HA-RNF34. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Ub-K27 antibody. Levels of the transfected proteins were analyzed by performing immunoblotting with an anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.(C) Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or K311R mutant together with HA-RNF34 and/or Myc-TRIM31 following a 3MA (0.2 mM) treatment. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Flag, anti-Ub-K63 or anti-Ub-K27 antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA or anti-Flag antibody. ell-based studies were performed independently at least three times with comparable results.(D) Co-immunoprecipitation analysis of the ubiquitination of endogenous MAVS in THP-1 cells transfected with siCtrl, siRNF34, or siTRIM31 oligos and infected with VSV for the indicated times. Cell-based studies were performed independently at least three times with comparable results.(E) Co-immunoprecipitation analysis of the endogenous interaction of MAVS-TRIM31 and MAVS-RNF34 in PBMCs infected with VSV for the indicated times. Cell-based studies were performed independently at least three times with comparable results.