EMBO/SourceData huggingface dataset:
Figure 3A Relative activity of NF-kB luciferase reporter was measured in A549 cells transfected with plasmids encoding PB1-F2. The mRNA levels of pro-inflammatory cytokines were determined by semi-quantitative RT-PCR. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).Cells were transfected with PB1-F2-expressing plasmids. Relative IFNβ mRNA levels were determined by semi-quantitative RT-PCR (top) and qPCR (bottom). All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).C U937 cells were co-transfected with the IFNβ luciferase reporter plasmid and the PB1-F2 or NS1 expressing plasmid and treated with polyI:C for 12 h before harvesting. IFNβ promoter activity was determined by luciferase reporter assay. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).Cells were infected for 24 h with each virus at 1 MOI. The mRNA level of IFNβ was determined by semi-quantitative RT-PCR, and PB1-F2 protein expression was assessed by Western blotting. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).E Cells were transfected with each plasmid encoding PB1-F2. 18 h after transfection, cells were treated with MG132 for 6 h and the level of IFNβ mRNA was determined by semi-quantitative RT-PCR (left) and qPCR (right). PB1-F2 protein expression was assessed by Western blotting. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).F A549 cells were transfected with PB1-F2 clones mutated at positions 68 and 69; 24 h after transfection, the IFNβ mRNA level and PB1-F2 protein expression were determined by semi-quantitative RT-PCR and Western blotting, respectively (left). The relative expression level of IFNβ mRNA was also analyzed by qPCR (right). All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).
Figure 4A Mice (n = 5 or 6 per group) were infected with IAV with indicated pfu. Survival was monitored daily for 2 weeks.B Mice (n = 6-9 per group) were infected with each IAV as indicated. Body weight was recorded daily after infection for 2 weeks.C Mice (n = 5 or 6 per group) were infected with viruses (1000 pfu) carrying PR8 PB1-F2 with the I68T, L69P, or I68T+L69P mutation. Body weight changes and survival rate were monitored daily. Detailed analyses on the virulence necessitated the monitoring of body weight below the standard level (80%) of mortality.Three cohorts of mice (5 or 6 per group) were infected with 500 pfu of one of the three IAVs. Viral protein expression in mouse lung was determined by Western blotting 2 days after infection.E Mice (6 per group) were infected with 500 pfu of one of the three IAVs. Viral titration by plaque assay was performed at 2 days post-infection using lung homogenates.F Mice (5-9 per group) were infected with 500 pfu of each virus. At 2 days post-infection, IFNβ mRNA levels were determined by qPCR.G Mice (5-9 per group) were infected with 1 × 105 pfu of each virus. At 2 days post-infection, IFNβ protein levels in lung homogenates were measured by ELISA.
Figure 5A SDS-PAGE analysis of proteins immunoprecipitated from A549 cell lysates using HA-antibody. Bands were stained by Coomassie Blue. Results were confirmed in three independent experiments, and representative data are shown.E Analysis of interaction between DDX3 and 1918 PB1-F2. Cells were transfected with indicated plasmids; 18 h later, cells were treated with MG132 for 6 h. Cell lysates were immunoprecipitated with anti-HA antibody. Proteins were detected by immunoblotting with the indicated antibodies (left panel). IAV-infected cell lysates were immunoprecipitated with anti-PB1-F2 antibody and immunoblotted with the indicated antibodies (middle panel). Purified recombinant 1918 PB1-F2 protein was incubated with recombinant LysRS or LysRS-DDX3, and the interaction was analyzed by immunoblotting (right panel). Results were confirmed in three independent experiments, and representative data are shown.F Analysis of interaction between DDX3 and PB1-F2 mutants. A549 cells were transfected with the indicated clones; 18 h after transfection, cells were treated with MG132 for 6 h and analyzed by immunoprecipitation. Results were confirmed in three independent experiments, and representative data are shown.G A549 cells were treated with IFNβ for the indicated time periods and subjected to Western blotting (top). A549 cells were infected with indicated IAV. At 24 h post-infection, cell lysates were analyzed by Western blotting (bottom). Results were confirmed in three independent experiments, and representative data are shown.H Representative fluorescence images showing expression level of GFP-DDX3 (green). A549 cells were co-transfected with plasmids encoding the GEP-DDX3 and Flag-tagged PR8 or 1918 PB1-F2. At 18 h after transfection, cells were treated with MG132 for 6 h and subjected to fluorescence analysis. Results were confirmed in three independent experiments, and representative data are shown.I Mice were infected with 1×105 pfu of each virus, and lung homogenates were examined for endogenous Ddx3 expression. Results were confirmed in three independent experiments, and representative data are shown.J A549 cells were transfected with the indicated plasmids or treated with poly I:C. Cytoplasmic and nuclear fractions were separated and subjected to Western blotting. Results were confirmed in three independent experiments, and representative data are shown.K A549 cells were transfected with the indicated plasmids, and IFNβ mRNA levels were determined by semi-quantitative RT-PCR (top) and qPCR (middle). IFNβ promoter activity was determined by luciferase reporter assay (bottom). Results were confirmed in three independent experiments, and representative data are shown.
Figure 6Groups of five mice were intranasally infected with IAV (1918), and recombinant DDX3 protein was administered intranasally. Survival was monitored daily for 2 weeks.Groups of five mice were intranasally infected with IAV (1918), and recombinant DDX3 protein was administered intranasally. Two days post-infection, virus titers in lung homogenates were determined by plaque assay. A p value of <0.05 was considered significant (** p < 0.01).Groups of five mice were intranasally infected with IAV (1918), and recombinant DDX3 protein was administered intranasally. Two days post-infection, the expression level of IFNβ in the lungs was analyzed by semi-quantitative RT-PCR (left panel) and qPCR (right panel). A p value of <0.05 was considered significant (** p < 0.01).
Figure 1(B) Left, orthogonal view of a 20μm tumor section labelled with antibodies against EpCAM (in red) and DNA in blue. Right, dot plot bar representing cell heights in cells from tumor sections, n=60 cells from 1 tumor.(C) On the left, images of low magnification views of healthy tissues labelled with antibodies against pericentrin (PCNT) and CDK5RAP2, shown in red and green, respectively. DNA is in blue. The white dashed squares represent the regions shown in higher magnifications on the right. One centrosome was considered as such when PCNT and CDK5RAP2 signals co-localized (Inset 1). Lack of co-localization (Insets 2 and 3) was noticed and discarded during quantification.(D) Super-resolution microscopy of healthy tissues labelled for the centriole marker CEP135 (in green) and PCNT (in red).(E-F) Representative images of two different fields of the same tumor (E) and of a tumor section to highlight different centrososome phenotypes, labelled as described in (C). The pink dashed line highlights an area of cells without centrosomes. The white dashed squares represent the regions shown in higher magnifications in insets in (G and I) and the pink dashed linesurrounds nuclei without centrosomes. (G) Insets from (F). (H) Examples of tumor sections showing small (left) and large (right) regions with nuclei without centrosomes.(I) Insets from (F) showing different types of centrosome amplification events.(J) Super-resolution microscopy of tumor tissues labelled as in (C).(K) Maximum Z-projection of a tumor section labelled with EpCAM antibodies (Red) and DNA in blue. The dashed line shows the absence of EpCAM staining, arrows show a disorganized region.
Figure 2(A) Images of EOC tumors labelled with antibodies against PCNT (red) and CDKRAP2 (green). Nuclei are shown in blue. The colored arrows reflect different centrosome number. The list of centrosome behaviors annotated it is not exhaustive to allow comprehension of the events. #TT72, shows a cyst-like arrangement of EOC nuclei where it is impossible to correspond centrosomes to individual nuclei. The white bars with arrows of different colors highlight possible interpretations of the same condition. In #TT79, regions without centrosomes can be identified. In the inset, a single centrosome localized in the lumen can be identified and it is not possible to know to which nucleus (white dashed lines) it corresponds to. #TT88 presents only very small clusters of centrosomes, few nuclei without centrosomes and many conditions with 2 centrosomes. In #TT61, regions without centrosomes can be distinguished, in addition to nuclei associated with single centrosomes. In #TT46, clusters containing large (inset 1) or small clusters (inset 3) can be identified. In cyst-like arrangements several centrosomes can be identified (inset 2), suggesting that all cells have at least one centrosome or, in contrast just one centrosome (inset 4), illustrating lack of centrosomes.(B-C) Plots showing the frequency of centrosome amplification events (B) and the frequency of nuclei without centrosomes (C) in tumor tissues. Note that in B and C, the order of the tumors is conserved between the two plots to allow for comparison. For each tumor, 10 random fields were chosen and analysed, with an average of 5248 nuclei counted per tumor.(D) Tumor sections labelled with CEP135 (green) and PCNT (red) on the left and CEP135 (green) and CEP192 (red) on the right. The white squares represent the regions shown in higher magnifications on the right.
Figure 3(B) Stills of a time-lapse movie of Ctrl spheroids. Time is shown in hours (hrs). (C) z- view of Ctrl cells as shown in (B). Note the red-colored cancer cells at the beginning of the movie on top of the mesothelial layer (green) while at later time points, they have cleared through the mesothelial cells.(D) Graph bars of the normalized clearance in A.U. of iOVCAR8 (left) and iSKOV3 (right) spheroids after the indicated treatments. The two cell lines show different clearance capacity. For each cell line, three independent experiments were performed. For iOVCAR8, we analyzed 47 spheroids in DMSO and 56 spheroids in centrinone. For iSKOV3 we analyzed 56 spheroids in DMSO and 48 spheroids in centrinone. Dots represent the mean and the shadow±SEM. Statistical significance was assessed with an ANCOVA test. Dots represent the mean and the shadow±SEM. Statistical significance was assessed with an ANOVA test.(F) Representative images of DMSO (left) and centrinone (right) spheroids. Mean±SD. Red nuclei represent false colored invading nuclei. (G) Dot plot showing the quantification of the number of nuclei detected on the bottom side of the BM. Three or four positions from three BM inserts were analyzed in each condition. Mean±SD. Statistical significance was assessed with the Mann-Whitney test. For all the experiments, the centrosome number was verified in parallel to confirm the decreased centrosome conditions compared to DMSO.
Figure 1Vero and Caco-2 cells were infected with SARS-CoV-2 at MOIs of 0.2 for 8 h. Infected cells were then permeabilized and immunostained for the intracellular SARS-CoV-2 nucleoprotein (NP, red). Nuclei were stained with Hoechst (blue) before imaging by fluorescence confocal microscopy. Scale bars: 100 µm.Infection of Vero and Caco-2 cells was monitored over 24 h using the same flow cytometry-based assay used for the experiment shown in panel D. Infection is given as the total fluorescence associated with the NP protein-positive cells. MFI, mean fluorescence intensity. n = 3.
Figure 2Cells were pretreated with the indicated concentrations of aprotinin (A) and SB412515 (B), which are inhibitors of TMPRSS2 and cathepsin L, respectively. Infection of Calu-3, Caco-2, A549*, and Vero cells with SARS-CoV-2 at MOIs of 0.3, 0.4, 0.2, and 0.3, respectively, was achieved in the continuous presence of the drug. Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where inhibitors had been omitted. n = 2-4 biological replicates.SARS-CoV-2 particles were bound to A549* and Vero cells (MOIs 0.2 and 0.3, respectively) (C) or Calu-3 and Caco-2 cells (0.6 and 0.5, respectively) (D) on ice for 90 min and subsequently warmed rapidly to 37°C to allow infectious penetration. SB412515 (10 µM, C) or aprotinin (30 µM, D) was added at different times post warming to block further proteolytic activation. Infection was analyzed by flow cytometry, and data were normalized to samples where protease inhibitors had been omitted. n = 2.
Figure 3Cells were pretreated with endosomal-pH interfering drugs at the indicated concentrations and subsequently infected with SARS-CoV-2 as described in Fig. 2A and 2B in the continuous presence of NH4Cl (A), chloroquine (B), bafilomycin A1 (C), or concanamycin B (D). The proportion of infected cells was quantified by flow cytometry as described in Fig. 1D, and data were normalized to control samples that were not treated with inhibitors. n = 2-6 biological replicates.
Figure 4A549* cells expressing or lacking TMPRSS2 were pretreated with the indicated concentrations of camostat mesylate (B), SB412515 (C), and bafilomycin A1 (D). Infection with SARS-CoV-2 (MOI ~0.2) was achieved in the continuous presence of the drug. Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where inhibitors had been omitted. n = 5-6 biological replicates.
Figure 5The timing of the MG-132-sensitive step during SARS-CoV-2 infectious entry into Calu-3 and Caco-2 cells was assayed as detailed in D but using 60 µM MG-132 for Caco-2 cells and MOIs of 0.5 and 0.6, respectively. n = 2.A549* cells expressing or lacking TMPRSS2 were pretreated with MG-132 at the indicated concentrations and subsequently infected with SARS-CoV-2 (MOI ~0.2) in the continuous presence of the drug. The proportion of infected cells was quantified by flow cytometry as described in Fig. 1D, and data were normalized to that from control samples for which MG-132 had been omitted. n = 6 biological replicates.
Figure 6SARS-CoV-2 was subjected to pretreatment with trypsin and furin for 15 min at 37°C prior to infection of Caco-2 and Vero cells (MOIs of 0.4 and 0.3, respectively). Infected cells were quantified by flow cytometry as described in Fig. 1D. Data were normalized to samples not pretreated with trypsin. n = 4-6 biological replicates.A549* expressing or lacking TMPRSS2 cells and Vero cells were first infected with SARS-CoV-2 at MOIs of 0.1 and 0.2, respectively, for 24 h and then cocultured for 5 h along with target cells, not infected but prestained with CMFDA, a cytosolic green dye. Cells were subsequently treated with trypsin or furin for 5 min at 37°C and left to coculture for an additional hour at 37°C. After fixation, nuclei were stained with Hoechst (blue), and infected cells were subjected to immunofluorescence staining against SARS-CoV-2 nucleoprotein (magenta). Samples were ultimately imaged by confocal fluorescence microscopy. White stars indicate syncytia with at least four nuclei. Scale bars: 100 µm. Images of microscope fields obtained in (E) were quantified [A549*: n (no virus) = 30, n (no protease) = 80, n (trypsin) = 80, and n (furin) = 60; A549* TMPRSS2: n (no virus) = 30, n (no protease) = 80, n (trypsin) = 79, and n (furin) = 60; Vero: n (no virus) = 40, n (no protease) = 112, n (trypsin) = 114, and n (furin) = 60]. The fusion index is given as f = 1 - [(number of cells in a field after fusion)/(number of nuclei)].
Figure 7SARS-CoV-2 particles (MOI of 0.3) were first subjected to trypsin treatment for 15 min at 37°C and then exposed to buffers at the indicated pH for 10 min at 37°C, and vice versa. Calu-3 and Vero cells were then infected at a MOI of 0.3 and analyzed by flow cytometry as described in Fig. 1D. n = 4-8 biological replicates.
Figure 8SARS-CoV-2 was first treated with trypsin or thermolysin and then allowed to infect A549* and Vero cells (MOIs ~0.2 and 0.3) in the continuous presence of SB412515 (cathepsin L inhibitor). Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where SB412515 had been omitted. n = 4 biological replicates.
Figure 1C. Tissue distribution of the Ldb2 protein. Representative western-blot image is shown. Yellow asterisks indicate uncharacterized bands.D. Expression of Ldb2 in the cerebral cortex. DAPI-stained nuclei visualized (right) in the cerebral cortex.E. Expression of Ldb2 in the amygdala. LA; lateral amygdala, BLA; basolateral amygdala, Ce; central nucleus of amygdala.F-G. Expression of Ldb2 in neurons. The merged images obtained with Ldb2 (stained in green) and NeuN (stained in red) antibodies are shown for cerebral cortex layers I-VI and the amygdala (F). most of Ldb2-positive cells are NeuN-positive. G. NeuN-positive cells/Ldb2-positive cells (left) and Ldb2-positive cells/NeuN-positive cells were counted in cerebral cortex layers I-VI.
Figure 2Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Home cage-activity test (A). Voluntary locomotor activities in the home cages were monitored for a week. Data of dark (left) and light (right) phases are indicated.Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Open-field test (B)Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Acoustic startle response (C)Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Prepulse-inhibition test (D)Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in , Morris water-maze test (E) where data in hidden test (escape latency and moving speed, path length and no movement time) and probe test (path length) are shownLdb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Y-maze test (F).Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Fear-conditioning test (G). Results of contextual (left) and cued (right) tests are shown.Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Hot-plate test (H).
Figure 3Locomotor activity was assessed in the same experimental set as in the open-field test. After acclimation for 120 min, mice (n = 10-12/group) were injected (arrows) with methylphenidate (MPD: 3 mg/kg, A) and their locomotor activity was monitored. In the right panels, cumulative values for pre- and post-injection times are also shown.Locomotor activity was assessed in the same experimental set as in the open-field test. After acclimation for 120 min, mice (n = 10-12/group) were injected (arrows) with MK-801 (0.2 mg/kg, B) and their locomotor activity was monitored. In the right panels, cumulative values for pre- and post-injection times are also shown.C. Popping behavior induced by the injection of MK-801 (0.5 mg/kg) was manually assessed in WT (n = 4) and KO (n = 6) animals. Typically, each episode is constituted by blocks of 5-15 successive jumping behaviors. Episode numbers in 20 min (left) and total duration (right) are shown in a box-whisker plot.
Figure 4A. Fear-conditioning test. Freezing (%) was monitored in the cued (top) and contextual (bottom) tests before (pre) and after (post) administration of clozapine (left) or haloperidol (right). Data are presented as means ± S.E.M. (n = 7-9/group). *, p < 0.05; ***, p < 0.001 (Tukey test); #, p < 0.05 (t-test, KO/saline vs. KO/clozapine).B. Home cage-activity test. Spontaneous locomotor activity of mice chronically fed chow with or without 0.24% (w/w) lithium carbonate for two months was monitored for 6 days in the home cage.
Figure 5A. Scatter plots showing Venus-fluorescent intensities and the median amplitude of miniature EPSCs (mEPSC) in the LA of the AV-KO mice and their littermates (AV-WT). B. Scatter plots showing Venus-fluorescent intensities and the mean frequency of miniature EPSCs in the LA of the AV-KO and their control littermates (AV-WT). Three typical pictures of Venus-positive cells with various fluorescent intensities are shown. Lines in each panel are regression lines showing inverse correlation of the fluorescent intensity with the mEPSC amplitude (A) or the mEPSC frequency (B). The black dotted lines are regression lines for AV-WT mice, the red dotted lines are those for AV-KO mice, and black solid lines are those for all cells.
Figure 6B. Numbers of Venus-positive neurons in the lateral nucleus (LA) and the lateral side of the basal nucleus (BAl) of Naive, Unpaired, and Paired groups. Open and filled bars represent AV-WT and AV-KO mice, respectively. Positive neurons of AV-WT was increased only in the Paired group compared to the Naïve and Unpaired groups in the LA (upper panel). AV-KO was increased in the Unpaired as well as the Paired groups compared to the Naïve group Positive neurons both in the Unpaired and Paired AV-KO mice were increased compared to the same groups of the AV-WT mice In the BAl , positive neurons were increased both in the Unpaired and Paired groups compared to the Naïve group in both the AV-WT and AV-KO mice The error bars represent S.E.M. (n =5/group).C. Numbers of Venus-positive neurons classified by fluorescence intensities in the LA of the AV-WT and AV-KO mice. Weak positive neurons were increased in the Paired group compared to the Naïve group in AV-WT Both Weak and Middle positive neurons were increased in the Paired group compared to the Naïve group in AV-KO The increment of the Middle Venus-positive neurons was larger in the AV-KO compared to AV-WT