EMBO/soda-nel huggingface dataset:
BRLF1 suppresses RNA Pol III-mediated RIG-I inflammasome activation in the early EBV lytic lifecycle (2020)
Figure 4

Figure 4. BRLF1 inhibits the activation of RIG-I inflammasome and immune responses.

(a) Stable 293T cells in the presence of pro-caspase-1, pro-IL-1β and ASC overexpression were transfected with control vector or BRLF1 with control vector, IFI16, NLRP3, RIG-I or AIM2 for 36 h. After the cells were infected with HSV-1 (MOI=1) for 12 h, the cells were collected, and the cell lysates were analyzed by western blotting to determine the levels of pro-caspase-1, cleaved caspase-1, pro-IL-1β and mature IL-1β as indicated.

(b) Scramble (sc) or shRNAs against RIG-I and empty control vector (ctr), BRLF1- or BRLF1 L578A-expressing plasmids were co-transfected into EBV-ΔBRLF1-harboring HNE1 cells. Thirty-six hours after the transfection, cell pellets were collected, after which the cleavage of caspase-1 and IL-1β was measured as indicated.

(c) Analysis of ASC oligomerization. ASC-expressing plasmid was transfected into 293T cells or HNE1 cells with empty vector or GFP-BRLF1 in the presence of AIM2- or RIG-I-expressing plasmid. Twelve hours after HSV-1 infection (MOI=1) in 293T cells, or 48 h after EBV-ΔBRLF1 infection (MOI=1000) in HNE1 cells, the cell pellets were collected, and cell lysates were treated with DSS to induce chemical cross-linking and analyzed by western blotting for ASC oligomerization.

(d) EBV-ΔBRLF1-harboring HNE1 cells were transfected with control vector, BRLF1 or BRLF1 L578A in the absence or presence of BZLF1 for 48 h. The total RNAs were extracted and subjected to real-time PCR analysis.

(e) A549 cells were transfected with control vector, BRLF1 or BRLF1 L578A for 36 h. After the cells were infected with HSV-1 (MOI=1) for 12 h, the total RNAs were extracted and subjected to real-time PCR analysis.

Data information: The values are shown as the mean ± standard deviations of triplicate determinations from three independent experiments. *, p < 0.05. **, p< 0.01. Tukey's multiple-comparison test.
BRLF1 suppresses RNA Pol III-mediated RIG-I inflammasome activation in the early EBV lytic lifecycle (2020)
Figure 5

Figure 5. BRLF1-mediated inhibition of inflammasomes attenuates T and NK cell activation during EBV primary infection.

(a-b) Jurkat cells (a) and NK-92MI cells (b) were infected with EBV-WT or EBV-ΔBRLF1 virus (MOI=500) for 36 h. Then, the cells were collected, and the total RNAs were extracted, reverse-transcribed and analyzed by real-time PCR. Color gradient depicts the mean mRNA expression normalized to the housekeeping gene GAPDH and compared to the mock group. Results are presented as the mean ± SD, n = 3 biological replicates, *, p < 0.05. **, p< 0.01, Student's t‐test.

(c-e) Jurkat and NK-92MI cells were cultured with conditional medium from EBV-WT or EBV-ΔBRLF1-infected Ramos cells (paracrine), or Jurkat and NK-92MI cells were directly infected with EBV-WT or EBV-ΔBRLF1 viruses (autocrine). (c) The cell pellets were collected and the whole cell extracts were analyzed by western blotting to detect TAK1, IKKα/β and IκB phosphorylation. The cell killing activity of Jurkat cells (d) and NK-92MI cells (e) were measured by the detection of the LDH release from the K562 target cells. Results are presented as the mean ± SD, n = 3 biological replicates, *, p < 0.05, **, p< 0.01, Tukey's multiple-comparison test.

(f-h) Anti-IL-1β and/or anti-IL-18 neutralizing antibodies (NA), TAT-Flag or TAT-N572 peptide were added into EBV-WT- or EBV-ΔBRLF1-infected Jurkat cells or NK-92MI cells for 24 h. (f) TAK1, IKKα and IκB phosphorylation were detected as indicated. In addition, the cell killing activity of Jurkat cells (g) and NK-92MI cells (h) were measured. Results are presented as the mean ± SD, n = 3 biological replicates, *, p < 0.05, **, p< 0.01, Tukey's multiple-comparison test.
BRLF1 suppresses RNA Pol III-mediated RIG-I inflammasome activation in the early EBV lytic lifecycle (2020)
Figure 6

Figure 6. BRLF1-deficient EBV primary infection activates T and NK cells in human lymphocytes.

(a-d) PBMCs were isolated and infected with EBV-WT or EBV-ΔBRLF1 virus (MOI=500). Thirty-six hours after infection, the cells were harvested and the total RNAs were extracted, reverse-transcribed and subjected to real-time PCR analysis. The differential expression of receptors and cytokines (a), cytotoxic genes (b), T cell-specific genes (c) and NK cell-specific genes (d) are shown. The values are shown as the mean ± standard deviations of five analyses of PBMCs from five different health donors. Results are presented as the mean ± SD, n = 5 biological replicates, *, p < 0.05. **, p< 0.01, Student's t‐test.

(e-h) FACS analysis of T and NK cell activation in PBMCs in the presence of EBV-WT or EBV-ΔBRLF1 primary infection. The PBMCs were left uninfected or infected with EBV-WT or EBV-ΔBRLF1 virus for 36 h, and the EBV-ΔBRLF1-infected lymphocytes were incubated with additional TAT-Flag or TAT-N572 peptide for 12 h. Immunofluorescent PE-CD3 and APC-CD25 antibodies were used to detect T cell activation (e, g), and PE-CD56 and APC-CD69 were used to detect NK cell activation (f, h). Representative images of the FACS analysis of T cell and NK cell activation are shown (e, f), and the percentages of activated T cells and NK cells were calculated and are shown as the mean ± standard deviation (g, h) for lymphocytes from 5 different health donors; Results are presented as the mean ± SD, n = 5 biological replicates, *, p < 0.05. **, p< 0.01, Student's t‐test.

Expanded View Figure Legends
POLO REGULATES SPINDLY TO PREVENT PREMATURE STABILIZATION OF KINETOCHORE-MICROTUBULE ATTACHMENTS (2019)
Figure 2

Figure 2. Expression of PoloT182D compromises mitotic fidelity.

(A) Representative immunofluorescence images of KT alignment efficiency in mitotic S2 cells expressing PoloWT-EGFP, PoloT182D-EGFP or lacking expression of any Polo transgene. Cells were treated with MG132 prior to fixation to prevent cells from exiting mitosis and increase the number of pre-anaphase figures. Insets display magnifications of the outlined regions, which highlight both low-tension misaligned (inset 1 and 2) and high-tension aligned KTs (inset 3).

(B) Graph represents the percentage of cells in each indicated mitotic state, as shown in (A) (n≥ 532 cells for each condition, n=2 independent experiments).

(C) Representative immunofluorescence images of calcium-stable KT-MT attachments in metaphase S2 cells expressing either PoloWT-EGFP or PoloT182D-EGFP. Insets display magnifications of the outlined regions. Asterisk highlights either an aligned KT pair attached to MTs in an end-on fashion (PoloWT-EGFP) or an aligned KT pair in which a sister KT is laterally attached to the end of a MT fiber (PoloT182D-EGFP). Plotted profiles show the overlap between Polo-EGFP and tubulin signals for the highlighted KT.

(D) Graph represents the percentage of metaphase cells showing at least 1 KT with a lateral interaction, as shown in (C) (asterisk) (n≥58 cells for each condition, n=2 independent experiments).

(E) Selected frames from live image analysis of chromosome segregation fidelity in neuroblasts expressing either PoloWT or PoloT182D. Neuroblasts without Polo overexpression were used as control. Jupiter-GFP was imaged for direct visualization of the mitotic spindle. Time 0 refers to anaphase onset.

(F) Representative immunofluorescence images of mitotic neuroblasts from squashed brains showing distinct chromosome content. Spc105 was used as reference for KTs. Chromosome content is shown for each neuroblast.

(G) Table shows the quantification of the percentage of asynchronous migration (n≥23 neuroblasts for each condition, n≥3 independent experiments) and of aneuploid cells shown in (E and F), respectively. For the quantification of aneuploidy, the whole brain was analyzed and prometaphase and metaphase neuroblasts were scored for chromosome number. An aneuploid cell was considered when >8 chromosomes (chr) were visualized (n≥360 neuroblasts from at least 7 larvae brains for each condition, n≥3 independent experiments). ND, not determined.

Data information: Data are shown as mean ± SD. Scale bar: 5 μm.
POLO REGULATES SPINDLY TO PREVENT PREMATURE STABILIZATION OF KINETOCHORE-MICROTUBULE ATTACHMENTS (2019)
Figure 3

Figure 3. RNAi-based screen identifies Rod as a suppressor of the PoloT182D phenotype.

(A) Schematics of the RNAi screen strategy. Females expressing UASPoloT182D under the control of eyless-Gal4 promoter were crossed with males carrying a specific UASRNAi transgene. Crosses were left either at 18°C or 25°C and adult offspring viability was assessed.

(B) Representative images of the eye phenotype observed in adult flies that were expressing UASPoloT182D in the eye imaginal disc during development at indicated temperatures. An example of an eye from an adult fly co-expressing UASPoloT182D with UASRodRNAi is also shown. Expression of UASlacZ transgene in the eye imaginal disc was used as control for overexpression and UAS dilution effect.

(C) Graph represents the mean percentage of males born at 25°C either expressing eyGal4-driven UASPoloT182D together with UASlacZ or in combination with UASRodRNAi (n≥3 independent crosses for each condition). The number of males with the genotype of interest born in each cross was normalized to the mean total number of males that are born in a control cross (eyGal4>UASlacZ, n=9 independent crosses).

(D) Selected stills from live cell imaging of neuroblasts expressing either UASPoloT182D alone or in Rod-depleted background (UASPoloT182D+UASRodRNAi). Neuroblasts without transgene expression were used as control and neuroblasts expressing UASPoloT182D together with UASlacZ were used as control for UAS dilution effect.

(E) Quantification of the time spent in prometaphase (from NEBD until last KT alignment at the metaphase plate) for neuroblasts shown in (D) (n≥17 neuroblasts for each condition, n≥4 independent experiments). Statistical analysis was calculated using an one-way ANOVA test for multiple comparisons. p values: ns, not significant; **, <0.01.

(F) Quantification of the percentage of prometaphase/metaphase mitotic figures per brain showing abnormal chromosome number (>8 chromosomes (chr) as shown in Fig.2F, G (n≥360 neuroblasts from at least 7 larvae brains for each condition, n≥3 independent experiments). Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ns, not significant; **, <0.01.

Data information: Data are shown as mean ± SD. Scale bar: 5 μm.
POLO REGULATES SPINDLY TO PREVENT PREMATURE STABILIZATION OF KINETOCHORE-MICROTUBULE ATTACHMENTS (2019)
Figure 4

Figure 4. Expression of constitutively active Polo kinase reduces Rod streaming from late congressing KTs.

(A) Selected stills from live cell imaging of neuroblasts expressing UASPoloWT, UASPoloT182D, or depleted of dynein heavy chain (UASDhcRNAi) and expressing Rod-GFP under control of its endogenous promoter. CID-RFP was used as a KT reference. Insets display magnifications of the outlined regions, which highlight late congressing KTs. Asterisks indicate direction of chromosome segregation (putative spindle pole positions).

(B) Graph represents the mean fluorescence intensity (MFI) for Rod-GFP determined relative to CID-RFP MFI for the KT pairs highlighted in (A). Time was measured from the first frame a KT pair moved away from the other congressing KTs, until metaphase alignment (vertical dashed lines).

(C) Graph represents the levels of KT Rod-GFP determined relative to CID-RFP measured throughout mitotic progression for control neuroblasts shown in (A) (UASPoloWT, n= 24 neuroblasts, n=4 independent experiments).

(D) Graph represents the levels of Rod-GFP determined relative to CID-RFP measured at late congressing KTs (as shown in A, insets) (n≥15 neuroblasts for each condition, n≥3 independent experiments).

(E) Selected stills from live imaging analysis of Rod-GFP streaming from KTs in neuroblasts expressing either PoloWT or PoloT182D. Asterisks indicate direction of chromosome segregation (putative spindle poles positions). Arrowheads highlight streaming of Rod-GFP from unaligned KTs. Representative kymographs are shown for Rod-GFP streaming from metaphase until anaphase onset (AO). Frames were acquired every 5sec.

Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: **, <0.01; ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.
POLO REGULATES SPINDLY TO PREVENT PREMATURE STABILIZATION OF KINETOCHORE-MICROTUBULE ATTACHMENTS (2019)
Figure 5

Figure 5. Expression of constitutively active Polo mimics the depletion of Spindly in the inhibition of stable KT biorientation by the RZZ complex.

(A) Representative immunofluorescence images of ZW10 levels at unaligned KTs in neuroblasts expressing UASPoloWT, UASPoloT182D or depleted of dynactin subunit p50 (UASp50RNAi) or Spindly (UASSpindlyRNAi). Spc105 was used as a KT reference. Insets display magnifications of the outlined regions which highlight both late congressing KT (inset 1) and sister KTs that are non-oriented along the spindle axis (inset 2, except for the UASPoloWT panel which displays congressed KTs). UASPoloWT neuroblasts were used as control.

(B) Graph represents ZW10 levels at late congressing and non-oriented KTs for neuroblasts shown in (A). Cells were selected when most KTs were congressed and late congressing KTs were clearly identified. Similarly, non-oriented KTs were considered when the KT-KT axis was not parallel to most congressed KTs (or a metaphase plate). ZW10 signal was determined relative to Spc105 and all values were normalized to the control mean fluorescence intensity, which was set to 100% (n≥69 KTs from at least 19 neuroblasts for each condition, n≥3 independent experiments).

(C) Selected stills from live imaging analysis of KT alignment in neuroblasts expressing UASPoloT182D, either alone or in a Rod-depleted background (UASPoloT182D+UASRodRNAi), depleted of dynactin (UASp50RNAi), Spindly (UASSpindlyRNAi), dynein (UASDhcRNAi) or Rod in a dynein-depleted background (UASDhcRNAi+UASRodRNAi). Outlined regions show KTs that are non-oriented (lateral attachment) relative to spindle axis and that were followed over time until stable biorientation (arrowhead). Cartoon depicts the behavior of the respective KT pair over successive frames relative to the spindle axis (dashed lines). Neuroblasts without transgene expression were used as control.

(D) Quantification of the time a KT pair takes from the first contact with MTs until stable biorientation for neuroblasts shown in (C) (n≥12 KTs from at least 8 neuroblasts for each condition, n≥5 independent experiments).

Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: *, <0.05; **, <0.01; ***, <0.001; ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.
POLO REGULATES SPINDLY TO PREVENT PREMATURE STABILIZATION OF KINETOCHORE-MICROTUBULE ATTACHMENTS (2019)
Figure 7

Figure 7. Polo phosphorylates Spindly at unattached and unaligned KTs.

(A) In vitro kinase assays with the indicated recombinant protein in the presence of [γ-32P]-ATP for 30 min. Phosphorylation was detected by autoradiography and levels of proteins were visualized by coomassie blue staining.

(B) ClustalW sequence alignment for Spindly orthologues. The region encompasses the Polo-dependent phosphorylation site identified in Drosophila Spindly (red box). Asterisk highlights a conserved residue identified in silico by the Eukaryotic Linear Motif (ELM) resource as a putative Polo-dependent phosphorylation site in human Spindly. Sequence alignment was coloured according to clustal colour scheme in Jalview (www.jalview.org, v2.10.5). Abbreviations: h, Homo sapiens; m, Mus musculus; x, Xenopus laevis; d, Drosophila melanogaster.

(C) Sequence alignment showing that the Polo-dependent phosphorylation site in Drosophila Spindly lies within a conserved region found in different dynein adaptors. Abbreviations: h, Homo sapiens; m, Mus musculus; d, Drosophila melanogaster. Sequence alignment was coloured according to clustal colour scheme in Jalview (www.jalview.org, v2.10.5). The binomial log10 sequence logo was created using the PSSMSearch software (Krystkowiak et al, 2018).

(D) Representative immunofluorescence images of Spindly-phospho(ph)Ser499 levels at unattached KTs in control Drosophila S2 cells and in Polo-depleted or BI2536-treated cells. Insets display magnifications of the outlined regions. Cells were treated with colchicine prior to fixation to generate unattached KTs. CID was used as a KT reference.

(E) Graph represents the levels of Spindly phS499 at unattached KTs for cells shown in (D). Spindly phS499 levels were determined relative to CID and all values were normalized to the control mean fluorescence intensity, which was set to 100% (n≥1246 KTs from at least 63 cells for each condition, n=2 independent experiments). Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ****, <0.0001

(F) Representative immunofluorescence images of Spindly phS499 levels at unaligned and aligned KTs in a PoloWT-EGFP expressing Drosophila S2 cell. Insets show magnifications of the outlined regions, which highlight either a unaligned (u) KT or an aligned (a) KT. CID was used as a KT reference. Plotted profiles of signal intensities of phS499 and CID are shown for the highlighted KTs.

(G) Graph represents the levels of Spindly phS499 at unaligned KTs versus aligned KTs for cells shown in (F). Spindly phS499 levels were determined relative to CID and all values were normalized to the mean fluorescence intensity of unaligned KTs, which was set to 100% (n≥34 KTs from 14 cells, n=2 experiments). Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: ****, <0.0001.

Data information: Data are shown as mean ± SD. Scale bar: 5 μm.
POLO REGULATES SPINDLY TO PREVENT PREMATURE STABILIZATION OF KINETOCHORE-MICROTUBULE ATTACHMENTS (2019)
Figure 8

Figure 8. Polo-mediated phosphorylation of Spindly on Ser499 impairs its binding to the RZZ and delays KT accumulation during chromosome congression.

(A) Western blot analysis of MBP-Zwilch pull downs of full length (FL) his6x-SpindlyWT and phosphomimetic mutant his6x-SpindlyS499D. Immobilized MBP was used as negative control. B:Beads, FT: Flow-through.

(B) Western blot analysis of MBP-Zwilch pull downs of his6x-SpindlyWT or his6x-SpindlyS499D fragments (aa1-510). Immobilized MBP was used as negative control. B:Beads, FT: Flow-through.

(C) Graph represents the levels of his6x-Spindly relative to MBP-Zwilch from at least three independent pull down experiments. The values obtained for his6x-SpindlyWT were set to 1.

(D) Selected frames from live imaging analysis of SpindlyS499A-EGFP or SpindlyS499D-EGFP behavior during KT alignment in Drosophila S2 cells expressing PoloWT-mRFP. SpindlyWT-EGFP expressing cells were used as control. All Spindly-EGFP constructs were resistant to the RNAi targeted against endogenous Spindly. Insets show magnifications of the outlined regions that highlight KTs that delay in congressing to the spindle equator (yellow dashed line). Time 0 is the first shift in KT direction from poleward to anti-poleward movement.

(E) Graph represents the variation in SpindlyWT-EGFP, SpindlyS499A-EGFP and SpindlyS499D-EGFP levels at KTs highlighted in (D) and its correlation with efficiency of alignment. All values were determined as described in Fig.6D. Time 0 is the first shift in KT direction from poleward to anti-poleward movement (n≥5 KTs from at least 4 cells for each condition, n≥2 independent experiments).

Data information: XXXX Data are shown as mean ± SD. Scale bar: 5 μm.
POLO REGULATES SPINDLY TO PREVENT PREMATURE STABILIZATION OF KINETOCHORE-MICROTUBULE ATTACHMENTS (2019)
Figure 9

Figure 9. Phosphorylation of Spindly Ser499 imposes an RZZ-dependent delay on chromosome congression.

(A) Representative immunofluorescence images of ZW10 localization to unaligned KTs in Drosophila S2 cells expressing either SpindlyWT-, SpindlyS499A- or SpindlyS499D-EGFP. Insets display magnifications of the outlined regions, which highlight late congressing KTs. CID was used as a KT reference.

(B) Graph represents the percentage of ZW10 levels at late congressing and non-oriented (not oriented parallel to the spindle axis) KTs for cells shown in (A). ZW10 levels were determined relative to CID and all values were normalized to the control mean fluorescence intensity, which was set to 100% (n≥72 KTs from at least 26 cells for each condition, n=4 independent experiments).

(C) Selected frames from live imaging analysis of chromosome congression in Drosophila S2 cells expressing SpindlyWT-EGFP, SpindlyS499A-EGFP, SpindlyS499D-EGFP or SpindlyS499D-EGFP in a ZW10-depleted background. The first frame shows EGFP-positive cells. Chromosome alignment was followed in vivo by direct visualization of KT-associated PoloWT-mRFP signal. Time 0 refers to nuclear envelope breakdown (NEBD), metaphase refers to full KT alignment and Anaphase onset refers to onset of chromosome segregation.

(D) Quantification of the time spent in prometaphase (from NEBD until last KT alignment at the metaphase plate) for cells shown in (C) (n≥10 cells for each condition, n≥2 independent experiments).

Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: *, <0.05; **, <0.01; ****, <0.0001 . Data are shown as mean ±SD. Scale bar: 5 μm.