EMBO/sd-character-level-ner huggingface dataset:
Fig.Figure 4(A) Quantification of EMSA assays evaluating the binding of protamine to 19 nt-long ssDNA and ssRNA molecules. Each probe (0.2 μM) was incubated with increasing concentrations of (PR)20 for 10´. Curve-fitting was performed using non-linear regression with the Hill equation. (B) Percentage of GAPDH levels quantified by qPCR in reactions containing increasing doses of protamine (n=3). Data represent mean values ± SEM. (C) Percentage of RNA (1 μg) remaining after a 15´ digestion with increasing doses of RNase A in the presence or absence of protamine (5 μM). Data derive from one representative experiment that was repeated twice. (D) Immunofluorescence of Cy3-labelled salmon protamine (red) and the nucleolar factor UBF1 (green) in U2OS cells treated with 30 μM Cy3-protamine for 4 h. Scale bar (white) indicates 2.5 μm. (E, F) HTM-mediated quantification of the EU levels per nucleus (E) and O-propargyl-puromycin (OPP) levels per cell (F) in U2OS cells exposed to 30 μM protamine for 16 (E) or 24 (F) h. EU and OPP were added 30´ prior to fixation. Black lines indicate median values (****, p<0.0001; t-test). (G) RT-PCR of NACA and GADD45A mRNAs in U2OS cells treated with protamine (30 μM) or (PR)20 (20 μM) using primers in non-consecutive exons to monitor alternative splicing events. A scheme of the primers used is provided on the right side of the panel. (H) RT-PCR of GADD45A mRNAs in U2OS cells treated with HA-tagged (PR)20, (GR)20, (PK)20 and (GA)20 peptides (20 μM) using the pipeline defined in (G).
a, 4HA–Sae2 was immunoprecipitated ± VPA with anti-HA and subsequently ± anti-acetyl-Lysine.f4a, 4HA-Sae2 was immunoprecipitated ± VPA with anti-HA and subsequently ± anti-acetyl-Lysine. Eluate was analysed using anti-HA. Lane 1: input Sae2; 2: input Sae2-HA -VPA; 3: as in 2 but +VPA; 4: 3 µl elution Sae2-HA -VPA after anti-HAimmunoprecipitation (IP; input AcK-IP); 5: double amount of 4; 6: IP anti-AcK elution from anti-HA IP of Sae2-HA -VPA; 7: as in 6 but +VPA. b, SAE2::PKerg6Δ cells were treated as in Fig. 2a. After 30 min induction, VPA and PMSF were added or not. Samples were processed for western blot using anti-PK. c, Wild-type SAE2::PK, SAE2::PKatg1Δ and SAE2::PKatg19Δ cells were grown as in b. After 30 min induction, VPA was added or not and samples treated as in b. d, Wild-type SAE2::PK cells were grown as in b. After 120 min induction, rapamycin (200 ng ml-1) was added or not and samples were treated as in b.
Figure 2ST2 cells were either left untreated (A) followed by stimulation with IL-17 (500 ng/ml) for 2 hours. Transcription response induced by IL-17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing. In red are transcripts considered to be significantly changed (log2 fold change > 1 or < -1, - log10 Benjamini-Hochberg adjusted p-value > 2, based on the analysis of three independent experiments; tringle is used for transcripts with - log10 adjusted p-value > 100). Names of several significantly upregulated transcripts are indicated. ST2 cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM) for 30 minutes (B), followed by stimulation with IL-17 (500 ng/ml) for 2 hours. Transcription response induced by IL-17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing. In red are transcripts considered to be significantly changed (log2 fold change > 1 or < -1, - log10 Benjamini-Hochberg adjusted p-value > 2, based on the analysis of three independent experiments; tringle is used for transcripts with - log10 adjusted p-value > 100). Names of several significantly upregulated transcripts are indicated. C. The Venn diagram representing the number of significantly changed transcripts upon IL-17 stimulation detected in (A) and (B). D-E. Comparison of IL-17-induced transcriptional response in presence or absence of TBK1/IKKε inhibitor. (D) Transcripts that are significantly changed in both conditions. (E) Transcripts that pass significance threshold for induction only in presence of TBK1/IKKε inhibitor. Dashed lines indicate significantly upregulated transcripts (log2 fold change > 1); red lines separates transcripts that are more upregulated upon IL-17 stimulation in the presence of TBK1/IKKε inhibitor as compared to IL-17 alone. F. ST2 cells pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) were left unstimulated or stimulated with IL-17 (500 ng/ml) for 2 hours and induction of mRNA for selected genes was analyzed by real time PCR. Mean + SEM from five independent experiments is shown, statistical significance was determined using unpaired two‐tailed Student's t‐test. G. ST2 cells were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL17 (500 ng/ml) for indicated time points and analyzed by immunoblotting. H. ST2 wild type, TBK1 KO, IKKε KO, or cells lacking both kinases (DKO) were stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting. I. TBK1/IKKε DKO cells reconstituted with either wild type or kinase dead mutant (K38A) version of both kinases were stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.
Figure 4 [A] DA induces genes involved in SA biosynthesis and signaling in Arabidopsis grown with Pi deficiency. Data information: qPCR results show values of means ± SE (n=3), two biological replicates were analyzed with similar results. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.  [B] DA elevates SA accumulation levels in Pi-deficient Arabidopsis. Means ± SE of three biological replicates. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.  [C] Images of plants grown with different treatments using DA (9.7 µg per mL free space) or SA (100 µM in plant growth medium). Exogenous application of SA mimics DA-induced anthocyanin accumulation (D) patterns in Pi-deficient plants. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test. Exogenous application of SA mimics DA-induced IPS1 gene induction (E) patterns in Pi-deficient plants. Data information: qPCR results show values of means ± SE (n=3), two biological replicates were analyzed with similar results. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test. Compared to the wild type plants, the NahG transgenic plants showed altered responses to DA under Pi-deficiency condition, as shown by plant images (F) Compared to the wild type plants, the NahG transgenic plants showed altered responses to DA under Pi-deficiency condition and quantification of anthocyanin accumulation levels (G). Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.
Model illustrating the pleiotropic changes of tamoxifen in PSCs and PDAC stroma.Figure 8Top panel: In PSCs tamoxifen reduces actomyosin contractility via GPER. This mechanically down regulates HIF-1A that is the unifying factor through which tamoxifen reduces LOX-L2, MMP-2, and FN. GPER activation reduces the synthesis and secretion of collagen by PSCs. Tamoxifen also impairs LOX-L2 collagen remodelling. The red colour has been used to highlight the two main pillars by which tamoxifen acts on PSCs. Bottom panel: In PDAC tissues tamoxifen reduces hypoxia levels and increases vascularisation. Also, reduces the amount and organization of collagen and FN in the ECM
The mitophagy receptor FUNDC1 is a novel substrate of ULK1 and FUNDC1 (N118A) that prevents its binding to ULK1, impairs ULK1 translocation to mitochondria, and inhibits mitophagy A HeLa cells were co-transfected with FUNDC1-Myc and FLAG-ULK1.Figure 2A HeLa cells were co−transfected with FUNDC1−Myc and FLAG−ULK1. Cells were lysed and immunoprecipitated with anti−Myc antibody 24 h after transfection. B FLAG−FUNDC1 was co−transfected with GFP−vector or GFP−ULK1 for 24 h in HeLa cells, and then, cell lysates were immunoprecipitated with anti−GFP antibody. CMEFs were exposed to hypoxia (1% O2) for 12 h or FCCP (20 μM) for 3 h, followed by immunoprecipitation using anti‐ULK1 antibody. D The expression of FUNDC1 is induced by 10 ng/ml tetracycline (Tet) in FUNDC1‐inducible HeLa cells. Cells were transfected with HA‐ULK1 or HA‐ULK1 (K46N) for 24 h. Then, the cell lysates were prepared for immunoblotting using indicated antibodies. EULK1+/+ cells were transfected with FUNDC1‐Myc, and ULK1-/- cells were transfected with FUNDC1‐Myc alone, FUNDC1‐Myc and HA‐ULK1, or FUNDC1‐Myc and HA‐ULK1 (K46N). Cells were harvested and lysed for immunoblots24 h after transfection. F Purified GST‐FUNDC1 and GST‐FUNDC1 (S17A) were subjected to an in vitro kinase assay with Myc‐ULK1 immunoprecipitated by anti‐Myc antibody from Myc‐ULK1‐transfected cells about 24 h post‐transfection. Phosphorylated FUNDC1 was detected by anti‐p‐S17‐FUNDC1 antibody. The red asterisks mark the target bands. G Western blotULK1, FUNDC1, and the phosphorylated FUNDC1 in MEFs exposed to hypoxia (1% O2) for the indicated times. HWestern blot analysis of the kinetics of ULK1, FUNDC1, and the phosphorylated in MEFs treated with FCCP(20 μM) for the indicated times.actin I, J The single mutant which can abolish the ULK1 and FUNDC1 interaction was identified. HeLa cells were co‐transfected with Flag‐ULK1 and the indicated constructs. 24 h after transfection, cells were lysed for immunoprecipitation by anti‐Flag antibody. KMEFs were transfected with FUNDC1‐Myc and its mutants after the endogenous FUNDC1 was knocked down by siRNA. 36 h after transfection, cells were harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1) and then lysed for immunoblotting with the indicated antibodies. L MEFs were co‐transfected with FUNDC1‐Myc or its mutants and MitoDsred after the endogenous FUNDC1 was knocked down by siRNA. 24 h post‐transfection, cells were treated with hypoxic (1% O2) conditions for 12 h or FCCP (20 μM) for 6 h before fixed by 4% paraformaldehyde and stained with indicated antibodies. Scale bar, 10 μm.
Figure 3Schematic model of cholesterol conversion into 24OHC catalyzed by CYP46A1. 24OHC levels in spent media from LN229 (left) and GBM#P3 (right) cells transduced with lenti-Ctrl or lenti-CYP46A1 measured using targeted LC-MS/MS IC50 curves for 24OHC in LN229 and GBM#P3 cells using the CCK-8 assay. Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of LN229 and GBM#P3 cells undergoing apoptosis after exposure to 0 - 20 μM 24OHC for 72 h. Data are shown as the mean ± SEM (n = 3). Colony forming ability of GBM cell lines treated with 24OHC (0 - 20 μM) for 14 days. Western blot analysis of the apoptosis marker c-PARP, c-caspase 3 and PCNA in lysates (20 µg) from GBM cells treated with 24OHC (0 - 20 μM) for 72 h. Tumoursphere formation assays for GSCs treated with different concentrations of 24OHC (0 - 20 μM). Scale bar = 100 μm. Graphic representation of the quantification of tumoursphere formation. Data are shown as the mean ± SEM (n = 3). GBM#P3: *P = 0.0192, ***P = 0.0006, ***P = 0.0002; GBM#BG7: **P = 0.0017, ***P < 0.0001, ***P < 0.0001; GBM#BG5: **P = 0.0027, ***P = 0.0001, ***P < 0.0001.
Figure 1(A) lid-1(xd288) and atgl-1(xd314) mutants show LD accumulation in most neurons in C. elegans, including neurons in the head region, tail and ventral nerve cord. xdIs109 is a stable transgenic line with pan-neuronal expression of the LD marker PLIN1::GFP. The white arrows indicate PLIN1::GFP-positive LDs in the ventral nerve cord of mutants. Scale bar: 20 μm. The right panels depict the head region. Scale bar: 5 μm. The insets in the right panels are enlarged views of PLIN1::GFP rings, representing LDs. (B) Quantification of the numbers of LDs in head region neurons (each dot represents one worm). The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to the control xdIs109. **P < 0.01, ***P < 0.001, ****P < 0.0001. ns: not statistically significant. Data show mean ± SEM. n ≥ 9. (C-D) The genetic loci of lid-1 and atgl-1. The coding regions are in light blue boxes and the noncoding regions are shown as lines. The UTRs are in red boxes. xd288 is a point mutant of lid-1 and causes a missense S111L mutation in the α/β hydrolase domain. xd314 and xd310 are point mutants of atgl-1. xd314 harbors a missense G19R mutation and xd310 harbors a missense G210E mutation. (E) Oil red O staining show dramatically increased neutral lipids in the head region of lid-1(xd288) and atgl-1(xd314) compared with N2. Scale bar: 10 μm. (F-H) EM images of neurons. The red dashed line marks the outline of neurons. The red arrows mark LDs. Scale bar: 0.5 μm. The scale bar in enlarged images is 100 nm. (I) Serial sections of atgl-1(xd314) show two LDs (indicated by blue and red arrows) in a neuron cell body. Scale bar: 0.5 μm.
Summary model for Ca2+ signaling outside and within the tether proteins-assembled ER/PM junctions.Figure 8In resting cells, the ER/PM junction tethers are confined mostly to the ER and the Ca2+ signaling proteins, the PM PMCA and Orai1 and the ER IP3Rs, SERCA2, SARAF and STIM1, are not assembled into complexes and function in house-keeping. The PM proteins are localized at a region with poor PI(4,5) P2 in the inner leaflet of the PM. STIM1 is bound with Ca2+ and is folded in a manner that its SOAR domain cannot access Orai1. Upon stimulation of GPCRs and generation of IP3, peripheral ER Ca2+ is depleted to unfold STIM1 and expose its SOAR and polylysine domains, which results in STIM1 clustering while still in the ER anchors STIM1 at the PM through the polylysine domain and clustering and activation of Orai1 by the SOAR domain. This takes place at a PI(4,5) P2-poor domain of the PM that is not sufficient to anchor the tethers at the PM. At this PI(4,5) P2-poor domain SARAF cannot interact with STIM1 and SERCA2 and PMCA are not close to the Orai1-STIM1 complex, which allow maximal Ca2+ influx. After the initial bolus of Ca2+ influx, the tethers ANO8 and E-Syt1 form a PI(4,5) P2-rich compartment to which the GPCRs complex, STIM1-Orai1 complex, SERCA2, PMCA and IP3Rs translocates to the PI(4,5) P2-rich domain, where they tightly communicate to control signaling intensity and the properties of the Ca2+ signal to maintain the fidelity of the physiological Ca2+ response. Failure to translocate to the tethers-assembled compartment results in a runaway Ca2+ response and pathology.
XIAP is a novel E3 ligase for Mdm2.Figure 3(A) Lysates from HCT116 cells were immunoprecipitated separately with anti‐XIAP antibody and an isotype‐matched control IgG. Immunoprecipitates and input were analysed by western blotting. The data are representative of two biological replicates. (B) Recombinant His‐XIAP protein was incubated separately with purified GST and GST‐Mdm2 fusion proteins on glutathione beads for 4 h followed by western blot analysis using anti‐His antibody. GST and GST‐Mdm2 were analysed by Coomassie blue staining. The data are representative of two biological replicates. (C) HCT116 cells were treated with XIAP‐specific or control siRNAs. Twenty‐four hours later, cells were cultured in the presence of 50 μg ml−1 cycloheximide for the indicated periods of time, and subsequently analysed by western blotting. We should mention that amounts of cell lysates were adjusted to achieve similar expression levels of Mdm2 at time 0, while the same amounts of cell lysates were used to examine levels of XIAP and actin. The data are representative of three biological replicates. The ratio of Mdm2 to actin is presented in Supplementary Figure S3B. (D) p53−/−Mdm2−/− MEF cells were co‐transfected with the indicated Mdm2, XIAP, and p53 constructs. Cell lysates were analysed by western blotting with the indicated antibodies. We should mention that XIAP H467A expressing plasmid was always used less than XIAP expressing plasmid to ensure expression levels of XIAP and XIAP H467A were similar. The data are representative of three biological replicates. (E) p53−/−Mdm2−/− MEF cells were transfected with the indicated plasmids. Twenty‐four hours after transfection, cells were treated with 20 μM MG‐132 for additional 4 h. Cell lysates were denatured before proteins conjugated to His‐ubiquitin were pulled down by Ni2+‐NTA beads. The bead‐bound proteins and total cell lysates (TCLs) were analysed by western blot with anti‐Mdm2 antibody. The data are representative of three biological replicates. (F) In all, 2 μM Mdm2 or its C464A mutant and 1 μM XIAP or its H467A mutant proteins were incubated with 100 nM E1, 2 μM E2 and 200 μM Ub in a total 20 μlin vitro ubiquitination reaction buffer at 37°C for 1 h. The reaction mixtures were analysed by western blotting with anti‐Mdm2 antibody. The data are representative of three biological replicates. (G) p53−/−Mdm2−/− MEF cells were transfected with the indicated plasmids. Cell lysates were analysed by western blotting. The data are representative of three biological replicates. (H) p53−/−Mdm2−/− MEF cells were transfected with the indicated plasmids. Twenty‐four hours after transfection, cells were treated with 20 μM MG‐132 for additional 4 h. Cell lysates were denatured before proteins conjugated to His‐ubiquitin were pulled down by Ni2+‐NTA beads. The bead‐bound proteins were analysed by western blotting with anti‐p53 antibody. The data are representative of three biological replicates.
a, Quantification of galectin-8-positive Salmonella in unprimed BMDMs at 4 h post-infection.f4a, Quantification of galectin-8-positive Salmonella in unprimed BMDMs at 4 h post-infection. b c, Unprimed BMDMs infected with S. typhimurium for 4 h and immunostained for galectin-8, GBP2 and LC3. Arrowheads, bacteria shown in insets. Scale bars, 10 µm. d, Quantification of galectin-8/LC3-double-positive Salmonella at indicated time points post-infection. e-h, Quantification of cytosolic and vacuolar bacteria by flow cytometry in BMDMs infected with mCherry-positive S. typhimurium (e-g) or S. flexneri (h, wild-type or ΔT3SS) for 4 h. Graphs show mean and s.d. or 5-95 percentile (Box plots) of technical triplicates. Data are representative of 2 (g, h), 3 (a-d) and 4 (e, f) independent experiments. *P  0.05, **P  0.01 (two-tailed t-test).
Figure 2(A-B) Representative confocal images of Gb3 accumulation, revealed by STX staining, in brain sections from CLN7Δex2 mice at 3 and 7.5 months of age compared with CLN7 WT mice. Scale bars: 80 µm. (C-D) Gb3, NeuN, and GFAP distribution in brain areas of CLN7Δex2 mice at 7.5 months of age. Scale bars: 40 µm, 20 µm. (E) Lipidomic analysis of neurons isolated from CLN7 WT and CLN7Δex2 mouse forebrain. Data are presented as mean ± SD, **: P ≤ 0.001, ***: P ≤ 0.0001, as determined by ANOVA (n= 3 biological replicas)
Figure 6A. SPATA2 regulates TNF-a-induced NF-κB activation. HEK293T cells were transfected with the SPATA2 siRNA (SMARTpool or with to individual siRNA) or a non-targeting control siRNA, and NF-kB activation was monitored with luciferase-based NF-kB reporter assays. Error bars specify the standard error of the mean (SEM) of three independent experiments. p** < 0.05 (two-tailed, unpaired Student´s t-test). B. Knockdown of SPATA2 enhances expression of the NF-κB target genes. HEK293T cells were transfected with SPATA2 siRNA or a non-targeting control siRNA and the expression of the indicated mRNAs was measured in cells stimulated with TNF-a for 0, 3 and 6 hours with qPCR. C. SPATA2 is involved in TNF-α-induced necroptosis. SPATA2 was knocked down in L929 cells using RNAi, and cells were pretreated with DMSO, Z-VAD, or Z-VAD+NEC1 for 1 hour followed by treatment with TNF-α for 2.5 hours. Cell viability was assessed with propidium iodide staining and flow cytometry. Knockdown of SPATA2 mRNA was verified using real time PCR. p** < 0.05 (two-tailed, unpaired Student´s t-test). D. Knockdown of SPATA2 reduces phosphorylation of MLKL in TNF-α-induced necroptosis. TNF-α-dependent necroptosis was induced in L929 as described in Figure 6C. Phosphorylation of MLKL was analyzed using S358 phospho-specific antibody. E. Model for SPATA2 function in TNF-α signaling. SPATA2 interacts with CYLD and HOIP under steady-state conditions, and TNF-α-stimulation results in their recruitment to the TNF-RSC. SPATA2-dependent recruitment of CYLD impacts activation of TNF-α-induced NK-B signaling and necroptosis.
Distinct Roles for the vBcl-2-mediated Inhibition of Autophagy and Apoptosis in γHV68 Chronic Infections In Vivo.Figure 7Splenic infectious centers measured at 12 dpi (A, left), 14 dpi (B, left), 21 dpi (C, left), 28 dpi (D, up), 35 dpi (E, up), or 42 dpi (F, up) and viral genome load measured by real-time PCR at 12 dpi (A, right), 14 dpi (B, right), 21 dpi (C, right), 28 dpi (D, down), 35 dpi (E, down), or 42 dpi (F, down), in the BALB/cmice intranasally infected with the WT or recombinant γHV68 mutants, as indicated (Mean±SEM of five mice per group/time point/experiment). Data of 14 dpi and 28 dpi is pooled from two and three separate experiments, respectively. Preformed infectious virus was negligible in all spleen samples. No significant difference was detected at 12 dpi (A), 14 dpi (B), and 21 dpi (C) with the Δα1 and ΔBH2 mutant viruses when compared to the WT in infectious center titers [for Δα1, P = 0.58 (day 12); P = 0.75 (day 14); P = 0.18 (day 21); for ΔBH2, P = 0.64 (day 12); P = 0.73 (day 14); P = 0.81 (day 21); unpaired t-tests] and in viral genome loads [for Δα1, P = 0.85 (day 12); P = 0.76 (day 14); P = 0.96 (day 21); for ΔBH2, P = 0.56 (day 12); P = 0.73 (day 14); P = 0.25 (day 21); unpaired t-tests]. At 28 dpi (D), 35 dpi (E), and 42 dpi (F), the decreased infectious center titers (top) of vBcl-2 mutant viruses, as compared to WT γHV68, were statistically significant [vBcl-2Δα1 versus vBcl-2AAA (42 dpi), P = 0.46; vBcl-2Δα1 versus WT γHV68 (42 dpi), P = 0.08; unpaired t-tests]. At 28 dpi (D), 35 dpi (E), and 42 dpi (F), the decreased viral DNA loads (bottom) of the vBcl-2Δα1, vBcl-2AAA, and vBcl-2 null mutants, as compared to WT γHV68 and the vBcl-2ΔBH2 mutant, were statistically significant as follows (unpaired t-tests): at day 28, vBcl-2Δα1 versus WT γHV68, P<0.01; vBcl-2AAA versus WT γHV68, P<0.01; vBcl-2-null versus WT γHV68, P<0.01; vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.001; vBcl-2AAA versus vBcl-2ΔBH2, P<0.001; vBcl-2-null versus vBcl-2ΔBH2, P<0.001; at day 35, vBcl-2Δα1 versus WT γHV68, P<0.05; vBcl-2AAA versus WT γHV68, P<0.05; vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.01; vBcl-2AAA versus vBcl-2ΔBH2, P<0.05; at day 42, vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.001; vBcl-2AAA versus vBcl-2ΔBH2, P<0.001; vBcl-2ΔBH2 versus WT γHV68, P = 0.33. I.C., infectious center. *, P<0.05; **, P<0.01; ***, P<0.001.