EMBO/sd-character-level-ner huggingface dataset:
a–c) Hematoxylin and eosin staining of the SVZ and dentate gyrus (DG) of P28 mice (a).figf5(a-c) Hematoxylin and eosin staining of the dentate gyrus and dentate gyrus (DG) of P28mice (a). Arrows mark cells in the SVZ. Mean ± s.e.m. of the SVZ cellularity (b) and DG area (c) per section are shown. Dotted lines indicate the boundaries of the SVZ and DG (a,i,j,p) or granular zone (GZ; d,e,p). E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. NS, not significant; *P 0.05; **P 0.01; ***P 0.001 (d-m) Immunofluorescence of the DG (d-h) of P28mice (d,e,i,j). Boxed areas are shown in more detail in insets (e,j) and/or panels below (i,j). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (d,e), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes. Mean ± s.e.m. of the number of GFAP+nestin+ and GFAP+SOX2+ radial glia (f,h), and NSCs (k,m), and GFAP+nestin− astrocytes (g,l) per section are shown. Dotted lines indicate the boundaries of the SVZ and DG (a,i,j,p) or granular zone (GZ; d,e,p). E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. NS, not significant; *P 0.05; **P 0.01; ***P 0.001 (d-m) Immunofluorescence of the SVZ (i-m) of P28mice (d,e,i,j). Boxed areas are shown in more detail in insets (e,j) and/or panels below (i,j). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (d,e), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes. Mean ± s.e.m. of the number of GFAP+nestin+ and GFAP+SOX2+ radial glia (f,h), and NSCs (k,m), and GFAP+nestin− astrocytes (g,l) per section are shown. Dotted lines indicate the boundaries of the SVZ and DG (a,i,j,p) or granular zone (GZ; d,e,p). E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. NS, not significant; *P 0.05; **P 0.01; ***P 0.001 (n,o) Mean ± s.e.m. of the number of TUNEL+ cells per 100 SVZ cells (n) or per 1 mm2 DG area (o) of P28 mice. (p-r) BrdU retention in the SVZ and DG of P31mice. BrdU+ cells are marked by arrows (p). Mean ± s.e.m. of the number of BrdU+ cells per section are shown (q,r). Dotted lines indicate the boundaries of the SVZ and DG (a,i,j,p) or granular zone (GZ; d,e,p). E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. NS, not significant; *P 0.05; **P 0.01; ***P 0.001 (s,t) Mean ± s.e.m. of percentage of GFAP+nestin+BrdU+ cells of total BrdU retained cells in the SVZ (s) and SGZ (t) of P31mouse. n = 5 mice, ≥4 sections per mouse, >500 cells counted per mouse in n, >20 BrdU+ cells counted per mouse.
Figure 8Diagram for deletion of MyD88 in macrophages using MyD88ΔMP mice. Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and MyD88ΔMP mice (n = 5 mice/group). Comparisons of mRNA levels of VEGF-C in sorted MHCII+ F4/80+ macrophages from jejunum and ileum of WT and MyD88ΔMP mice. The mean of transcription levels of VEGF-C in WT mice was normalized to 1, and the relative level in MyD88ΔMP mice were presented as fold change (n = 5 mice/group). Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and MyD88ΔMP mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm. Images and comparisons of number of Prox1+ LECs within initial 100 μm portion (upper) and VEGFR3 expression (lower, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from WT and MyD88ΔMP mice. Each dot indicates mean value of 5-10 villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm. Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between WT and MyD88ΔMP mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.
Figure 5A. Crystal structure of the menin-JunDMBM complex (Adapted from (Huang et al., 2012)). MI potentially blocks the interaction of menin and JunD. B. Co-IP in HEK293T cells transfected with vector or a flag-JunD expressing plasmid upon treatment with DMSO, MI-503 (30, 100, 300 nM from lane 3 to lane 5, respectively), or MI-463 (30, 100, 300 nM from lane 6 to lane 8, respectively). C, D. ChIP assay to detect the binding of menin, HDAC3, JunD, and histone3 acetylation at the Pbk locus in vehicle or MI-503 treated PIME cells. Three independent experimets (n=3). *P = 0.0132 (Menin), *P = 0.0153 (HDAC3), **P = 0.0092 (Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference. E. ChIP assay to detect the binding of menin, HDAC3, JunD, and histone3 acetylation at the Pbk locus in vehicle or MI-503 treated INS-1 cells. Three independent experimets (n=3). ***P = 0.0008 (Menin), *P = 0.0183 (HDAC3), *P = 0.0485 (Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference. F. Mouse primary islets treated with MI-503 for 3 days were subjected ChIP assay to detect the binding of menin, HDAC3, JunD, as well as histone acetylation level at the Pbk locus. Three independent experimets (n=3). *P = 0.0218 (Menin), *P = 0.0145 (HDAC3), *P = 0.0184 (Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference.
Figure 4(A) Live imaging in NIH3T3 cells expressing ACTN4 constructs stained with SNAP488 provided positions of mitotic cells. After fixation, cells were stained with phalloidin-AlexaFluor647, rechecked for proper processing and transferred to dSTORM imaging. (B) Upper panel shows expression of ACTN4 constructs (green, confocal image) and phalloidin-AF647 staining in the same cells (orange, dSTORM image) scale bar 10 µm; lower panel represents zoom of the indicated areas (white boxes); scale bars 5 µm. (C) Phalloidin-AF647 localizations per post-mitotic nuclei; data shown as mean ± 2.5-97.5 percentile: 52 ACTN4-wt nuclei, 55 ACTN4ΔCH1-NLS nuclei and 40 nuclei of non-dividing cells (ACTN4-wt) from 5 biological replicates. ***p(ACTN4ΔCH1-NLS post-mitotic) < 0.001, ****p(ACTN4-wt non-dividing) < 0.0001, *p (ACTN4ΔCH1-NLS post-mitotic vs. ACTN4-wt non-dividing) < 0.05 by One Way ANOVA. (D) Number of filaments in the post mitotic nuclei; as in (C); data shown as mean ± 2.5-97.5 percentile: 52 ACTN4-wt nuclei, 55 ACTN4ΔCH1-NLS nuclei from 5 biological replicates, ***p < 0.001 by t-test. (E) Actin filament widths for ACTN4-wt and ACTN4ΔCH1-NLS in early G1 nuclei; ); data shown as mean ± 2.5-97.5 percentile with 96 filaments (ACTN4-wt) and 36 filaments (ACTN4ΔCH1-NLS) from 5 biological replicates, **p < 0.01 by t-test.
TLR8 agonist inhibition of HIV dependent upon a functional vitamin D pathway.Figure 6(A) Macrophages transduced with non-specific scrambled shRNA (shNS), TLR8 shRNA (shTLR8), CYP27B1 shRNA (shCYP27B1), or VDR shRNA (shVDR) were treated with CL097 for 24 h before infection with HIV with continuous CL097 treatment. Top, immunoblot analysis performed using antibodies raised to TLR8, CYP27B1, VDR and β-actin. Bottom, ELISA performed for extracellular release of HIV p24 antigen over 10 d. Results are reported as mean ± s.e.m., n = 4. (B) Macrophages were incubated with ssRNA40 or CL097 in the presence of 45 or 100 nmol/L 25D3 for 24 h before infection with HIV. Cells were then washed and incubated with 25D3 and ssRNA40 or CL097 for 10 d Extracellular release of HIV p24 antigen was detected by ELISA. Results are reported as mean ± s.e.m., n = 6. * p<0.05.
Figure 4Network of putative interactors, ranked as described in the text, and relative GO analysis. Interactors expressed in the epidermis are shown GO analysis of the predicted interactors. RT-qPCR showing the validation of uc.291-ACTL6A interaction by RNA cross-linking immunoprecipitation (CLIP) in HEK293E cells, as fold enrichment over IgG. SMARCC2 was also included. Data shown are the mean ± s.d.; n=3 (technical); *p<0.05, Student's t-test. On the right, control WB for immunoprecipitation of both antibodies. RT-qPCR showing the validation of uc.291-ACTL6A interaction by RNA immunoprecipitation (RIP) in differentiated HEKn as fold enrichment over IgG. Data shown are the mean ± s.d.; n=3 technical, on 1 human donor; *p<0.05, Student's t-test. On the right, control WB for immunoprecipitation. Secondary structure of uc.291 predicted binding region (nt 1700-2000) using the CROSS Global Score as a soft constraint inside RNA structure [43]. Uc.291 predicted binding region to interact with ACTL6A (nucleotide 1763-1912) by Global Score (score of 0.59). Dotted line define the CROSS score threshold [43].
Figure 4Limited proteolysis. Recombinant purified proteins were incubated with increasing concentrations of trypsin. Remaining protein levels were analyzed by quantitative immunoblotting. Data are means ± SEM (* p < 0.05, **p < 0.01, ****p < 0.0001, by 2-way ANOVA and Dunnett's multiple-comparison test; n = 4 independent experiments; exact p values are shown in Appendix Table S1). Direct binding of compounds to Munc18-1. Bead-bound fluorescence of compounds 9, 10 and 13 was quantified upon incubation of compounds with bead-immobilized GST, or GST-tagged WT, R406H or G544D. Data are means ± SEM (**p < 0.01, ***p < 0.001, by Kruskal-Wallis test and Dunn's multiple comparison test, or by one-way ANOVA followed by Dunnett's post-hoc test; n = 9 independent experiments; exact p values are shown in Appendix Table S1). Predicted binding sites of compounds 9, 10 and 13. The top set of images gives a global view of the binding sites. The middle set of images gives a 3D view of residue intermolecular interactions with the compounds of interest. The bottom set of images gives a diagrammatic 2D representation of the same interactions. Hydrogen bonds are represented by arrows (arrowhead pointing to acceptor) and salt bridges by a red to blue gradient line (color change red:negative, and blue:positive).
Epg5 knockout mice show muscle atrophy.Figure 4.(A and B) Electromyography of the gastrocnemius muscle from a 3-mo-old Epg5−/− mouse showing fibrillations (A) and positive sharp waves (B). These defects were absent in control mice. (C) At 10 mo, Epg5−/−mice showed high-amplitude and long-duration action potentials when conducting MUAP tests. The blue lines show the start and end of an action potential. Dur, duration; Amp, amplitude. (D-F) H&amp;amp;E staining of gastrocnemius muscles showed features of muscle degeneration in Epg5−/−mice. The arrow in E indicates centrally nucleated fibers. (G) The transcription levels of the atrophy-related genes were up-regulated in gastrocnemius muscles of Epg5−/−mice. (A) Levels of LC3 and p62 in brain and spinal cord extracts from Epg5+/− and Epg5−/−mice. SC, spinal cord. (J) Levels of LC3-II and p62 in muscle extracts from Epg5+/− and Epg5−/− mice. (K) Percentage of p62 aggregate-positive myofibers in Epg5+/− and Epg5−/− mice. Means ± SEM of three mice are shown. (L) Accumulation and colocalization of p62 and ubiquitin aggregates in the gastrocnemius muscles of Epg5−/− mice but not controls. Bars: (D-F and L) 10 µm; (H and I) 1 µm.
Figure 2A Representative kymographs of LysoTracker-labeled lysosomes in proximal dendrites of hippocampal neurons infected with scrambled shRNA (shSc), LAMTOR2 shRNA (shLAMTOR2), or Raptor shRNA (shRaptor). Scale bar, 5 µm. B Quantitative analysis of lysosomal movement from kymographs (N = 44, 24, and 19 neurons for shSc, shLAMTOR2, and shRaptor, respectively, from 3-10 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Sidak's post-test ***P < 0.001 as compared to shSc; n.s., not significant. C, D FRAP analysis of dendritic lysosomal movement in Raptor KD neurons. C Representative images of a neuron at 0 and 4 min after photobleaching. D Quantification of the lysosomes in C undergoing retrograde (red) or anterograde (cyan) transport (N = 7 and 8 neurons for shSc and shRaptor, respectively, from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Mann-Whitney U test ***P < 0.001 as compared to shSc; n.s., not significant. E LAMTOR1 KD did not affect trafficking of vesicles labeled with Alexa 594-conjugated transferrin (red, Tf 594) in dendrites. Shown are dendritic segments (upper) and kymographs (lower). Scale bar, 5 µm. F Quantitative analysis of vesicular movement from kymographs (N = 10 neurons from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Mann-Whitney U test ***P < 0.001 as compared to shSc; n.s., not significant.
Fig.Figure 3(a) Control males were injected with either tesaglitazar (grey syringe) or vehicle twice per week for 4 weeks. Males were paired with control females to generate offspring. (b) Adult weight in the offspring of tesaglitazar-injected males (Tesa-inj, n = 16) compared to the offspring of vehicle-injected males (Vehicle-inj, n = 23). Two-tailed Student's t-test, P = 0.019, t = 2.44, df = 37. (c) Glucose level in the offspring of Tesa-inj (n = 14) and Vehicle-inj (n = 21) males during a glucose tolerance test. Repeated measures ANOVA, treatment effect P = 0.0083, F (1, 33) = 7.877, time effect P < 0.0001, F (4, 132) = 347.7, interaction P = 0.0776, F (4, 132) = 2.155 Conc.; concentration.
Cellular mechanisms of AGR2 secretion.Figure 5A) Formation of GFP-LC3 autophagic puncta in TMED2 overexpressing HEK239T cells as monitored using confocal microscopy (right panel). DAPI was used for nuclear staining visualization. Percentage of GFP-LC3 puncta in control (CTL) and TMED2 overexpressing HEK293T cells as quantified from three independent experiments by counting 240 GFP-LC3 positive cells for each condition (left panel). (**): p=0.0032. B) Western blot detection (left panel) and quantification (right panel) of LC3 level in control and TMED2 overexpressing HEK293T cells treated or not with 50 µM chloroquine for 2 hours. Actin (ACT) served as a loading control. (*): p=0.0498. C) Western blot analysis of AGR2 expression in control and TMED2 overexpressing HEK293T cells upon autophagy inhibition with 20 µM and 10 µM chloroquine treatment. Actin (ACT) served as a loading control. D) HEK293T cells were transfected with control (ev) or TMED2 plasmid and then used in the ERMIT assay with AGR2 WT the presence of gradual amounts of chloroquine. E) Western blot analysis of secreted AGR2 upon TMED2 overexpression. eAGR2, extracellular AGR2; iAGE2, intracellular AGR2. F) Representative pictures of extracellular vesicles (EVs) heterogeneity purified from conditioned media of HEK293T cells transfected with control (ev) or TMED2 plasmid and analyzed by cryo-electron microscopy. Right upper panel: western blot analysis of CD63 in total medium (left) and in the purified extracellular vesicles fraction purified from media conditioned by control and TMED2 overexpressing cells (middle) and western blot analysis of AGR2 levels (right) in extracellular vesicles enriched from culture media conditioned by control and TMED2 overexpressing cells. G) Western blot analysis of secreted AGR2 upon TMED2 silencing eAGR2, extracellular AGR2; iAGE2, intracellular AGR2. H) Quantification of extracellular AGR2 level (eAGR2) in cells silenced for TMED2 (siTMED2), compared to control (siGL2). Data are representative of three independent experiments and are presented as extracellular-to-intracellular AGR2 ratio. (**): p=0.0029. I) Secretion of AGR2 wild-type (wt), E60A and ∆45 mutant as determined by Western blot. eAGR2, extracellular AGR2; iAGE2, intracellular AGR2.
a) prApe1 propeptide recruitment to Atg8-harbouring GUVs in the presence and absence of Atg19.figf5(a) prApe1 propeptide recruitment to Atg8-harbouring GUVs in the presence and absence of Atg19. The experimental set-up is shown on the right. (b) Atg19-dependent envelopment of prApe1-coated beads with Atg8-positive membranes. The experimental set-up is shown on the right. The membrane was labelled by incorporation of a rhodamine-labelled lipid. ( (c) Quantification of the percentage of GUV-associated eGFP-Ape1-coated beads showing detectable membrane bending (N = 3; 40 GUVs per condition). The experimental set-up is shown in b. The density of Atg8-6×His on the membrane was titrated by different amounts of nickel lipids in the GUV membrane. The graph is based on at least three independent experiments. Shown are the averages and s.d. ( (d) Quantification of the percentage of GUV-associated eGFP-Ape1-coated beads showing detectable membrane bending. The quantification is based on three independent experiments for the mutant Atg19 versions and 16 independent experiments for wild-type Atg19 and the no-Atg19 control (Atg19, N = 16, total GUVs = 382; Atg19 W412A, N = 3, total GUVs = 55; Atg19 F376A, F379A, N = 3, total 66 GUVs; Atg19 F376A, F379A, W412A, N = 3, total GUVs = 84; Atg19 F376A, F379A, P385A, E386A, W412A, N = 3, total GUVs = 60; Atg19-34, N = 4, total GUVs = 110; no Atg19, N = 16, total GUVs = 382). Shown are the averages and s.d. The experimental set-up is as shown in b; the p-values in c and d were calculated using a two-tailed Student t-test. (e) Visualization of Atg19 during the envelopment of prApe1-coated beads with Atg8-containing membranes. Note that Atg19 directly binds the prApe1-coated beads (arrows). The experimental set-up is shown on the right. Scale bars, 5 μM. Abbreviations: 8, Atg8; 19, Atg19; DIC, differential interference contrast. All experiments have been conducted at least three times.
Assessment of cone photoreceptor and glial cell types in human retina .Figure 5A) Feature expression heatmap showing the expression of 11 known cone photoreceptor markers across 16 retinal cell clusters. Brown colour indicates ≥10 nTrans (number of transcripts). B) The proportion of cone photoreceptor subtypes identified in population C10, based on expression of OPN1LW / OPN1MW (L/M cones) and OPN1SW (S cones). C) Scatter plots showing expression of OPN1LW/OPN1MW or OPN1SW in individual cone photoreceptors for population C10. The colour depicts expression level for OPN1LW/OPN1MW in individual cells. D) Heatmap of top 20 differentially expressed genes between L/M cones and S cones. The colour depicts normalised gene expression (z-score capped at 2.5). E) Expression pattern of glial markers in Muller glia (C9) and retinal astrocytes (C16). x-axis depicts normalized transcript levels.
Long-term losartan treatment reduces Tgf-β signaling and expression of fibrosis-associated proteins in C7-deficient forepaws.Figure 5Representative Western blots of whole forepaw skin lysates from age-matched wild-type, untreated C7-hypomorphic, and C7-hypomorphic mice treated with losartan for 7 weeks, probed for proteins analyzed in Figs3 and 4. Erk1/2 or β-tubulin was used as a loading control. While losartan did not increase the expression of C7, it effectively attenuated fibrosis by reducing Tgf-β signaling and subsequent Tgf-β-regulated protein expression. Arrows point to bands corresponding to Smad2 and Smad3.Densitometric quantification of Western blots as in (A) from multiple different mice per group (n ≥ 3). Expression was normalized to a loading control (Erk1/2 or β-tubulin) and protein level expressed as percentage of wild-type. Values represent mean ± S.E.M. The data were analyzed by Student's paired t-test; P-values wild-type vs. C7-hypomorph: Tsp1 *P = 0.0490, Tgfbr2 P = 0.0812, P-Smad2/3 *P = 0.0346, fibronectin *P = 0.0115, αSma *P = 0.0038, tenascin-C *P = 0.0228; P-values C7-hypomorph + losartan vs. C7-hypomorph: Tsp1 *P = 0.0415, Tgfbr2 *P = 0.0483, P-Smad2/3 *P = 0.0272, fibronectin **P = 0.0025, αSma **P = 0.0038, tenascin-C *P = 0.0134; P-values wild-type vs. C7-hypomorph + losartan: all not significant. Quantitative real-time PCR (qPCR) analysis of RNA isolated from whole forepaw skin of mice as in (A). Expression of Asma (Acta2), Tgfbr2, Tsp1 (Thsb1), and Tnc normalized to the expression of Gapdh and shown as the percentage of wild-type expression. Losartan treatment downregulated the expression of all four genes that were elevated in untreated C7-hypomorphic mouse paws. The reduction of Asma, Tgfbr2, and Tsp1 did not reach statistical significance in one or two conditions due to large variation in the samples. Values represent mean ± S.E.M., unpaired t-test with Welch's correction used; P-values wild-type vs. C7-hypomorph: Asma P = 0.059, Tgfbr2 P = 0.17, Tnc *P = 0.019, Tsp1 P = 0.098; P-values C7-hypomorph + losartan vs. C7-hypomorph: Asma *P = 0.048, Tgfbr2 P = 0.089, Tnc *P = 0.028, Tsp1 P = 0.087; P-values wild-type vs. C7-hypomorph + losartan: Asma P = 0.81, Tgfbr2 P = 0.36, Tnc P = 0.33, Tsp1 P = 0.81; n ≥ 5 different paws per group.
Figure 4A Top: Sequence alignment of human H2A.B and H2A. Residues not conserved in H2A.B and H2A are colored in red. Secondary structures are indicated above. H2A.B N-terminal domain (NTD) and the docking domain (DD) are indicated. H2A.B residues subject to mutagenesis analyses are indicated as boxes. Bottom: Schematic view of mutants used for MNase analysis. The H2A and H2A.B sequences are indicated by blue and gray, respectively. B,C Effects of H2A mutations (B) and H2A.B mutations (C) on nucleosome resistance to MNase digestion. MNase digested nucleosomal DNA are analyzed by 10% Native-PAGE. All MNase digestion experiments are repeated twice. D,E Graph presentation of effects of H2A mutations (D) and H2A.B mutations (E) on nucleosome resistance to MNase digestion. MNase digestion efficiency for each sample is calculated from quantitation of the digested DNA against total DNA. All experiments are repeated twice. Data are mean ± SD, n = 2. F Structural comparison of H2A.B α2-α3 helices with their H2A counterparts. Structures of H2A-H2B dimer in the canonical nucleosome (yellow, PDB: 3LZ0) and H2A.B-H2B dimer in the H2A.B-NCP (orange) were superimposed for comparison. The dashed lines indicate the ~15 degrees kink of C-terminal region of H2A.B α2 helix.
Figure 1(a) THP-1 cells were transiently infected with BRLF1-expressing or empty control lentivirus. Twenty-four hours later, the control or BRLF1-expressing THP-1 cells were primed with 40 ng/ml TPA overnight and then were either uninfected or infected with HSV-1 (MOI=1) in serum-free medium for 12 h. The supernatants and whole cell lysates were collected and analyzed by western blotting to detect pro-caspase-1 (p45), cleaved caspase-1 (p20), pro-IL-1β, mature IL-1β, pro-IL-18 and mature IL-18 as indicated. The release of mature IL-1β (b) from THP-1 cells were measured by enzyme-linked immunosorbent assay (ELISA) following HSV-1 infection. The release of IL-18 (c) from THP-1 cells were measured by enzyme-linked immunosorbent assay (ELISA) following HSV-1 infection. Ramos cells were infected with wild type EBV-WT or BRLF1-deficient EBV-ΔBRLF1 virus at a high titer (MOI=100). After being transduced with empty control or BZLF1-expressing lentivirus, the supernatants and cell pellets of EBV-WT- or EBV-ΔBRLF1-harboring Ramos cells were collected after 12 h in serum-free culture, and then the levels of EBNA1, BRLF1, BZLF1, pro-caspase-1 (p45), cleaved caspase-1 (p20), pro-IL-1β, mature IL-1β, pro-IL-18, mature IL-18, GSDMD and cleaved N-GSDMD in the supernatants and whole-cell lysates were analyzed as indicated (d). The release of mature IL-1β (e) from Ramos cells were measured by enzyme-linked immunosorbent assay (ELISA). The release of mature IL-18 (f) from Ramos cells were measured by enzyme-linked immunosorbent assay (ELISA). (g) After being transduced with control or BZLF1-expressing lentivirus for 48 h, EBV-WT- and EBV-ΔBRLF1 infected Ramos cells were stained with PI and measured using fluorescent flow cytometry. Representative images of pyroptotic cells are shown and the percentages of pyroptotic cells were calculated in duplicate.