EMBO/soda-nel huggingface dataset:
Inclusion of cGAMP within virus-like particle vaccines enhances their immunogenicity (2021)
Figure 7

Fig 7: cGAMP-VLPs containing the SARS-CoV-2 S protein induce an enhanced antibody response.

A. cGAMP-S-VLPs and Empty-S-VLPs incorporate SARS-CoV-2 S. Lysates from VLP producer cells and VLP preparations were analysed by western blot for the presence of the S2 subunit of SARS-CoV-2 S, Gag-GFP and actin using the indicated antibodies.

B, C. Immunisation with cGAMP-S-VLPs augments anti-Spike and anti-RBD antibody titres. Mice were immunised via the intra-muscular route with 5x105 IU of cGAMP-S-VLPs, Empty-S-VLPs, or PBS as a control. Three weeks after immunisation, sera were collected, heat-inactivated, and titres of antibodies capable of binding to SARS-CoV-2 S (B) or its RBD (C) were determined by ELISA. The antibody response was expressed as Endpoint titre defined as the reciprocal of the highest serum dilution that gives a positive signal (blank+10SD). The dotted line shows the limit of detection (LOD).

D. Immunisation with cGAMP-S-VLPs induces neutralising antibodies. Using serum samples from (B), antibody titres capable of neutralising SARS-CoV-2 were determined by microneutralisation (MN) assay. Calculated IC50 doses from multiple serum dilutions are shown. The dotted line shows the LOD.

Data information: Panel (A) is representative of two independent experiments. In (B-D), data were pooled from two independent experiments including a total of 12 mice per condition; symbols show data from individual animals. Horizontal lines indicate the mean. Statistical analyses were done using a Kruskall-Wallis test followed by Dunn's multiple comparisons test (B, C, D). *p<0.05; ****p<0.0001. See also Fig EV5.

Expanded View figure legends
Sororin loads to the synaptonemal complex central region independently of meiotic cohesin complexes (2016)
Figure 1

Figure 1. Sororin distribution during prophase I.

A-E Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread spermatocytes at (A) zygotene (Zyg.), (B) pachytene (Pac.), (C) diplotene (Dip.), (D) late diplotene (L. dip.), and (E) prometaphase I (ProM I).

F-H Double-immunolabeling of Sororin (green) and SYCP1 (pseudocoloured in pink) on spread spermatocytes at (F) zygotene, (G) pachytene, and (H) diplotene.

I-K Double-immunolabeling of Sororin (green) and HORMAD1 (pseudocoloured in blue) on spread spermatocytes at (I) zygotene, (J) pachytene, and (K) diplotene. The sex bivalent (XY) and their pseudoautosomal regions (arrows), as well as the round body (arrowheads) are indicated. Scale bar: 10 µm.
Sororin loads to the synaptonemal complex central region independently of meiotic cohesin complexes (2016)
Figure 2

Figure 2. Sororin distribution in comparison with cohesins during prophase I.

A,C Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread spermatocytes at (A) early zygotene (E. zyg.) and (C) late zygotene (L. zyg.).

B,D Double-immunolabeling of SMC3 (pseudocoloured in cyan) and SYCP3 (red) on spread spermatocytes at (B) early zygotene and (D) late zygotene.

E Double-immunolabeling of Sororin (green) and REC8-myc (pseudocoloured in purple) on a spread mid zygotene spermatocyte. The sex univalents (X,Y) are indicated. Scale bar: 10 µm.
Sororin loads to the synaptonemal complex central region independently of meiotic cohesin complexes (2016)
Figure 3

Figure 3. Sororin distribution in Syce3-/-, Sycp1-/-, Rec8-/-, Smc1β-/- and Spo11-/- spermatocytes.

Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread pachytene-like (Pac.-like) and zygotene-like (Zyg.-like) spermatocytes of different genotypes. Unsynapsed axial elements of sex chromosomes (X,Y) are indicated. Arrowheads indicate nucleolar-associated round bodies. Scale bar: 10 µm.
Sororin loads to the synaptonemal complex central region independently of meiotic cohesin complexes (2016)
Figure 4

Figure 4. Sororin distribution during the meiotic divisions.

Double-immunolabeling of Sororin (green) and SYCP3 (red), and counterstaining of the chromatin with DAPI (blue) on spread spermatocytes at (A) metaphase I (M I), (G) anaphase I (A I), (H) interkinesis (Int.), (I) prophase II (Pro. II), and (J) metaphase II (M II). The sex bivalent (XY) is indicated. Arrows indicate the centriolar area at cell poles. Arrowheads indicate the Sororin labeling at the centromere of Y chromosome.

B-F Selected metaphase I autosomal (B and C) and sex (E and F) bivalents. The sex chromosomes (X,Y) are indicated.

K,L Enlarged metaphase II centromeres. Scale bar: 10 µm.
Sororin loads to the synaptonemal complex central region independently of meiotic cohesin complexes (2016)
Figure 5

Figure 5. Relative distribution of Sororin and kinetochores during the meiotic divisions.

Double-immunolabeling of Sororin (green) and kinetochores (ACA serum, red), and counterstaining of the chromatin with DAPI (blue) on spread spermatocytes at (A) metaphase I (M I), (B) metaphase II (M II), and (C) anaphase II (A II). Arrowheads indicate the cell poles. The centromere of the Y chromosome and chromatids (Y) is indicated. Scale bar: 10 µm.
Sororin loads to the synaptonemal complex central region independently of meiotic cohesin complexes (2016)
Figure 6

Figure 6. Distribution of Sororin in Sgo2-/- knockout spermatocytes.

Double-immunolabeling of Sororin (green) and SYCP3 (red), and counterstaining of the chromatin with DAPI (blue) on spread Sgo2-/- spermatocytes at (A) pachytene (Pac.), (B) diplotene (Dip.), (C) metaphase I (M I), and (D) metaphase II-like (M II-like). The sex bivalent (XY) is indicated. Arrowheads indicate round bodies. Arrows in (A) indicate the pseudoautomal region in the sex bivalent, and the centriolar area at cell poles in (C,D). Scale bar: 10 µm.
Sororin loads to the synaptonemal complex central region independently of meiotic cohesin complexes (2016)
Figure 7

Figure 7. Distribution of Sororin in okadaic acid treated spermatocytes.

Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread cultured wild-type control (WT Control), and okadaic acid treated wild-type (WT + OA) and Smc1β-/- (Smc1β-/- + OA) spermatocytes.

A-B WT control cultured spermatocytes in (A) late zygotene (L. zyg.) and (B) metaphase I (M I). Arrows indicate the cell poles.

C-D WT cultured spermatocytes treated with okadaic acid in (C) late zygotene (L. zyg.) and (D) metaphase I-like (M I-like).

E-F Smc1β-/- cultured spermatocytes treated with okadaic acid in (E) pachytene-like (Pac.-like) and (F) metaphase I-like (M I-like). Arrowheads indicate nucleolar-associated round bodies. The unsynapsed axial elements of the sex chromosomes (X,Y) in prophase I, as well as the sex bivalent (XY) in metaphases I are indicated. Scale bar: 10 µm.
A novel P2X2-dependent purinergic mechanism of enteric gliosis in intestinal inflammation (2020)
Figure 1

Fig 1: ATP induces a gliosis in msEGCs

A: Volcano plot showing significantly regulated genes between control and ATP-treated msEGCs.

B: Visual representation of GO terms associated with enriched genes in ATP-treated msEGCs compared to control.

C-F: Heat maps of indicated GO terms in ATP-treated msEGCs compared to control.

G-I: qPCR analysis of indicated gliosis genes in ATP-treated EGCs.

J: qPCR analysis of IL-6 in msEGCs that were treated for 6h with ATP.

K: IL-6 protein levels in supernatants from msEGCs collected after 24h treatment with ATP.

Data information: In (A) data is shown as fold change > 1.5, p-value < 0.05; (n=5 for untreated and n=6 for ATP-treated EGCs). In (G-K) data are shown as fold change +SEM. (G-I) n=6-9, msEGCs. (J) n=4-9, msEGCs. (K) n=6-18, msEGCs. In (A-I): ATP concentration was 100µM. In (J, K) ATP concentration was 1, 10 or 100µM, Statistics were performed by applying unpaired Student´s-t-test (G-K) and/or one-way ANOVA with a subsequent Bonferroni test (J and K). In (A) a limma-trend pipeline model and in (B) the Fisher´s exact test were performed. * indicates significance to control, # indicates significance to ATP-treatment with * / # =p<0.05, ** / ## =p<0.01 and *** / ### =p<0.001.
A novel P2X2-dependent purinergic mechanism of enteric gliosis in intestinal inflammation (2020)
Figure 2

Fig 2: ATP-induced gliosis is mediated by p38-MAPK and selective purinergic signaling

A: Effect of P2 receptor antagonism on ATP induced IL-6 release. Cells were treated with P2-antagonist suramin (1, 10 and 100µM) alone or together with ATP (100µM) for 24h.

B: ATP induced IL-6 release in msEGCs measured by ELISA. Cells were treated with the indicated concentrations of ATP-and ATPɣS for 24h.

C: Effects of P2X antagonists on ATP induced IL-6 release. Cells were treated for 24h alone or together with ATP (100µM) in absence or presence of P2X2, P2X4 and P2X7-antagonists PSB-1011, 5-BD-BD and A740003, respectively.

D: P2X2 antagonism of ATP induced mRNA expression of IL-6, GFAP and RCAN by qPCR in msEGCs. Cells were treated with the P2X2-antagonist PSB-1011 (20µM) alone or together with ATP (10µM) for 6h.

E: Representative confocal images of P2X2 (green) and GFAP (violet) positive msEGCs in vivo and in vitro. White arrows mark double positive (white) cells. Scale bar 50µm.

F: P2X2-siRNA reduces P2X2-mRNA and dampens the gliosis gene expression after ATPɣS (100µM) treatment for 6h.

G: P2X2-siRNA reduces IL-6 release after ATPɣS treatment (10µM, 100µM) for 6h.

Data information: In (A-D and F) data is shown as fold induction +SEM and (G) as IL-6 pg/ml +SEM, (A): n=10-15, msEGCs; (B): n=3-15, msEGCs; (C): n=8-17, msEGCs; (D): n=4, msEGCs; (E): n=3-5, msEGCs; (F): n=3-5, msEGCs; (G): n=3-5, msEGCs. Statistics in (A-D, F, G) were performed by applying unpaired Student´s-t-test and/or one-way ANOVA with a subsequent Bonferroni test. * indicates significance to control, # indicates significance to ATP-treatment with * / # =p<0.05, ** / ## =p<0.01 and *** / ### =p<0.001.
A novel P2X2-dependent purinergic mechanism of enteric gliosis in intestinal inflammation (2020)
Figure 3

Fig 3: An ATP-induced enteric gliosis in postoperative bowel inflammation

A: ATP measurement at the indicated postoperative time points in peritoneal lavages of mice that underwent intestinal manipulation (IM) or were naïve.

B: PCA plot of gene expression from POI mice at different disease stages and naïve mice; n=3 for each group.

C: Visual representation of p-value (-log10) against fold enrichment of GO terms associated with enriched genes in mice that underwent IM.

D: Heat map of enriched genes connected with GO term gliosis in mice that underwent IM or in naïve animals.

E: Venn diagram of gliosis genes expressed in vitro and in vivo.

F-H: qPCR analysis of indicated gliosis marker in mice that underwent IM (n=7).

I: Histological analysis of EGC proliferation in vivo. Representative confocal images and quantification show Sox10 (green) and Ki67 (violet) positive EGCs (white arrows) in the small bowel muscularis externa at naïve, IM6h and 24h. Scale bar 50µm.

J: Experimental setup for glia specific RNA analysis. GFAP-cre+ x Ai14-floxed mice underwent IM and small intestine ME was digested and sorted for tdTomato by FACS to provide glia-specific RNA for qPCR measurements seen in (K-M).

K-M: qPCR analysis of indicated gliosis markers in td+ glia cells from naïve mice and mice that underwent IM (n=5-7, POI mice).

Data information: In (A) data are represented as mean +SEM; n=7-12, POI mice. In (F-H and K-M) data are represented as fold induction +SEM. In (I) data are represented as mean of double positive cells per total Sox10 positive cells +SEM; 9-20 whole mount specimens per conditions; n=8, POI mice per group. Statistics were performed by applying unpaired Student´s-t-test (A, C, F-I, K-M). In (C) the Fisher´s exact test was performed. * indicates significance to naïve animals, # indicates significance to the indicated time point with * / # =p<0.05, ** / ## =p<0.01 and *** / ### =p<0.001.
A novel P2X2-dependent purinergic mechanism of enteric gliosis in intestinal inflammation (2020)
Figure 4

Fig 4: The novel P2X2 antagonist ambroxol prevents IL-6 release from EGCs

A: Ambroxol treatment scheme. Mice were treated with ambroxol or vehicle and underwent a sham operation (laparotomy) or intestinal manipulation (IM). Small bowel muscularis externa (ME) was isolated and analyzed 3h or 24h after surgery.

B-D: Postoperative gene expression analyses of indicated gliosis marker in the ME.

E: Histological analysis of EGC proliferation by quantification of Ki67 (violet) and Sox10 positive EGCs at IM24h. Mice were treated as seen in (A).

F: Histological counting of infiltrating myeloperoxidase (MPO)-positive leukocytes in the postoperative ME (n=6-8).

G: FACS analysis of infiltrating cells in the ME of mice treated with ambroxol or vehicle. CD45, Ly6C and CX3CR1 were used to distinguish resident macrophages (CD45+/Ly6C-/CX3CR1+), infiltrating monocytes (CD45+/Ly6C+/CX3CR1-) and infiltrated monocyte-derived macrophages (CD45+/Ly6C+/CX3CR1+).

H: Postoperative in vivo GI transit measurement in mice treated with ambroxol or vehicle.

Data information: In (B-D) data are represented as fold change +SEM versus the sham groups (n=6-8, POI mice per group). In (E) data are represented as mean of double positive cells per total Sox10 positive cells +SEM; 6 whole mount specimens per conditions; n=11 mice per IM group and n=3 per sham group. In (F) data is presented as mean +SEM MPO+ cells per mm² small intestine ME tissue. In (G) data is presented as cells per 100mg ME tissue +SEM n= 3-5 POI mice per group. In (H) data is presented as mean +SEM; n= 12 mice per group. Statistics were performed by applying unpaired Student´s-t-test and one-way ANOVA with a subsequent Bonferroni test (B-H). * indicates significance compared to sham animals, # indicates significance between vehicle and ambroxol treatment with * / # =p<0.05, ** / ## =p<0.01 and *** / ### =p<0.001.
A novel P2X2-dependent purinergic mechanism of enteric gliosis in intestinal inflammation (2020)
Figure 5

Fig 5: ATP induces gliosis in hEGC

A: Schematic workflow on the collection and processing of surgical specimens collected during a pancreaticoduodenectomy. Samples were provided on ice directly from the operation room and the ME was separated from lamina propria mucosae.

B: Visual representation of p-value (-log10) against fold enrichment of GO terms associated with enriched genes in patient specimen that underwent IM.

C: Heat map of enriched genes connected with GO term gliosis in patient specimens that underwent IM at two time points: early and late; n=3 human patients.

D: Venn diagram of gliosis genes expressed in human (red) and murine (blue) ME-tissue.

E: IL-6 protein release in hEGC cultures upon stimulation with ATP (200µM) or ATPɣS (100µM) after 6h and 24h treatment (n=4-6, hEGCs).

­F: IL-6 protein release in hEGC cultures upon stimulation with NTPDase inhibitor ARL67156 at indicated concentrations; n=4-6, hEGCs.

G: Immunofluorescence microscopy revealed P2X2 expression (green) in a majority of s100β+ (violet) hEGCs in intact myenteric ganglia of the human colon. White arrows mark double positive glia cells and green arrows mark P2X2 positive neurons. Scale bar, 50µm.

H: Effect of the P2X2 antagonism on ATP-induced IL-6 release. ELISA measurement of IL-6 in hEGCs upon treatment with P2X2-antagonist PSB-1011 (20µM) alone or together with ATP (200µM) treated for 24h (n=6, hEGCs).

I: PSB-1011 (20μM) treatment inhibited ATP-triggered calcium responses in HEK cells transfected with P2X2. Data are represented as ΔF/F0 +SEM; n=102 HEK cells.

J: The P2X2 receptor antagonist PSB-0711 (20μM) nearly abolished the ATP-triggered calcium response in HEK cells transfected with P2X2. n=219 HEK cells.

K: Ambroxol blocks ATP-induced IL-6 release in hEGCs; Protein release measurement by ELISA of IL-6 in hEGCs. Cells were treated with ATP (200 µM) alone or together with ambroxol (20 µM) for 24h; n=6, hEGCs.

L-N: qPCR analysis of several gliosis and ATP-target genes in the mechanically manipulated surgical specimens incubated in the presence or absence of ambroxol (20µM). (n=7, 4 human patients).

Data information: In (E, F, H and K) data are represented as mean IL-6 release +SEM. In (I and J) data are represented as ΔF/F0 +SEM. In (L-N) data is shown as fold induction +SEM. Statistics were performed by applying unpaired Student´s-t-test and one-way ANOVA with a subsequent Bonferroni test (E and F, H-N), and in (B) the Fisher´s exact test was performed. * indicates significance compared to controls, # indicates significance to ATP treatment or between vehicle and ambroxol with * / # =p<0.05, ** / ## =p<0.01 and *** / ### =p<0.001.
Myeloid leukemia with transdifferentiation plasticity developing from T-cell progenitors (2016)
Figure 1

Figure 1. Single DN2 cells possess malignant myeloid and T-lymphoid potential.

A. Representative FACS plot (left) showing a mixed-lineage clone with a T-lymphoid (CD25+/CD11b–Gr1–), myeloid (CD11b+Gr1+/CD25–), and biphenotypic (CD11b+Gr1+/CD25+) fraction derived from a single Myc/Bcl2-transformed DN2 cell grown on 1:1 OP9:OP9-Dll1 co-culture with lineage-promiscuous cytokines IL-2, IL-3, IL-6, IL-7, SCF,GM-CSF, Flt3. Morphology of the indicated flow-sorted cell fractions was analyzed by Giemsa staining (right). B. Phenotype frequencies of clones from Myc/Bcl2-transformed single cells (grown and phenotyped as mentioned in A). Analyzed were 31 clones from LSKs, 107 from GMPs and 28 from DN2 cells. C. Cumulative survival of 31±2 and 27±6 days of mice that received 5104 to 5105 T-lymphoid cells or myeloid cells, respectively, that were flow-sorted from mixed Myc/Bcl2+ DN2-derived clones. The summarized data are from 3 mice injected with the T-lymphoid and 4 mice injected with the myeloid fraction of two different clones. D. Representative FACS plots demonstrating predominant outgrowth of myeloid clones from single Myc/Bcl2-transformed LSK or GMP cells on OP9:OP9-Dll1 co-cultures. E. (top panels) Serial replating of Myc-Bcl2+ DN2 cells in methylcellulose generated myeloid clones with stable genomic reassembly of the Dβ1 TCR locus in vitro (gel: left two lanes). Upon transplantation, the same clones induced myeloid leukemia that retained the initial rearrangement in vivo (gel: right two lanes) as assessed by nested PCR. GL = germline. (bottom panels) Methylcellulose-based replating of Myc-Bcl2+ GMPs generated myeloid clones that exclusively displayed the TCRß locus in germline configuration. F. Cumulative survival of mice that received 106 cells of TCRβ-rearranged myeloid DN2-derived clones leading to death within 33±4 days after transplantation. Two DN2 clones were transplanted into 5 recipient mice.
Myeloid leukemia with transdifferentiation plasticity developing from T-cell progenitors (2016)
Figure 2

Figure 2. Myc/Bcl2+ DN2 cells give rise to clonal myeloid and T-lymphoid leukemia in vivo.

A. Representative FACS plots of spleens from leukemic recipient mice that had received LSK, GMP or DN2 cell grafts immediately after retroviral Myc/Bcl2 transduction. Numbers indicate frequencies (%) of CD45.2 donor cells within the indicated gates summarizing the mean±SEM of at least 8 recipients per group from a total of 10 independent experiments. B. Cumulative survival of mice that received 2104 to 2105 freshly Myc/Bcl2-transduced LSK, GMP or DN2 cells; Graph shows n=11 Myc-Bcl2+ LSK-, n=14 DN2-, and n=8 GMP-transplanted animals. C. Histological spleen sections of Myc-Bcl2+ LSK-, GMP- or DN2-transplanted mice displaying myeloperoxidase staining. Leukemic cell engraftment in spleens ranged from 75.1%-95.5%. D. Multiplex quantitative RT-PCR demonstrating strong expression of PU.1 in the myeloid (M3L: CD11b+Gr1+/CD3–CD4–CD8–) and biphenotypic T/myeloid (BL: CD11b+Gr1+/CD3+CD4+CD8+) fractions but not in the T-lymphoid (TL: CD11b–Gr1–/CD3+CD4+CD8+) fraction that were flow-sorted from diseased Myc-Bcl2+ DN2-transplanted mice. Myeloid progeny of Myc/Bcl2+ GMP-transplanted or T-lymphoid progeny of Myc/Bcl2+ DN3-transplanted animals as well as healthy thymocytes (Thy) and BM cells from WT mice served as controls. Results are shown as fold over actin transcripts. E. D-J rearrangement PCR of flow-sorted
T-lymphoid (TL), biphenotypic (BL) and myeloid (ML) fractions of donor splenocytes of a diseased Myc/Bcl2+ DN2-transplanted mouse. All three fractions revealed a stable genomic reassembly of the Dβ1 TCR locus (Dβ1Jβ1.5) demonstrating clonality in DN2 leukemia. thy = thymus. F. FACS plot of diseased intrathymic grafts (CD45.2-gated), demonstrating that Myc/Bcl2+ DN2 cells could produce multi-lineage leukemia within a T-cell supporting environment; Myc/Bcl2+ GMPs remained myeloid restricted under these conditions. Numbers indicate cell frequencies within the gates (mean±SEM) representing six independent experiments. G. Intrathymic transplantation of mock-infected non-leukemic DN2 cells produced predominantly T-lymphoid but also myeloid progeny 23 days after injection. This differentiation outcome resembles that of freshly isolated DN2 cells (Bell and Bhandoola, 2008; Richie Ehrlich et al., 2011), and indicates that the short-term culture necessary for retroviral transduction did not interfere with the physiological lineage potential of DN2 cells.
Myeloid leukemia with transdifferentiation plasticity developing from T-cell progenitors (2016)
Figure 4

Figure 4. Bcl11b and Gata3 are required for DN2-leukemia.

A. Western blot illustrating abundant Bcl11b and Gata3 protein expression in Myc/Bcl2+ DN2- but not LSK- or GMP-derived blasts. Thymus (Thy) and bone marrow (BM) extracts from healthy WT mice served as controls. Detection of α-tubulin or valosin-containing protein (VCP) ensured comparable loading. B,C. Proliferation assays illustrating growth of Myc/Bcl2+ DN2- (B) or GMP-derived (C) blasts after lentiviral transduction with the indicated shRNAs. The shRNA-transduced cells were flow-sorted based on co-expression of the RFP-reporter and seeded on OP9/OP9Dll1 1:1 layers. Growing cells were RFP-sorted for 3 rounds within 21 days of culture and percent growth was calculated over the initially seeded cell number in relation to the Luc control. Shown are results of 4 independent experiments with cells from 4 different DN2- and 3 different GMP-leukemic animals. Luc= control shRNA targeting luciferase (Suzuki et al., 2003). At least 2 different shRNAs were used against Bcl11b or Gata3 as indicated. * = p≤0.05, Student’s t-test. D. Representative FACS plots showing CD45.2-donor splenocytes of a secondary (CD45.1) recipient 23 weeks after transplantation with 4103 RFP+ Myc-Bcl2+ DN2-leukemia blasts carrying the indicated shRNAs. Luc: luciferase control shRNA. The data are representative of 2 Gata3, 4 Bcl11b, and 2 luciferase shRNA-transplanted animals.