EMBO/soda-nel huggingface dataset:
SARS-CoV-2 nucleocapsid suppresses host pyroptosis by blocking Gasdermin D cleavage (2021)
Figure 6

Figure 6. SARS-CoV-2 inhibits IL-1β secretion in human monocytes.

(A-D) GSDMD-/- THP-1 cells rescued with wild-type or linker substituted GSDMD were transfected with plasmids encoding nucleocapsid or control vector and then stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were immunoblotted with antibodies against the indicated proteins (A) and supernatants were subjected to an ELISA assay to determine IL-1β levels (B). Otherwise, cell viability in cell pellets was examined using a cell viability assay through checking ATP levels inside cells (C). Cells were stained with propidium iodide (PI) and the percentage of PI positive cells was calculated (D). For B-D, Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results.

(E, F) GSDMD-/- THP-1 cells rescued with wild-type or linker substituted GSDMD were infected with SARS-CoV-2 at an MOI of 1 for 1h, followed by washing away extracellular viruses and further culture for 3 h. Otherwise, cells were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants were subjected to an ELISA assay to determine IL-1β levels (E). Otherwise, cell viability in cell pellets was examined using a cell viability assay through checking ATP levels inside cells (F). For E, F, Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results.

(G) Sorted CD14+ monocytes from donors were infected with SARS-CoV-2 at an MOI of 1 for 1h, followed by washing away extracellular viruses and further culture for 3 h. Cells were immunoblotted with antibodies against the indicated proteins. CD14+ monocytes from healthy donors were treated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h and served as a positive control.

(H) Donor monocytes were infected with SARS-CoV-2 at an MOI of 1 for 1h, followed by washing away extracellular viruses and further culture for 3 h. Supernatants were collected and subjected to a proteome profiling using an antibody array. Intensities for each cytokine were normalized individually and shown together as scores for both dot size and color. LPS and nigericin treated healthy donor monocytes served as a positive control. n=6 for each group.

Data information: For A-D, Control vector used here was empty vector corresponded to the nucleocapsid-containing vector. For B-F, Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. For A-G, experiments were repeated for three time with similar results.
TATA and paused promoters active in differentiated tissues have distinct expression characteristics (2021)
Figure 3

Figure 3: scRNA-seq reveals differences in expression characteristics of TATA and paused genes.

(A) The differences in cell-to-cell gene expression variability for the TATA and the paused effector gene groups were analyzed using the scRNA-seq data. The coefficient of variation (standard deviation/mean) of gene expression was calculated for all genes in the tissue with the highest expression for each gene. The median coefficient of variation was consistently lower for the paused genes compared to the TATA genes.

(B) The frequency of cells with any detectable expression (> one read) was calculated in tissues with the highest expression for each gene (expressing tissue) and in five other tissues with the least expression for each gene (other tissues). The frequency of cells with detectable expression in the expressing tissues, a measure of expression robustness, is lower for the TATA genes compared to the highly paused genes (left) (Wilcoxon two-sided test, *P <10-15). The frequency of cells with detectable expression in the other tissues, a measure of background expression, is also lower for the TATA genes compared to the paused genes (right) (Wilcoxon two-sided test, *P <10-15).

(C,D) Normalized gene expression levels (read count normalized for each cell, log2) in different tissues, from the scRNA-seq experiment, for a TATA group gene, and a highly paused group gene, are shown. (C) The TATA gene, Ccp84Aa, shows noisy expression in the epidermis, without detectable background expression in non-expressing tissues. (D) The highly paused gene, Gip, shows very robust expression in crystal cells, but has high background expression in the non-expressing tissues.

(E) The number of annotation terms associated with each gene in the BDGP in situ database. This is a measure of whether the expression of a gene is restricted to specific subsets of tissue (Wilcoxon two-sided test, *P <10-15).

(F) The coefficient of variation across different isogenic lines of Drosophila from DGRP, after being corrected for dependence on expression (loess regression) is plotted for different effector gene groups (Sigalova et al. 2020). Genes from the TATA enriched group show high variability compared to the paused genes (Wilcoxon two-sided test, *P <10-15). Boxplots in all panels show the median as the central line, the first and the third quartiles as the box, and the upper and lower whiskers extend from the quartile box to the largest/smallest value within 1.5 times of the interquartile range.
TATA and paused promoters active in differentiated tissues have distinct expression characteristics (2021)
Figure 4

Figure 4: Paused genes are more accessible than the TATA genes.

(A) Average read-count normalized MNase signal (RPM) from 2-4 h and 14-17 h embryos are shown at the different effector gene promoter groups. The grey area highlights the changes in nucleosome occupancy, which are prominent at highly paused genes (arrow).

(B) Chromatin accessibility is shown as the average read-count normalized ATAC-seq signal (RPM) from 14-17 h for each promoter group. Paused genes show higher accessibility at the promoter region than the TATA genes.

(C, D) ATAC-seq chromatin accessibility was measured in different tissues isolated from 14-17 h embryos using INTACT. (C) Read-count normalized ATAC (RPM) signal at individual genes from the TATA group (Ccp84Aa) and highly paused gene group (ect) are shown. (D) Read-count normalized ATAC signals (RPM) from different tissues were calculated for each gene from 150 bp upstream of the TSS to the TSS. Paused genes show higher average accessibility across all tissues compared to the TATA genes (Wilcoxon two-sided test, *P <2.2*10-16).
Endophilin-A regulates presynaptic Ca2+ influx and synaptic vesicle recycling in auditory hair cells (2018)
Figure 1

Figure 1. Endophilin-A expression in the cochlea

(A) Schematic domain overview of endophilins-A1-3, highlighting the BAR and SH3-domains.

(B-B') Schematic overview of the sample collection procedure for single-cell PCR (scPCR). Single IHC cytoplasms from acutely dissected organs of Corti of C57BL/6J (Wt) mice after hearing onset were aspirated and processed for scPCR as depicted. (B') Expression analysis of endophilins-A1-3 from individually isolated IHC cytoplasms using RT-PCR from a representative experimental run. Please note that for these experiments, negative bath control samples from before and after the isolation procedure were an essential requirement to ensure lack of contamination from cellular debris in bath solution. HPRT was used as a housekeeping gene.

(C-C') Immunoblotting of tissue lysates from p15 Wt (C57BL/6J), 1-SKO, 1/2-DKO and 1/3-DKO revealed protein expression of endophilins-A1 and -A2 in the murine cochlea and ensured antibody specificity. Unfortunately, none of the commercially available endophilin-A3 antibodies we tested in these experiments gave a specific signal for A3 in cochlear extracts, but rather appeared to (also) detect A1. γ-adaptin was used as loading control. All antibody epitopes localize to the distinct C-terminal regions of the different endophilin-A family members. Hi, hippocampus; Ce, cerebellum; Co, pooled cochleae from a single individual of the indicated genotype; Md, modioli (micro-dissected and pooled from 10 Wt animals); OC, organs of Corti (micro-dissected and pooled from 10 Wt animals).
Endophilin-A regulates presynaptic Ca2+ influx and synaptic vesicle recycling in auditory hair cells (2018)
Figure 3

Figure 3. Disruption of endophilins reduces Ca2+-influx of IHCs after hearing onset

(A) Ca2+-current-voltage relationships in response to 10 ms step-depolarizations. (A') The peak of Ca2+-influx was significantly reduced in endophilin 1-SKO (**p = 0.0024) and 1/3-DKO (***p < 0.0001) when compared to Wt. (Wt n = 30/ N = 20; 1-SKO n = 15/ N = 9; 1/3-DKO n = 39/ N = 20; Non-parametric K-W with post-hoc Dunn's correction).

(B) Quantification of CtBP2-labeled ribbons revealed a number of approximately 14-15 ribbon synapses per IHC in all groups, that remained comparable across all genotypes (Wt n = 12/ N = 9; 1-SKO n = 6/ N = 4; 1/3-DKO n = 8/ N = 6; one-way ANOVA, p = 0.717).

(C) Quantification of the cross-sectional area of CaV1.3 immunofluorescence revealed approx. 34 % smaller clusters in 1-SKOs and 24 % smaller clusters in 1/3-DKOs. (Wt n = 153/ N = 2; 1-SKO n = 102/ N = 2; 1/3-DKO n = 441/ N = 4; ***p < 0.0001; K-W and Dunn's post-hoc test).

(D) Representative maximum projections of confocal sections from organs of Corti of p15 Wt, 1-SKO, and 1/3-DKO mice stained for CtBP2 to label synaptic ribbons. Please note that CtBP2 expression is also found in the nucleus (nuclei are highlighted by dashed circles in the individual panels). Scale bars: 10 µm.

(E) 2D STED images of IHC ribbon synapses stained for CtBP2 (magenta) and CaV1.3 (green). CaV1.3 Ca2+-channels remain clustered at AZs of 1-SKO and 1/3-DKO IHCs. Scale bars: 1 µm.

Data information: box plots in (A'-C) illustrate the median with the interquartile range, whiskers indicate 10-90 % of data points, the squares present the respective mean value.
Endophilin-A regulates presynaptic Ca2+ influx and synaptic vesicle recycling in auditory hair cells (2018)
Figure 5

Figure 5. Disruption of endophilins causes a reduction of IHC otoferlin levels

(A) Confocal maximum projections of otoferlin-immunolabeled organs of Corti from p15 Wt and 1/3-DKO mice illustrated with an intensity-coded look-up table where brighter colors indicate higher intensity. Scale bars: 5 µm.

(B) Otoferlin levels were reduced by approx. 28.4 ± 3.5 % in 1/3-DKO IHCs compared to Wt IHCs (relative intensities normalized to Wt; Wt n = 31 images (229 IHCs)/ N = 5 organs of Corti (3 animals); 1/3-DKO n = 20 images (196 IHCs)/ N = 5 organs of Corti (4 animals); unpaired student's t-test; ***p < 0.001; box plots illustrate the median with the interquartile range, whiskers indicate 10-90 % of data points, the squares present the respective mean value).

(C) Normalized otoferlin intensity line profiles through single IHCs of the respective genotypes at a longitudinal central plane through the nucleus from apical (0.0) to basal (1.0) revealed similar distribution patterns of otoferlin in IHCs of Wt and 1/3-DKO and an overall reduction of otoferlin levels throughout the entire cell in 1/3-DKOs. Traces indicate the mean pixel intensity ± SEM; Wt n = 123/ N = 3; 1/3-DKO n = 124/ N = 3.

(D-E) Otoferlin interacts with endophilin-A1. (D, D') Interaction of otoferlin and endophilin-A1 detected by co-IP in HeLa cells co-expressing GFP-otoferlin and RFP-endophilin-A1. Otoferlin-GFP was immunoprecipitated (IP) by GFP-Trap beads, and blots were probed with a KO-validated anti-endophilin-A1 antibody. (E, E') Exogenously overexpressed GFP-otoferlin was immunoprecipitated using GFP-Trap beads and incubated with purified endophilin-A1 (pA1). IP was then followed by immunoblotting with an anti-endophilin-A1 antibody.
Endophilin-A regulates presynaptic Ca2+ influx and synaptic vesicle recycling in auditory hair cells (2018)
Figure 9

Figure 9. High K+ stimulation leads to accumulations of ELVs and increased occurrence of coated pits at ELVs in endophilin 1/2-DKOs

(A) Representative electron micrographs of Wt and 1/2-DKO specimen indicate accumulations of ELVs and high numbers of coated pits forming from the ELVs in ribbon proximity (radius r = 1 µm from the center of the ribbon) in the DKO after 15 min stimulation with extracellular solution supplemented with 50 mM K+. Scale bars: 200 nm.

(B) Significantly reduced numbers of RA-SVs in IHCs of 1/2-DKO compared to Wt after stimulation, as quantified using random ultrathin sections of ribbon-occupied AZs from several IHCs per genotype. Mann-Whitney test; ***p < 0.001.

(C) Numbers of MP-SVs were slightly reduced in stimulated Wt compared to stimulated 1/2-DKOs, where SV numbers closely matched the ones of unstimulated 1/2-DKO. Mann-Whitney test; *p < 0.05.

(D) Reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1/2-DKOs compared to Wt after stimulation. RA-SVs were excluded from this quantification. Student's t-test; ***p < 0.001.

(E) Stimulated 1/2-DKOs exhibited accumulations of ELVs in comparison to stimulated Wt. Mann-Whitney test; ***p < 0.001.

(F) Increased overall number of coated structures in stimulated endophilin 1/2-DKOs. (F') Both, coated vesicles and coated pits forming at ELVs were significantly increased in 1/2-DKO after stimulation. Mann-Whitney test; **p < 0.01; ***p < 0.001.

Data information: stimulated Wt n = 36 sections from N = 2 animals; stimulated 1/2-DKO n = 76/ N = 3; dashed lines indicate mean values of the unstimulated conditions from the respective genotypes for reference. Box plots illustrate the median with the interquartile range, whiskers indicate 10-90 % of data points, the squares present the respective mean value. For panels (B-F), please also refer to Appendix Table S13 for detailed statistical analysis.
Cobalt protoporphyrin IX increases endogenous G-CSF and mobilizes HSC and granulocytes to the blood (2019)
Figure 1

Fig 1. CoPP increases number of granulocytes and upregulates a set of cytokines. C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. (A) Chemical structures of heme (HO-1 substrate) and CoPP (HO-1 in vivo inducer). (B) Heme degradation reaction catalyzed by HO-1. (C) Heme oxygenase activity is increased by CoPP and decreased by SnPP in the liver as measured by gas chromatography. (D) Total leukocyte and red blood cell count in PB. CoPP increases the number of all WBC populations. Granulocyte and monocyte percentage are increasing, whereas lymphocyte percentage is decreasing among PB leukocytes after CoPP. (E) Heatplot of the cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP increases the concentrations of a group of cytokines (red box). (F) Selected cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP increases concentrations of G-CSF, MCP-1, IP-10, IL-6 and IL-5. Results are shown as mean + SEM, one-way Anova with Bonferroni post-test, n = 5 mice per group.
Cobalt protoporphyrin IX increases endogenous G-CSF and mobilizes HSC and granulocytes to the blood (2019)
Figure 2

Fig 2. Both G-CSF and CoPP mobilize myeloid cells, but only G-CSF mobilizes T cells. C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. (A) Cell numbers of main leukocyte populations in PB measured by flow cytometry. Both G-CSF and CoPP increase numbers of CD45+ cells in blood. G-CSF and CoPP similarly increase monocyte numbers, but the increase of granulocytes is higher after G-CSF. Lymphocytes, including T cells, are increased only by G-CSF and not by CoPP. (B) Cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP induces a group of cytokines (red box) which are not induced by G-CSF. (C) Plasma concentrations of selected cytokines and growth factors. CoPP highly increases concentration of endogenous G-CSF and IL-6 that are not increased by G-CSF. (D) Treatment with G-CSF and CoPP decreases percentage of Ly6G+ macrophages in the BM and (E) increases relative spleen weight. (F) viSNE maps of CD11b+ CD11c- blood cells, colored by SSC value. Three independent experiments were performed and the results were pooled. Results are shown as mean + SEM, one-way Anova with Bonferroni post-test, n = 7 mice per group.
Cobalt protoporphyrin IX increases endogenous G-CSF and mobilizes HSC and granulocytes to the blood (2019)
Figure 3

Fig 3. CoPP and G-CSF mobilize granulocytes with different phenotype. C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. (A) Representative flow cytometry plots show higher granularity (SSC) and Ly6G expression in CoPP-mobilized granulocytes than in G-CSF mobilized granulocytes. Relative abundance of different granulocyte phenotypes in blood (B) or BM (C) of mice treated with G-CSF and CoPP. Mice treated with CoPP have higher proportion of granulocytes with mature phenotype, than mice treated with G-CSF. (D) The percentage of cells isolated from C57BL/6xFVB mice treated with G-CSF or CoPP that are producing reactive oxygen species after incubation with indicated stimuli. Results are shown as mean + SEM, one-way Anova with Bonferroni post-test, n = 7 mice per group (B-C) or two-way Anova with Bonferroni post-test, n = 6 mice per group (D).
Cobalt protoporphyrin IX increases endogenous G-CSF and mobilizes HSC and granulocytes to the blood (2019)
Figure 4

Fig 4. CoPP mobilizes more HSPC than G-CSF in C57BL/6xFVB mice. (A) Gating strategy of KLS (c-Kit+Lin-Sca-1+) cells in blood, using CD48 and CD150 to distinguish HSC (hematopoietic stem cells), MPP (multipotent progenitors) and HPC (hematopoietic progenitors). (B) CoPP mobilizes higher numbers of HSC (KLS CD48-CD150+), MPP (KLS CD48-CD150-), HPC-1 (KLS CD48+CD150-) and HPC-2 (KLS CD48+CD150+) cells than G-CSF to the PB. (C) Treatment with CoPP increases percentage of KLS cells in BM, what is related to increase in HPC populations, but not in LT-HSC fraction. In contrast, frequency of MPP tends to decrease (D) Scheme of common myeloid progenitor (CMP) differentiation towards granulocytes/monocytes, erythrocytes and platelets. (E) The number of KLS- (c-Kit+Lin-Sca-1-) is higher in mice treated with CoPP than in mice treated with G-CSF. Numbers of lineage committed progenitors: GMP (granulocyte-macrophage progenitors, KLS- CD48+CD150-CD34+) and MEP (megakaryocyte-erythroid progenitors, KLS- CD48+CD150+CD34-) were higher after CoPP administration than after G-CSF. G-CSF and CoPP similarly increase EP numbers (erythrocyte progenitors, KLS- CD48-CD150-CD34-). (F) Both G-CSF and CoPP treatments decrease the KLS- percentage in the BM, however the decrease after CoPP is smaller. G-CSF and CoPP decrease percentages of MEP and EP, but only G-CSF decreases the percentage of GMP. Results are shown as mean + individual values, one-way Anova with Bonferroni post-test, n = 7 mice per group.
Cobalt protoporphyrin IX increases endogenous G-CSF and mobilizes HSC and granulocytes to the blood (2019)
Figure 5

Fig 5. CoPP mobilizes functional HSC that provide faster hematopoietic recovery and higher chimerism upon transplantation than G-CSF-mobilized HSC. Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. (A) Effect of G-CSF and CoPP-induced mobilization in C57BL/6-Tg(UBC-GFP)30Sch/J donor mice (mean + individual values, one-way Anova with Bonferroni post-test, n = 16 mice per group): Donor mice treated with CoPP had the highest number of mobilized granulocytes (i) and KLS cells (ii), compared to control or G-CSF-treated donor mice (B) Recipient mice which were transplanted with CoPP-mobilized PBMC had the highest number of WBC, PLT and the highest hematocrit values 2 weeks after PBMC transplantation, compared to G-CSF-mobilized and control PBMC recipients (mean + individual values, one-way Anova with Bonferroni post-test, NaCl: n = 8, G-CSF: n = 7, CoPP: n = 9 mice per group). (C) Recipient mice which were transplanted with CoPP-mobilized PBMC had the highest chimerism among CD45+ cells, granulocytes and B cells 2, 4 and 18 weeks after transplantation. Chimerism among T cells was the highest in recipients of G-CSF-mobilized PBMC 2 weeks after transplantation, but 18 weeks after transplantation it was the highest in CoPP-mobilized PBMC recipients (mean + SEM, two-way Anova with Bonferroni post-test, NaCl: n = 8, G-CSF: n = 7, CoPP: n = 9 mice per group). (D) The majority of primary recipients transplanted with G-CSF and CoPP-mobilized PBMC have detectable GFP chimerism among BM KLS cells. (E) Fraction of secondary recipient mice with chimerism in PB CD45+ cells exceeding 1% tends to be higher after CoPP-mobilized PBMC transplant than G-CSF-mobilized PBMC transplant. (F) PB chimerism in secondary recipients is higher in CoPP group than in the control group (mean + individual values, Kruskal-Wallis test with Dunn's post-test, NaCl: n = 7, G-CSF: n = 6, CoPP: n = 8 mice per group). (G-H) Percentage of secondary recipient mice with GFP chimerism in BM KLS (G) and KLS-(H) cells higher that 1% is similar in G-CSF and CoPP groups. (I-J) GFP chimerism in KLS (I) and KLS- (J) cells in the BM of secondary recipients (mean + individual values, Kruskal-Wallis test with Dunn's post-test, NaCl: n = 7, G-CSF: n = 6, CoPP: n = 8 mice per group).
Cobalt protoporphyrin IX increases endogenous G-CSF and mobilizes HSC and granulocytes to the blood (2019)
Figure 6

Fig 6. CoPP-induced mobilization depends on G-CSF, but not depend on Nrf2/HO-1 pathway. (A-B) Wild type C57BL/6 mice were injected with anti-G-CSF antibody alone or in combination with anti-IL-6 antibody one hour prior to CoPP treatment; the injections were repeated daily for 5 days and mice were sacrificed 6 hours after the last CoPP injection. (C-E) HO-1 deficient and control C57BL/6xFVB mice were injected with CoPP three times, every second day and sacrificed 24 hours after last injection. (F-H) Nrf2-deficient and control C57BL/6 mice were injected with CoPP daily for 5 days and sacrificed 6 hours after last injection (DMSO: n = 4, CoPP: n = 5 mice per group). (A) G-CSF and IL-6 concentration in plasma (B) CD45+ cells, granulocytes and HSC numbers in PB measured by flow cytometry. (C) Total leukocyte counts in PB (HO-1+/+: n = 5, HO-1-/- DMSO: n = 6, HO-1-/- CoPP: n = 4 mice per group). (F) CD45+ cell numbers in PB measured by flow cytometry. (D, G) Granulocyte percentage among PB leukocytes. (E, H) Cytokine and growth factor concentrations in plasma (E - HO-1+/+ n = 5, HO-1-/- DMSO: n = 5, HO-1-/- CoPP: n = 3 mice per group). Results are shown as mean + SEM, two-way Anova with Bonferroni post-test.