EMBO/soda-nel huggingface dataset:
A new patient derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan (2019)
Figure 5

Fig. 5.

High-throughput screen identifies compounds increasing glycosylation of α-dystroglycan.

(A) Schematic of the screening procedure. Compounds were dispensed in matrigel coated 384 well plates prior to seeding of murine myoblasts. Myoblasts were incubated for 24 h with 10 µM of the individual compounds, 5 µM of the positive control Trichostatin A or with the negative control DMSO. After fixation and staining with the IIH6 antibody, detecting glycosylated α-dystroglycan, the cells were imaged using the confocal PE Opera LX high-throughput microscope. The intensity of the α-dystroglycan glycosylation staining was determined, values normalized and a hit list generated. Scale bar, 20 µm.

(B) Compound validation strategy. After the primary screen, the α-dystroglycan glycosylation staining pattern of initially identified compounds was examined to exclude autofluorescent as well as toxic compounds. From the remaining compounds 11 hit compounds were nominated after acquired images were compared to controls in the respective screening plate. A dose response assay followed by flow cytometry assays further narrowed down hits to 1 compound.

(C) Dose response curve. H2K 2B4 cells were treated with serial dilutions of 11 hit compounds for 24 h and fixed, stained and imaged as described above. The average values and standard deviations of the mean fluorescent intensities of technical triplicates were calculated and a non-linear regression curve was fitted for the 3 compounds showing the highest increase in α-dystroglycan glycosylation (IIH6 immunostaining). Representative images of cells treated with 5 µM of the respective compounds as well as EC50 values are shown. HPFPTzone: (5Z)-5-[(3-ethoxy-4-hydroxy-phenyl)methylene]-3-(4-fluorophenyl)-2-thioxo-thiazolidin-4-one. 4BPPNit: 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile. DPPTzone: (5E)-5-[(2,5-dimethyl-1-phenyl-pyrrol-3-yl)methylene]-3-phenyl-2-thioxo-thiazolidin-4-one. Regression curve and EC50 values were calculated using GraphPad Prism 7 software. In images shown, green represents glycosylated α-dystroglycan and blue Hoechst staining. Scale bar, 50 µm.

(D) Structures of the 3 compounds showing the highest increase in α-dystroglycan glycosylation. Structure 1: HPFPTzone. Structure 2: 4BPPNit. Structure 3: DPPTzone.

(E) Flow cytometry analysis confirmed that 4BPPNit treatment (20 µM) significantly increased IIH6-reactive glycan in mouse H2K 2B4 cells, compared with untreated cells. There was no significant difference in the percentage of IIH6-positive cells. Values indicate mean ± s.e.m. (n= 6, triplicates of 2 independent experiments; t-test; NS, not significant; ** p<0.01).
A new patient derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan (2019)
Figure 6

Fig. 6.

Hit compound 4BPPNit validation in the FKRP dystroglycanopathy human iPSC models.

(A) FKRP-NSCs treated with 4BPPNit showed increased IIH6 reactivity, compared with untreated control cells. Note that the augmented IIH6 reactivity by 4BPPNit is much weaker than the that in CRISPR-corrected NSCs. Under the same exposure time, the signal intensity of IIH6 reactivity is saturated in corrected-NSCs.

(B) Representative immunoblots with the IIH6 antibody using FKRP- and corrected-NSCs treated with 4BPPNit for 24, 48 and 72 h, and DMSO only controls.

(C) Quantification of α-dystroglycan glycosylation (IIH6), compared with DMSO only controls.

(D) Representative immunoblots of laminin-binding analysis using FKRP- and corrected-NSCs treated with 4BPPNit and DMSO only controls.

(E) Quantification of laminin-binding activities, compared with DMSO only controls.

(F) Representative immunoblots with the AF6868 antibody detecting both α-dystroglycan core protein and β-dystroglycan in FKRP- and corrected-NSCs treated with 4BPPNit and DMSO only controls.

(G) Quantification of α-dystroglycan core protein and β-dystroglycan expression, compared with DMSO only controls.

Data information: Intensities of glycosylation, laminin-binding activity or protein expression are normalized to GAPDH protein expression. Note that the left and right panels of the immunoblots in (B) and (D) are not equivalent exposure time. See also Appendix Fig. S5. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).
A new patient derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan (2019)
Figure 7

Fig. 7.

Compound 4BPPNit induces upregulation of LARGE1 gene expression.

(A) DAG1 gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls.

(B) FKRP gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls.

(C) LARGE1 gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls.

Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).
Asymptomatic COVID-19: disease tolerance with efficient anti-viral immunity against SARS-CoV-2 (2021)
Figure 3

Figure 3 - Asymptomatic COVID-19 patients exhibit more robust SARS-CoV-2 specific Th17 responses compared to symptomatic patients.

A. Expressions of genes associated with T cell functionality were compared between acute asymptomatic (n=30) and symptomatic (n=26) COVID-19 patients. Heatmap of DEGs with FDR adjusted P value < 0.05, scaled based on log2RPKM values, with blue and red colors indicating low and high expressions, respectively.

B. Mass cytometry was performed on PBMCs obtained from acute symptomatic (n=37) and acute asymptomatic (n=19) COVID-19 patients and healthy controls (n=10). Naïve, TEMRA, Central memory (CM) and effector memory (EM) T cells were characterized based on CD45RA and CCR7 expressions. Data are presented as Mean ± SD *P<0.05; **P<0.01 (Mann-Whitney U test).

C. SARS-CoV-2 specific CD4+ non T follicular helper (TFH) cells from symptomatic (n=5) and asymptomatic COVID-19 (n=5) patients were characterized with flow cytometry based on the expression of IL-17a, IFN-γ, TNF-⍺, IL-4 and IL-10 upon SARS-CoV-2 peptide stimulation. Representative plot overlay of unstimulated (black) on peptide stimulated (red) was performed on asymptomatic patient. Data are presented as Mean ± SD **P<0.01 (Mann-Whitney U test).
Asymptomatic COVID-19: disease tolerance with efficient anti-viral immunity against SARS-CoV-2 (2021)
Figure 5

Figure 5 - Asymptomatic COVID-19 patients present less inflammatory monocyte, activated neutrophils and muted inflammatory response

A. Flow cytometry acquisition of patient whole blood was performed and number of mature and immature neutrophils were compared between asymptomatic (n=16) and symptomatic (n=52) COVID-19 patients and healthy controls (n=17). Data are presented as Mean ± SD *P<0.05; **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).

B. Mass cytometry was performed on PBMCs obtained from acute asymptomatic (n=19) and acute symptomatic (n=37) COVID-19 patients and healthy donors (n=10). Monocytes were characterized based on their CD14 and CD16 expression. Data are presented as Mean ± SD **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).

C. Classical, intermediate and non-classical monocytes were further defined in acute asymptomatic (n=19) and acute symptomatic (n=37) COVID-19 patients and healthy donors (n=10) with inflammatory marker CD169. Data are presented as Mean ± SD **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).

D. Expression data of genes associated with inflammatory monocytes, activated neutrophils and pro-inflammatory cytokines were compared between acute asymptomatic (n=30) and asymptomatic (n=26) COVID-19 patients and were represented by ratio with respect to expression in asymptomatic patients. Bar graphs show DEGs with FDR adjusted P value < 0.05 and fold change values are represented in the log2 scale.

E. Pro-inflammatory associated immune mediator levels in plasma fraction samples from first collection timepoint during hospital admission were quantified with 45-plex microbead-based immunoassay. Immune mediator levels were compared between asymptomatic patients (n=48) and symptomatic patients stratified by COVID-19 severity (mild, n=61; moderate, n=43; severe, n=68). Immune mediator levels for healthy controls (HC) (n=23) are indicated by the black dotted line. Patient samples with concentrations out of measurement range are presented as the value of Limit of Quantification (LOQ). Data are presented as Mean ± SD *P<0.05; **P<0.01; ***P<0.001 (One-way ANOVA with post hoc t test).
Asymptomatic COVID-19: disease tolerance with efficient anti-viral immunity against SARS-CoV-2 (2021)
Figure 6

Figure 6 - Asymptomatic patients express higher levels of markers associated with cellular repair and leukocyte migration.

A. Growth factors in the plasma of asymptomatic (n=48), symptomatic patients (n=173) and healthy controls (n=23) were analyzed by Luminex. A ratio of VEGF-A/VEGF-D was calculated and used to perform receiver operating characteristics analysis. The area under the curve was shown. Data are presented as Mean ± SD **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).

B. RNAseq analysis was performed on whole blood and selected transcripts that are overexpressed in asymptomatic patients over symptomatic patients are shown. Genes with false discovery rate less than 0.05 (*), 0.01 (**) or 0.001 (***) are indicated.

C. Cell counts of DCs, CD4 T cells, CD8 T cells and B cells that express specific chemokine markers between asymptomatic (n=19), symptomatic patients (n=37) and healthy controls (n=10) are analyzed by CyTOF and shown. Data are presented as Mean ± SD. *P<0.05; **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).

Expanded View Figure Legends
DSCR1-mediated TET1 splicing regulates miR-124 expression to control adult hippocampal neurogenesis (2019)
Figure 1

Figure 1. DSCR1 mutants show impaired adult neurogenesis

(A, B) Actively dividing cells labelled by BrdU and Ki67 are reduced in the SGZ of DSCR1 mutants compared to that of wild type mice. Brain sections were prepared 1 day after BrdU injection. The white box area is magnified in the lower panels: DAPI (blue), Ki67 (green), and BrdU (red). Arrow heads indicate BrdU and Ki-67 double positive cells in the SGZ.

(C, D) Differentiating cells are identified by staining with BrdU and DCX. Brain sections were prepared 10 days after BrdU injection.

(E, F) Maturation of progenitor cells are assessed by staining with BrdU and NeuN. Brain sections were prepared 21 days after BrdU injection Each hippocampal section was 40 μm in thickness, and a total of 24 sections were obtained from one hippocampus. Scale bars:100 μm in the large panel, and 10 μm in the magnified images. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animals for each condition, *P< 0.05, **P< 0.01.
DSCR1-mediated TET1 splicing regulates miR-124 expression to control adult hippocampal neurogenesis (2019)
Figure 2

Figure 2. DSCR1 regulates miR-124 by mediating methylation in the promoter of miR-124

(A) miR-124 expression is altered in DSCR1 mutants. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animals for each condition, *P< 0.01.

(B) Promoter activity of miR-124 in Neuro2 A cells with DSCR1 reduction or overexpression. Firefly luciferase reporter under the control of miR-124 promoter is measured. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05, **P< 0.01.

(C) Bisulfite sequencing analyses of the miR-124 promoter. Each CpG site is indicated, and the methylation status of two different regions are shown. Open and filled circles represent unmethylated and methylated CpG sites, respectively. The percentage of methylated CpGs among total number of CpGs is also shown.

(D) Neuro2A cells containing DSCR1shRNA show increased activity of miR-124 promoter measured. The miR-124 promoter was inserted in front of the luciferase reporter. Site-directed mutation of two methylation sites (31 and 58) in the promoter shows the luciferase activity similar to that of DSCR1 reduction. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05.

(E) Overexpression of DSCR1 reduced the luciferase activity, while removing all methylation sites in the miR-124 promoter increases it. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05.
DSCR1-mediated TET1 splicing regulates miR-124 expression to control adult hippocampal neurogenesis (2019)
Figure 3

Figure 3. DSCR1 regulates TET1 splicing

(A) DSCR1 binds to TET1 introns. The binding affinity of DSCR1 to the intron 8 of TET1 decreased with increased dosage of U1 snRNA and U2 snRNA.

(B) DSCR1 missing RNA recognition motif (△RRM) does not bind to the TET1 intron 8.

(C, D) Schematic diagram of the luciferase reporter separated by TET1 intron 8 and 9 or GAPDH intron 3 and 4. DSCR1 reduction increases the activity of this luciferase construct, while DSCR1 overexpression decreases its activity. In contrast, altering DSCR1 levels did not affect luciferase activity of the construct containing GAPDH introns. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05, **P< 0.01. ***P< 0.001.

(E) TET1 protein expression in the dentate gyrus region of hippocampus of DSCR1 mutants. TET1 is increased in DSCR1 knockout, while it is decreased in DSCR1 transgenic mice. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animal for each condition, *P< 0.05, **P< 0.01.
DSCR1-mediated TET1 splicing regulates miR-124 expression to control adult hippocampal neurogenesis (2019)
Figure 4

Figure 4. TET1 reduction restores abnormal adult neurogenesis in DSCR1 knockout mouse

(A) Schematic of the experimental timeline (left). Lentivirus containing TET1 shRNA and GFP or random shRNA and GFP is injected in to the contralateral dentate gyrus of hippocampus. 2 months old wild type or DSCR1 knockout mice were used for analyses.

(B) 3 weeks after lentivirus injection, neural progenitor cells are identified by double staining of GFP and Sox2 in dentate gyrus region. The white arrow indicates Sox2 and GFP double positive cells. Scale bar, 10 μm.

(C) The number of GFP and Sox2 double positive cells is restored to that of wild type. Scale bar: 10μm. Values are shown as mean ± SEM and tested for statistical significance by paired t-test. N = 3 animal for each condition, *P< 0.05.
DSCR1-mediated TET1 splicing regulates miR-124 expression to control adult hippocampal neurogenesis (2019)
Figure 5

Figure 5. Correcting DSCR1 dosage prevents impaired adult neurogenesis and learning and memory found in Ts65Dn mouse

(A) Genotype of Ts65Dn/DSCR1+/- mouse was confirmed by detecting markers for Ts65Dn and DSCR1 KO as shown previously (Duchon et al., 2011) (Ryeom et al., 2003) (Baek et al., 2009).

(B) Expression of the DSCR1 mRNA in Ts65Dn mouse was increased about 2.5 folds compared to that of wild type. However, Ts65Dn/DSCR1+/- mouse showed normal level of DSCR1 mRNA transcript. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N=3 for each condition, *P< 0.05.

(C, D) As expected, the levels of TET1 and miR-124 are decreased in Ts65Dn mouse, however Ts65Dn/DSCR1+/- mouse restores the TET1 mRNA and miR-124 expression. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. ß-actin was used for normalization. N = 3 (Diploid), 3 (Ts65Dn), 4 (Ts65Dn/DSCR1+/-) mice, *P< 0.05, **P< 0.01.

(E, F) The number proliferating progenitor neurons identified by BrdU and Ki-67 double staining is restored in the SGZ of Ts65Dn/DSCR1+/- mouse compared to that of Ts65Dn mouse. The white box area is magnified in the lower panels: DAPI (blue), Ki67 (green), and BrdU (red). Arrow heads indicate BrdU and Ki-67 double positive cells in the SGZ. Scale bars: 100 μm in the large image and 10 μm in magnified image. Each hippocampal section was 40 μm in thickness, and total 6 hippocampi were used for analysis. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 6 (control), 5 (Ts65Dn), 3 (Ts65Dn/DSCR1+/-) animals, *P< 0.05.

(G-J) Ts65Dn has learning and memory defects, but Ts65Dn/DSCR1+/- mouse clearly rescues learning and memory defects to control levels. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 8 (control), 5 (Ts65Dn), 10 (Ts65Dn/DSCR1+/-) animals, *P< 0.001 (G, H). *P< 0.05 (I), **P< 0.01 (I). (J) Open-field analysis shows no defects in movement of tested animals. Values are shown as mean ± SEM, N = 6 (control), 5 (Ts65Dn), 3 (Ts65Dn/DSCR1+/-) animals.
IKKbeta is required for the formation of the NLRP3 inflammasome (2021)
Figure 1

Figure 1. Inhibition of IKKβ or its activator TAK1 blocks the processing of caspase-1.

(A) WT BMDM were incubated for 1 h without (-) or with (+) the TAK1 inhibitor NG25 (2 μM), the IKKβ inhibitor BI605906 (5 μM) or the IKKβ inhibitor PS1145 (10 μM). The cells were then co-stimulated for 30 min with 100 ng/ml LPS and 4 mM ATP. Cell lysates (10 μg protein) were denatured in SDS, subjected to SDS-PAGE and immunoblotted with the antibodies indicated.

(B) As in A, except that the cells were co-stimulated for 1 h with 100 ng/ml LPS and 5 μM nigericin. Similar results were obtained in three independent experiments in A and B.

(C, D) As in A, B, except that primary human monocyte-derived macrophages were used instead of mouse BMDM. Similar results were obtained in two independent experiments.
IKKbeta is required for the formation of the NLRP3 inflammasome (2021)
Figure 2

Figure 2. IKKβ but not IKKα is required for the activation of caspase-1.

(A, B) The Mouse macrophage J774A.1 cell line was transfected with control siRNA or siRNA against IKKβ (A) or IKKα (B). 72 h post-transfection, the cells were co-stimulated for 60 min without (-) or with (+) 100 ng/ml LPS and/or 5 μM nigericin. Cell lysates (10 μg) were denatured in SDS, subjected to SDS-PAGE and immunoblotted with the antibodies indicated. Similar results were obtained in three (A) or two (B) independent experiments.

(C, D) As in (A, B) except that BMDM from IKKβ-LysM-Cre (flox/flox) (IKKβ (fl/fl)) or WT control mice were co-stimulated without (-) or with (+) 100 ng/ml LPS and/or 4 mM ATP (C) or with 100 ng/ml LPS and/or 5 μM nigericin (D). Similar results were obtained in two independent experiments.
IKKbeta is required for the formation of the NLRP3 inflammasome (2021)
Figure 3

Figure 3. IKKβ activity is required for the cleavage of gasdermin D and the secretion of caspase-1 fragments and IL-18.

(A, B) WT BMDM were incubated for 1 h without (-) or with (+), the IKKβ inhibitors BI605906 (5 μM), TPCA-1 (5 μM) or PS1145 (10 μM), the TAK1 inhibitor NG25 (2 μM) or the NLRP3 inhibitor MCC950. The cells were then co-stimulated for 30 min without (-) or with (+) 100 ng/ml LPS and/or 4 mM ATP (A) and/or 5 μM nigericin (B). The cell culture medium was removed and the cells lysed. Protein in the culture medium was precipitated (see Methods), dissolved in SDS. and subjected to SDS-PAGE, along with cell lysates. After transfer to PVDF membranes, immunoblotting was performed with antibodies recognizing both full length (FL) and cleaved (CL) gasdermin D (GSDMD), the p20 and p10 fragments of caspase-1 and IL-18. Similar results were obtained in two independent experiments.

(C) As in A, except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl) and control WT cells were used. Similar results were obtained in three independent experiments.
IKKbeta is required for the formation of the NLRP3 inflammasome (2021)
Figure 4

Figure 4. Oligomerisation of ASC requires IKKβ activity and is independent of caspase-1.

(A) WT BMDM were incubated for 1 h without (-) or with (+) 5 μM BI605906, then stimulated for 1 h with (+) 100 ng/ml LPS and/or 5 μM nigericin or left unstimulated. The cells were lysed in buffer containing 1% (v/v) Triton-X-100, and the Triton-X-100-soluble and Triton-X-100-insoluble fractions were prepared as in Methods. The Triton-X-100-insoluble fraction was dissolved SDS subjected to SDS-PAGE and immunoblotted with the antibodies indicated. The nuclear protein Histone H3B (H3B) was used as a loading control in the Tritin X-100 insoluble fraction. Similar results were obtained in three independent experiments.

(B) As in A, except that the Triton-X-100-insoluble fraction was first subjected to crosslinking for 45 min at 37oC with 2.0 mM DSS.

(C, D) As in A, B except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) mice (IKKβ (fl/fl)) were used. The U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) was used as a loading control. Similar results were obtained in two independent experiments.

(E, F) As in A, B except that BMDM from caspase-1 KO mice were used. Similar results were obtained in two independent experiments.
IKKbeta is required for the formation of the NLRP3 inflammasome (2021)
Figure 5

Figure 5. IKKβ induce interaction between NLRP3 and TGN38.

(A-H) WT BMDM were incubated for 1 h without or with the IKKβ inhibitors TPCA-1 (5 μM) (C, G) or BI605906 (5 μM) (D, H). The cells were then stimulated for 1 h with 100 ng/ml LPS (E) or 5 μM nigericin (F, G, H), or co-stimulated with 100 ng/ml LPS and 5 μM nigericin (B, C, D) or left unstimulated (control) (A). The cells were fixed and processed for PLA using a rabbit polyclonal antibody against TGN38 and a mouse monoclonal antibody against NLRP3. A PLA signal is only if the NLRP3 antibody and the TGN38 antibody come into close proximity. (A-H) the panel images show the PLA signal (red) and DAPI staining for nuclei (blue). Images were acquired by sequential laser scanning on a confocal microscope. Similar results were obtained in three independent experiments and representative images are shown. Scale bar = 50 μm.

(I-L) As in A-H, except that IKKβ inhibitors were omitted and iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl)) or WT mice were co-stimulated for 1 h with 100 ng/ml LPS and 10 μM nigericin or left unstimulated (control). Scale bar = 50 μm.

(M) The graphs show the mean + SEM of the average intensity of the PLA signal per cell acquired from 20 different fields and two separate experiments. The statistical significance was calculated using Kruskall-Wallis test with Dunn's multiple comparison; *denotes p<0.05, ***p<0.001 and ****p<0.0001; ns, non-significant.

(N) as in M, except that the PLA signal per cell acquired from 10 different fields from a similar experiment and statistical significance was calculated using two-way ANOVA with Sidak's multiple comparison; ****p<0.0001; ns, non-significant. Similar results were obtained in a second independent experiment. The graph shows the mean + SEM