EMBO/soda-nel huggingface dataset:
Loss of genomic integrity induced by lysosphingolipid imbalance drives ageing in the heart (2019)
Figure 6

Figure 6: in vivo recapitulation of the molecular defects caused by elevated DHS levels lead to severe decline of cardiac functionality which can be prevented by HAT inhibition: (A) Schematic representation of the experimental setup employed in the in vivo mouse experiments. Representative micrographs depicting (B) γH2A.X and (C) H3K27ac staining on the ventricle sections for the indicated cohorts. (D) Quantification of γH2A.X+ CMs nuclei represented as percentage bar graph. (E) Box-plot depicting the distributions of the corrected total nuclear fluorescence of the indicated cohorts. (F) Representative micrographs depicting ventricular view from the echocardiography of the indicated cohorts. (G-H) Quantification of fractional shortening and ejection fraction of the indicated groups represented as bean plot and percentage bar graph respectively (n=6 animals per condition). Scale bars for B,C = 50 µm.

Acknowledgements

The Kurian lab is supported by the NRW Stem Cell Network Independent Group Leader Grant (Grant No: 3681000801 and 2681101801, CECAD (Grant No:3624720211), German Heart Association (DZHK; Grant No: 3632009221), Else Kröner-Fresenius-Stiftung, Deutsche Forschungsgemeinschaft grant (KU 3511/4-1), and University of Cologne (Grant No: 3622801511). M.J. and A.A. are supported by National Institutes of Health grants 5R03AG053287 and 1K01HL135464 respectively. The Valenzano lab is funded by the Max Planck Institute for the Biology of Ageing. We acknowledge the central facilities at CECAD, CMMC and MPI Ageing for technical assistance. We would like to thank Dr. Janine Altmueller, CGI-Cologne, for assistance with whole transcriptome sequencing and all members of D.V and L.K laboratory for intellectual contributions at various stages of this project. We thank Dr. Thomas Langer and Dr. Björn Schumacher for their valuable comment on the manuscript.

Authorship

L.K. conceived the project and supervised the experiments. L.K, D.V & G.A designed all the experiments. G.A, D.B, W.Y, S.F, J.E.M, N.R, N.E.V, K.L & S.B performed the experiments. Killifish handling and sample preparation was done by J.D. G.A, J.S.M. G.A, A.P, D.V, M.J, M.P, A.A, S.F, A.S, J.H, A.T, C.D & L.K analysed and interpreted the data. S.G, G.A, L.K, A.K and St.B designed, performed and evaluated the in vivo mouse experiments. G.A, & L.K drafted the illustrations. G.A, & L.K prepared the manuscript.

Supplementary information

Supplementary information includes 7 Figures, one Text file, one Data file and 6 Movies.
From Chemoproteomic-Detected Amino Acids to Genomic Coordinates: Insights into Precise Multi-omic Data Integration (2021)
Figure 4

Figure 4. Association between amino acid reactivity and CADD score.

A, B. Distribution of the max CADD38 PHRED (model for GRCh38) scores for (A) cysteine (n=1,401) and (B) lysine (n=4,363) CpDAAs of low, medium, and high intrinsic reactivities, defined by isoTOP-ABPP ratios, low (R10:1>5), medium (2<R10:1<5), high (R<2) (Weerapana et al. 2010; Hacker et al. 2017). Kruskal-Wallis nonparametric test to examine reactivity group difference, Wilcox test used for pairwise comparisons (BH-adjusted p-values, *p. adj = 0.04, **p. adj = 0.0037, ***p. adj = 0.00013). The boxplot boxes represent the lower and upper quartiles, with the central band as median. Notches show the confidence interval based on median ± 1.58*IQR/sqrt(n), and whiskers mark observations that satisfy quartiles ± 1.5*IQR. Median CADD38 max codon scores with bootstrapped 95% confidence intervals for reactive groups are: low CpD Cys 27.3 [26.9, 28.0], medium CpD Cys 28.55 [27.80, 29.05], high CpD Cys 31 [28.8, 32.0], low CpD Lys 29.5 [29.3, 29.6], medium CpD Lys 29.25 [28.85, 29.50], high CpD Lys 29.05 [28.50, 29.55].

C. Shows CADD38 max codon scores for nonsynonymous SNVs at residues 220-479 of CASP8 (UniProt ID Q14790). Dashed horizontal line marks deleterious threshold of 25.

D. Crystal structure of CASP8 (PDB ID: 3KJN) highlighting C360 and C409. Bound covalent inhibitor B93 in yellow, with distance between detected cysteines and inhibitor measured in Angstroms. Protein surface color represents CADD38 max codon scores. Image generated in PyMOL (DeLano and Others 2002).

E. Activity of recombinant caspase-8 protein assayed using fluorogenic IETD-AFC substrate. Percentage activity shown relative to wild-type (WT) protein for three replicate experiments, bars and error bars as mean ± SD.

Table 1. Recommended best practices for inter- and intra-database integration for chemoproteomics datasets.

Map data to the appropriate genome assembly for downstream applications.

Genome assembly updates can introduce or refine genome resolution and in doing so alters the genomic coordinates of codons. Not all downstream pathogenicity predictors are compatible with both GRCh37 and GRCh38 (Appendix Table S14).

EXPANDED VIEW FIGURES
Structural basis of bacterial σ28-mediated transcription reveals roles of the RNA polymerase zinc-binding domain (2020)
Figure 3

Figure 3. Role of β′ ZBD in activating σ28-dependent transcription

A. Specific interactions between the upstream DNA (−25 and −26 nucleotides) and β′ ZBD.

B, C. Effects of deleting β′ ZBD on transcriptional activities of RNAP on different promoters. The fliCp (B) was used as template for σ28-dependent transcription, and the lacUV5p (C) was used in σ70-dependent transcription. RNA products indicated by solid triangles were quantified and the signal obtained from 100 nM wild-type RNAP was normalized to 1 in each test respectively.

D. Transcriptional activities of ZBD mutated σ28-RNAP on fliCp. RNA products indicated by solid triangles were quantified.

E. Binding of σ28-RNAP with fliCp as detected by EMSA (left). The heparin-resistant RNAP-promoter open complex is indicated as "RPo", and the heparin-sensitive RNAP-promoter closed complex is shown as "RPc". Percentages of RNAP-promoter open complex (RPo) and closed complex (RPc) in EMSA performed using σ28-RNAP and fliCp (right).

F. DNase I footprinting of the fliCp complex with σ28-RNAP. Promoter DNA was labeled on the non-template strand. The promoter region protected by RNAP is indicated by a red box at the bottom panel.

Data Information: In (B)-(E), data are mean ± SD from three determinations. Individual values of replicates are shown as dots. Statistical analyses were performed using the unpaired Student's t-test (two-tailed). * p<0.05, ** p<0.01.
Structural basis of bacterial σ28-mediated transcription reveals roles of the RNA polymerase zinc-binding domain (2020)
Figure 4

Figure 4. Importance of β′ ZBD in controlling transcription of σ28-dependent genes

A. Schematic diagram of constructing chromosomal mutations in rpoC gene in E. coli based on a CRIPSR/Cas9 system. A 20-nt guide RNA targeting a region between K74 and K87 on β′ subunit is shown in purple. Double-strand DNA fragments, which contains synonymous mutations (yellow background) in sgRNA targeting region to facilitate clone selection and aimed mutations at K74 or K87 or both residues (only example for constructing the Ec-K74A-K87A strain is shown), were used as donor templates.

B, C. Promoter activities of tarp (B) and fliCp (C) in E. coli wild-type (Ec-WT) and its mutants expressing ZBD-mutated RNAP (Ec-K74A, Ec-K87A and Ec-K74A-K87A) shown as mean ± SD from three replicates.

D. Volcano plot showing log2-fold change of mRNA abundance as a ratio of Ec-K74A-K87A and Ec-WT strains. Blue dots represent genes with significant difference between these two strains. Red dots represent flagellar genes. Data are shown as mean values of three independent colonies.

E. Relative mRNA levels of flagellar genes in Ec-K74A-K87A strain comparing with that in Ec-WT (normalized to 1 for each gene). Diagram for the role of σ28 (fliA gene) in flagella regulon hierarchy Clarke & Sperandio, 2005Fitzgerald, 2014(; ) is shown in the left panel. Data are mean ± SEM from three replicates.

F. Motilities of Ec-K74A-K87A strains with over-expression of wild-type rpoC gene (Ec-K74A-K87A-C) or carrying the control plasmid (Ec-K74A-K87A-V).

G. Activities of fliCp in Ec-K74A-K87A-C and Ec-K74A-K87A-V strains. L-arabinose was used at final concentration of 0.002%. Data are mean ± SD from three colonies.

Data Information: In (B), (C), (E) and (G), statistical analyses were performed using the unpaired Student's t-test (two-tailed). * p<0.05, ** p<0.01.
Structural basis of bacterial σ28-mediated transcription reveals roles of the RNA polymerase zinc-binding domain (2020)
Figure 5

Figure 5. β′ ZBD contributes to transcription initiated by σ factors with weak σ4/−35 element interactions

A. Diagram for mutations in σ28 and tarp. Conserved domains in σ factor are shown as numbering. The region 4 of σ28 was replaced by that from σ70 to obtain the chimeric σ28-R4m, and the sequence of −35 element in tarp was replaced by "TTGACA" in the chimeric tarp-35m. The proposed strength for interaction between σ4/−35 in different σ/promoter pairs is indicated by the thickness of arrow.

B. Activities of chimeric σ28-R4m in initiating transcription from tarp-35m, when reconstituted with wild-type (WT) or ZBD mutated RNAP core enzyme.

C-E. Effects of mutating β′ ZBD on transcriptional activities of σ38-RNAP (C, D) or σ70-RNAP (E) on osmYp.

Data Information: In (B)-(E), quantifications of RNA products indicated by solid triangles from three determinations are shown as mean ± SD at the bottom panel. Statistical analyses were performed using the unpaired Student's t-test (two-tailed). ns, not significant, * p<0.05,** p<0.01.
Dermal αSMA+ myofibroblasts orchestrate skin wound repair 
via β1 integrin and independent of type I collagen production (2022)
Figure 5

Figure 5. β1 integrin facilitates αSMA+ myofibroblasts-mediated wound repair.

(A) Violin plot of Itgb1 expression levels in fibroblasts from murine wounds. Raw scRNA-seq data are derived from (Haensel et al., 2020, Data ref: Haensel et al 2020).

(B) UMAP plots of ACTA2 and ITGB1 expression of fibroblasts in human wounds. Raw scRNA-data are derived from (Davis et al. 2020, Data ref: Davis et al, 2020).

(C) IF for β1 integrin (red) and αSMA (green) on wound tissue sections at day 17 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar: 20 μm.

(D) Quantification of the number of αSMA+ β1 integrin+ cells. WT, N=3; TK, N=3 biological replicates. Unpaired t-test performed comparing WT to TK.

(E) Wound area measurements in WT and Itgb1cKO mice. WT, N=5; Itgb1cKO, N=6 biological replicates. Two-way ANOVA performed comparing WT and Itgb1cKO mice.

(F-K) Reepithelization, granulation tissue formation, and vascularization in WT and Itgb1cKO mice. (F) Representative images of keratin 5 (Krt5, red) stained wounds at day 14 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 500 µm. (G) Quantification of epidermal thickness. WT, N=4; Itgb1cKO, N=6 biological replicates. (H) Immunofluorescent staining for CD31 (red) and nuclei (blue) on wound tissue sections at day 14 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 20 μm. (I) Quantification of CD31+ area per visual field normalized to WT mice. WT, N=6; Itgb1cKO, N=6 biological replicates. (J) Polarized light images of picrosirius red stained wound tissues at day 14 post-wounding. Red: collagen fibers. White boxes denote zoomed regions depicted in right panels. Scale bar, 50 µm. (K) Quantification of collagen fiber percent area normalized to the average area in WT controls. WT, N=6; Itgb1cKO, N=6 biological replicates.

(L) Quantification of collagen fiber width normalized to the average width in WT controls. WT, N=6; Itgb1cKO, N=6 biological replicates.

Data information: Data are reported as mean ± s.e.m. for (D, E, G, I, K, and L). Data are reported as median (open circle) with interquartile range indicated with bar for violin plots (A). One sample t-test performed comparing values to WT average for (G, I, K, and L). Exact p-values are reported.

Expanded View Figure Legends
FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma (2016)
Figure 1

Figure 1. SOX9 interacts with FBW7α through a conserved degron motif phosphorylated by GSK3.

(A) General sequence alignment of SOX9 high affinity CDC4 phosphodegron (CPD) motif against other affinity motifs present in known FBW7α substrates including Cyclin E, cMYC, and c-Jun.

(B) Immunoblot analysis of the immunoprecipitated HA-SOX9 or FLAG- FBW7α and their pull-down products. The corresponding Western blotting from the whole cell extract (WCE) of the transfected HEK293 showed the levels of exogenous HA-SOX9 or FLAG-FBW7α proteins in the total input. Cells were treated with 10 µM MG132 for 4 hours prior to harvesting for the immunoprecipitation. Blots are representative from 3 independent experiments.

(C) FBW7α immunoblot eluted from the agarose bead-bound peptide, which had been incubated with the recombinant SCF-FBW7α for 1 hour at 37◦C. The agarose bead-bound peptide contains either the non-phosphorylated SOX9 amino acid sequence spanning from residue number 231 to 245 (SOX9 peptide), or those with the threonine corresponding to residue number 236 and 240 being phosphorylated (pSOX9 peptide). The input (10%) showed the level of the supplemented recombinant SCF-FBW7α in the in vitro binding reaction. Shown blot is representative from 2 independent experiments.

(D) Representative Western blotting set from 3 independent repeats of pT236-SOX9 from lysates of HEK293 transfected with either HA-EV or HA-SOX9. Each lysate were immediately divided and left untreated or subjected to lambda phosphatase (λ-PPase) treatment for 1 hour at 37◦C prior to gel electrophoresis. SOX9 blot showed the presence of SOX9 protein in both the untreated and the phosphatase-treated SOX9-transfected cell lysates.

(E) Bead-immobilized in vitro translated (IVT) HA-SOX9 wild type (WT) or T236/240 mutant were either untreated or subjected to in vitro GSK3 kinase reaction for 90 minutes at 37◦C prior to elution and gel electrophoresis. The SOX9 blot showed total SOX9 protein eluted from the beads from each reaction. Shown immunoblots are single representative experiment from 3 independent repeats.

(F) GSK3-mediated phosphorylation of threonine 236 promoted SOX9 interaction with recombinant SCF-FBW7α in vitro. Bead-immobilized IVT HA-SOX9 were either untreated or phosphorylated with GSK3 as described in (E) prior to further incubation with recombinant SCF-FBW7α for in vitro binding assay. Immunoblot showed FBW7α protein in the in vitro binding reaction (input) as well as those which had been eluted from the untreated and GSK3-treated immobilized SOX9. The set of blots shown is representative from 3 independent experiments.
FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma (2016)
Figure 3

Figure 3. SOX9 protein stabilization correlates with low levels of FBW7α in medulloblastoma patients

(A) FBW7 mutations across medulloblastoma molecular subgroup as identified by exome sequencing. In the SHH medulloblastoma subgroup, FBW7 mutations occurred approximately 11 % in a cohort containing 133 SHH medcases (Kool et al., 2014) and can be categorized as stop-gain (n=3), non-synonymous (n=7). One case of splicing error mutation is detected in the Group 3 medulloblastoma.

(B) Immunoblotting of HA-SOX9-WT protein following expression of various FLAG-FBW7α constructs including wild type (WT), or those with R465A or R479A mutation in Daoy. The cells were transfected with equal amount of HA-SOX9-WT and FLAG-FBW7α and the whole cell lysates were collected 24 hours following the transfection. GAPDH proteins were shown to indicate total protein loading for each sample. The set of blots shown is representative from 2 independent experiments.

(C) Co-immunoprecipitation of HA-SOX9 with either wild type (WT) or R465A- FLAG-FBW7α. Unlike the wild type protein, FLAG-FBW7α containing arginine 465 mutation did not significantly co-immunoprecipitate with HA-SOX9 (IP with anti HA antibody) and vice versa (IP with anti FLAG antibody). The expressions of HA-SOX9 and FLAG- FBW7α- WT or R465A mutant were assessed in the whole cell extract (WCE) of the transfected HEK293 with GAPDH protein used to indicate total protein loading.

(D) Comparison of FBW7 mRNA expression across the medulloblastoma subgroups (WNT, SHH, Group 3, and 4) in a cohort containing 423 medulloblastoma cases. Expression of FBW7 in both normal fetal (n= 5) and adult cerebellum (n = 13) were used for comparison against the medulloblastoma.

(E) Overall survival analysis of medulloblastoma patient based upon FBW7 expression level. Analysis was performed on 383 out of the 423 cases from which the survival data were available. Cohort was divided into high- (blue line) and low- FBW7 (red line) expressing subgroup using FBW7 median expression as group classifier. Statistical analysis was determined by using a LogRank test.

(F) Correlation analysis between SOX9 protein/mRNA and FBW7 mRNA expression level from medulloblastoma tissue microarray consisting of 142 tissue samples. SOX9 protein level was scored following immunohistochemistry staining, while SOX9 and FBW7 mRNAs expression by the RNA Scope as described in Materials & Methods. A modest negative correlation was determined by Kendall’s correlation test (τ = - 0.223, p<0.001) between SOX9 protein/mRNA and FBW7 expression. Affiliation of samples with medulloblastoma subgroups was indicated by different colored circle (blue=WNT, red=SHH, yellow=Group 3, green=Group 4, grey=Undetermined).
FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma (2016)
Figure 5

Figure 5. RNA-Seq profiling of MB002 expressing SOX9-WT and FBW7-insensitive SOX9 mutant

(A) Scatter plot depicting the relationship between transcriptional changes measured as log2 FC between EV to SOX9-WT (x-axis) and EV to SOX9-T236/T240A (y-axis). Each dot represents one transcript, with colored dots indicating transcripts upregulated (log2 FC>0.585; p<0.05; red dots) or downregulated (log2 FC<-0.585; p<0.05; blue dots) in both comparisons. The transcript corresponding to the SOX9 gene is highlighted as a red filled dot. Indicated r-value indicates Spearman's rank correlation coefficient between the two FC profiles.

(B) Box-and-Whisker plot displaying the sample specific expression of three FBW7 isoforms; FBW7α (NM_033632), FBW7β (NM_018315) and FBW7γ (NM_001013415'). Each box represents the expression distribution over three replicates.

(C) Venn diagram illustrating the overlap of differentially expressed (FC>1.5, p<0.05) transcripts between any of the three comparisons: EV to SOX9-WT (upregulated: 145+308+21; downregulated 146+333+44), EV to SOX9-T236/T240A (upregulated: 294+308+95+21; downregulated 345+333+95+44), or SOX9-WT to SOX9- T236/T240A (upregulated: 112+95+21; downregulated 114+95+44). Each oval represents the number of transcripts either upregulated or downregulated in the respective comparison, where the direction of differential expression is indicated by the inequality sign in the label. Numbers in overlapping regions signify the number of transcripts commonly differentially expressed in all contributing comparisons.

(D) Heat map of the row-wise z-scores of 16 EMT hallmark genes in SOX9-WT and -T236/T240A samples. Heat map was generated using the GenePattern software (Reich et al., 2006).

(E) Quantitative PCR (qPCR) of SNAI2 (SLUG), VIM and POU3F1 (OCT6) (normalized to GAPDH levels) in MB002 cells stably transduced with EV (set to 1), SOX9-WT and SOX9-T236/240A. Cells were induced for 8 or 24 hours with doxycycline.

(F) Western blot of SOX9, HA-SOX9, pSOX9-T236, SNAI2 (SLUG), VIM and POU3F1 (OCT6) in MB002 cells stably transduced with EV, SOX9-WT and SOX9-T236/240A. Cells were induced for 24 and 48 hours with doxycycline and GAPDH were used as a loading control.

(G) Heat map of the row-wise z-scores of 14 pro-metastasis genes differentially expressed between SOX9-WT and -T236/T240A samples and previously identified as SOX9 target genes (Kadaja et al., 2014, Larsimont et al., 2015, Oh et al., 2014). Heat map was generated using the GenePattern software (Reich et al., 2006).
FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma (2016)
Figure 6

Figure 6. SOX9 confers cisplatin resistance in medulloblastoma

(A) Cisplatin dose response curves of (i) MB002, (ii) Daoy, and (iii) MIC in the absence (EV) or presence of SOX9 by Alamar blue. Cells were pre-conditioned with doxycycline to induce expression of SOX9 (or EV) prior to treatment with increasing concentrations of cisplatin. The IC50 were calculated following 5 (MB002 and MIC) or 3 days (Daoy) of treatment. Data are mean + standard deviation from 3 independent repeats, each containing 5 technical replicates.

(B) Cisplatin dose response curves of SOX9-expressing (i) Daoy and (ii) MIC in the absence or presence of FBW7α. Experiments and data analysis were performed as described in (A)

(C) Overall survival analysis of mice bearing Daoy or Daoy-expressing dox-inducible SOX9 treated with cisplatin. The dox-preconditioned cells (105 cells) were orthotopically xenografted to Nude-Foxn1nu mice and left for 1 week to prior to being treated with vehicle control or cisplatin (2mg/kg) intraperitoneally for every other day for a total of 6 doses.

(D) Heat map of the row-wise z-scores of 11 genes associated with cisplatin resistance in MB002 expressing Sox9-WT or Sox9-T236/T240A. Heat map was generated using the GenePattern software.

(E) Quantitative analysis of ATP7A, DUSP2, and TTK mRNAs in MB002 following expression of SOX9-WT or SOX9-T236/240A. Total RNA were collected 24 hours following doxycycline treatment, from which cDNA were generated for qPCR. Data are mean mRNA level (normalized to B2M transcript) + standard deviation from 3 independent experiments with statistical significance were determined by Multiple comparisons 2-way ANOVA with Bonferroni’s post-test.

(F) Time course western blotting of HA-SOX9, ATP7A, DUSP2, ERK1/2 pThr202/Tyr204 and total ERK1/2 in MB002 cells following doxycycline induction of either EV, SOX9-WT or SOX9-T236/240A. GAPDH was used as a loading control.