EMBO/soda-nel huggingface dataset:
Mechanism and function of DNA replication-independent DNA-protein crosslink repair via the SUMO-RNF4 pathway (2021)
Figure 5

Figure 5.

Unresolved DPCs undermine faithful mitotic chromosome segregation and cellular fitness

A. HeLa cells transfected or not with indicated siRNAs were synchronized in early S phase by double thymidine block, released and pulse-labeled 5,5 h later with 5-azadC for 30 min in the presence or absence of SUMOi. Following 5-azadC removal, cells were subjected to live-cell imaging analysis, and the duration of mitosis (nuclear envelope breakdown (NEBD) to anaphase onset) was quantified (red bars, median; at least 72 cells, pooled from three independent experiments, were analyzed per condition; ****p<0.0001, ns: not significant, Mann-Whitney test).

B. Representative time-lapse sequences of mitotic progression in cells in (A). NEBD corresponds to t=0. White arrowheads indicate cells undergoing division. Scale bar, 10 μm.

C,D. Quantification of mitotic defects in cells in (A) (mean; n=3 independent experiments; >97 cells quantified per condition; *p<0.05, **p<0.01, paired t-test).

E. HeLa cells transfected with RNF4 siRNA were synchronized in early S phase by double thymidine block. Six h after release from the block, cells were pulse-labeled with 5-azadC for 30 min. Mitotic cells were isolated by shake-off 8 h later, washed and incubated or not with MPS1 inhibitor (MPS1i) and collected at the indicated times. Cells were then processed for immunoblotting with indicated antibodies.

F. Clonogenic survival of 5-azadC-treated HeLa cells transfected with indicated siRNAs (mean±SEM; n=2 independent experiments).

G. Clonogenic survival of 5-azadC-treated WT HAP1 cells and derivative cell lines expressing a truncated form of RNF4 lacking the RING domain (∆RNF4; Fig. EV1H) (mean±SEM; n=2 independent experiments).
A Genome-Scale Transcriptional Regulatory Network of Arabidopsis Primary and Specialized Metabolism (2021)
Figure 5

Figure 5. Characterization of TCA cycle function in transcription factor mutant alleles.

(A) Representative 5-day-old seedlings of wild-type Arabidopsis thaliana Col-0 and TF mutants gata12 and wri3 grown in the dark. Scale bar, 1 mm.

(B) Heat map indicates which TFs significantly affects hypocotyl length, root length and the composite trait of root to total length in dark-grown seedlings and whether the effects of TFs are dependent on condition. TFs are hierarchically clustered using Euclidean distance.

(C) Heat map of the average relative effects of TF mutant alleles on hypocotyl length, root length and the ratio of root to total length reveal that TF lesions significantly perturbed TCA cycle-dependent growth. Mutant alleles are listed in rows and traits in columns. Cells of TF mutant alleles in heat map are colored if the AGI or AGI:TCA Metabolite linear model terms for each trait are statistically significant (P < 0.05, two-way ANOVA, 16-20 seedlings per genotype per condition across two experiments per genotype). Mutant alleles are hierarchically clustered using Euclidean distance.

(D-F) Hypocotyl length, root length, and root to total length is dependent on TF and exogenous TCA cycle metabolites. Radar plots presents mutant phenotypes relative to Col-0 (black). Ratio of mutant:WT traits were determined using the estimated marginal means of each genotype calculated from 16-20 seedlings per genotype per condition across two experiments.
A Genome-Scale Transcriptional Regulatory Network of Arabidopsis Primary and Specialized Metabolism (2021)
Figure 6

Figure 6. Phenotypes of mutant alleles are genotype by salt treatment-dependent.

(A) Heat map indicates which TFs significantly affects rosette area, growth rate, shoot biomass, flowering time and seed yield and whether the effects of TFs are dependent on salt treatment. TFs are hierarchically clustered using Euclidean distance.

(B) Heat map summarizing the relative effect of TF mutant alleles under control and salt treatment on rosette area, growth rate, shoot biomass, flowering time and seed yield. Mutant alleles are listed in rows and traits under both control and salt treatment are in columns. Cells of TF mutant alleles in heat map are colored if the AGI or AGI:Condition term in the linear model of each trait is statistically significant (P < 0.05, two-way ANOVA, N = 6-10 plants per genotype per condition). Mutant alleles are hierarchically clustered using Euclidean distance.

(C) Rosette area of mutant allele of BPC4 from day 7 to day 21 post-germination. Linear model for two-way ANOVA considers AGI, salt treatment, day post-germination and their interactions. The AGI and AGI:Salt Condition terms are statistically signification (P < 0.001, P < 0.01, respectively). Heat map under line plot indicates which term in linear model is statistically significant (P < 0.05) using two-way ANOVA for each day from 9 biological replicates per condition. Solid lines, Col-0; dashed line, bpc4; circle, control condition; triangle, 50 mM NaCl.

(D) Venn diagram of transcription factors in which the natural abundance of 13C and 15N and the ratio of 13C to 15N that were perturbed in their respective mutant alleles. Transcription factors are listed if the AGI or AGI:Salt Treatment linear model terms are statistically significant (P < 0.05) as determined by a two-way ANOVA from 3-5 biological replicates per genotype per condition.

EXPANDED VIEW FIGURE LEGENDS
Molecular basis for the bifunctional Uba4-Urm1 sulfur relay system in tRNA thiolation and ubiquitin-like conjugation (2020)
Figure 2

Figure 2 Functional analysis of structural elements in Uba4.

Analysis of the interaction between CtUba4 and GST-CtUrm1 (ratio 2:1 or 0.5:1 when marked with an asterisk) in the presence of nucleotide derivates by GST pull-down resolved by SDS-PAGE and visualized with Coomassie stain.

Close up (for reference see full structure on upper left) of the CtUba4 linker (aa 286-321) highlighting important residues and the coordination of the zinc site (inset, lower left).

Tabular summary of ATP hydrolysis by CtUba4 WT and mutants in the presence of CtUrm1. The amount of released PPi was measured using a fluorometric pyrophosphate assay kit. After PPi hydrolysis, Pi was also detected by a Malachite Green kit. Changes in enzymatic activity are indicated as a color gradient from blue to red, normalized to CtUba4 WT. PPi: pyrophosphate; Pi: inorganic phosphate; SD: standard deviation; n≥3

Functional analyses of ScUba4 mutant yeast using APM-gel retardation (top) and viability in response to rapamycin (bottom). PAGE was supplemented with APM to retard the migration of thiolated tRNAs and allow their visualization by northern blot using an anti-tRNAUUCGlu probe. APM: ([N-Acryloyl-amino]phenyl)mercuric chloride; YPD: yeast extract peptone dextrose. All residue numbering follows the CtUba4 sequence, but the respective ScUba4 numbering is added in subscript.
Molecular basis for the bifunctional Uba4-Urm1 sulfur relay system in tRNA thiolation and ubiquitin-like conjugation (2020)
Figure 3

Figure 3 Crystal structure of the Uba4C202K-Urm1 complex at 3.1 Å resolution.

Interaction analysis of WT and mutated CtUba4 with GST-CtUrm1 by GST pull-down in the presence of 1mM ATP. CtUba4C202K covalently linked to GST-CtUrm1 is marked by an asterisk.

Comparison of the structures of the CtUba4C202K-CtUrm1 complex and apo CtUba4 focusing on the arrangement of Urm1 and RHD in relation to the AD dimers. The symmetry operator of the crystallographic two-fold axis is indicated.

Overall structure of the CtUba4C202K-CtUrm1 complex. The AD dimer is shown as surface representation, the Uba4 linker (light blue), crossover loop (light brown) and Urm1 (salmon) are shown as cartoon. The C-terminus of Urm1 (dark red), Lys202 (green) and the formed covalent peptide bond between the two components (red) are highlighted. The ATP molecule (transparent red) is positioned in the nucleotide binding site based on the ATP-bound EcMoeB-MoaD complex (PDB ID: 1JWA). Uba4 and Urm1 molecules from the adjacent asymmetric unit which form the dimer are shown in transparent grey.

Close up view of the CtUba4 active site and the CtUrm1 C-terminus (top). A superposition of the linker from apo CtUba4 structure is shown (bottom). Crucial residues are shown as ball-and-stick model and the tentative positions of Cys202 Cys305 are indicated.
Molecular basis for the bifunctional Uba4-Urm1 sulfur relay system in tRNA thiolation and ubiquitin-like conjugation (2020)
Figure 4

Figure 4 The active cysteine of the Uba4 AD protects against covalent attachment of thiocarboxylated Urm1.

Analysis of covalent adduct formation between CtUba4 WT or C202 mutants and carboxylated (-OH) or thiocarboxylated (-SH) CtUrm1 in the presence or absence of TBH, DTT, TCEP and HA, respectively. ATP: Adenosine triphosphate TBH: tert-Butyl hydroperoxide HA: Hydroxylamine; DTT: 1,4-Dithiothreitol; TCEP: Tris(2-carboxyethyl)phosphine.

Analysis of covalent adduct formation between CtUba4 WT/cysteine mutants and CtUrm1-COSH in the presence of oxidizing agents. HP: Hydrogen peroxide; D: Diamide.

Phenotypic analysis of ScUba4 mutant yeast strains in response to rapamycin.

MS/MS spectrum of the K202-ε-GGH containing peptide from CtUba4C202K. A representative annotated fragmentation spectrum is shown with the b- and y-ions marked in red and blue, respectively. The precursor ion at m/z 591,98 is labelled in green. The peptide sequence is shown at the top with the collision-induced fragmentation pattern. The Urm1 conjugation site was identified by detection of the GGH remnant motif generated by chymotryptic digestion.

The table shows parameters of mass spectrometry identification of CtUba4C202K and CtUba4C202S with lists of detected peptides containing the K-ε-GGH remnant motif of Urm1. In CtUba4C202K, the conjugation site on K202 was confirmed. In the TBH treated samples of CtUba4C202S several potential conjugation sites were found. We present only high confidence peptides reaching a Mascot score higher than 40, which were detected in all analyzed samples. Asterisks indicate ambiguous site localization of K-ε-GGH. #PSMs: number of peptide-to-spectrum matches; m/z: mass to charge ratio of precursor ion.

Localization of all lysine residues (shown as green sticks) on the structure of CtUba4C202K-Urm1. CtUrm1 C-terminus and the lysine residues of CtUba4C202S that become covalently linked to Urm1-COSH in oxidizing conditions are presented in red.

Analysis of covalent adduct formation between CtUba4 WT or Cys202 mutants and thiocarboxylated (-SH) CtUrm1 in the presence or absence of TBH and HA, respectively.
EphA2 Phosphorylates NLRP3 and Inhibits Inflammasomes in Airway Epithelial Cells (2020)
Figure 1

Figure 1

Expression of EphA2 in human and murine cells

(A) Human myeloid cells and lymphoid cells were purified from PBMCs using a cell sorter. Total RNA was isolated from these primary cells and primary AECs to perform chip hybridization and microarray analysis.

(B-C) The profile of EphA2 expression in different cells is indicated. (B) Murine B cells and T cells were purified from spleens using a cell sorter, total RNA was isolated from murine B cells, T cells, BMDCs, BMDMs, AMs and AECs. Shown is mean ± SD. Data represent three independent biological replicates.

(C) Immunoblot analysis of EphA2 and β-actin in BMDCs, BMDMs, AMs, and AECs.
EphA2 Phosphorylates NLRP3 and Inhibits Inflammasomes in Airway Epithelial Cells (2020)
Figure 2

Figure 2

EphA2 regulates the production of inflammasome related cytokines in AECs

(A-D) ELISA of IL-1β, IL-18, IFN-β and IL-6 in supernatant of AEC cells, which were stimulated for 16 h with RNA virus reovirus (MOI=5) or DNA virus HSV-1 (MOI=5). n = 3 primary samples.

(E) Immunoblot analysis of IL-1β and caspase-1 in AECs cell lysate or cell supernatant, β-actin served as a loading control throughout.

Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. Mock indicated unstimulated cells. **P <0.01 and ***P<0.001 (unpaired t test). NS means no statistical difference. Data represent three independent biological replicates.
EphA2 Phosphorylates NLRP3 and Inhibits Inflammasomes in Airway Epithelial Cells (2020)
Figure 3

Figure 3

EphA2 controls airway allergy inflammation in reovirus infection murine model

(A-B) ELISA of IL-1β and IL-18 in BALF samples were from wild-type (WT) and EphA2-kockout (KO) mice (n=3 per strain) on day 1 and day 3 after intranasal infection with reovirus (4×104 PFU per mouse).

(C-E) Cell number and category of BALF samples were from wild-type (WT) and EphA2-kockout (KO) mice (n=3 per strain) on day 1 and day 3 after intranasal infection with reovirus (4×104 PFU per mouse). Shown is mean ± SD.

(F) Hematoxylin and eosin (H&E)-staining of lung sections were from EphA2+/+ and EphA2−/− mice intranasally infected with reovirus (4×104 PFU per mouse) on day 0, day 1 and day 3. Scale bars represent 200μm.

(G) Immunoblot analysis of EphA2 and IL-1β in lung homogenates were from wild-type (WT) and EphA2-kockout (KO) mice on day 1 and day 3 of intranasal infection with reovirus (4×104 PFU per mouse), β-actin served as a loading control throughout.

Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.
EphA2 Phosphorylates NLRP3 and Inhibits Inflammasomes in Airway Epithelial Cells (2020)
Figure 4

Figure 4

EphA2 functions as a negative regulator in ova-induced airway inflammation

(A-B) Quantitative RT-PCR analysis of mRNA encoding various cytokines (IL-4, IL-5, IL-13, IL-33) in lung samples from wild-type (WT) and EphA2-knockout (KO) mice (n=4 per strain) sensitized with ova; results presented relative to β-actin. Shown is mean ± SD.

(C) ELISA analysis of IL-33 in lung tissue homogenate samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova.

(D-G) ELISA analysis of IL-4, IL-13, IL-1β and IL-18 in lung tissue homogenate samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova.

(H) Cell number and category of BALF samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova. Shown is mean ± SD.

(I) H&E staining and PAS staining of lung sections from wild-type (WT) and EphA2-knockout (KO) mice sensitized with ova. Scale bars represent 200μm.

Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates.
EphA2 Phosphorylates NLRP3 and Inhibits Inflammasomes in Airway Epithelial Cells (2020)
Figure 5

Figure 5

EphA2 binds and colocalizes with NLPR3 in the cytosol

(A) Immunoblot analysis of proteins precipitated with anti-EphA2 or IgG (control) were from whole-cell lysates of AECs from wild-type mice stimulated with reovirus (MOI=5) for 3 h. Input, 10% of the AECs lysate.

(B) Full-length EphA2 and serial truncations of EphA2 with deletion of various domains.

(C) The indicated plasmids of HA-tagged full-length EphA2 and EphA2 truncation mutants were respectively transfected into HEK293T cells along with Myc-tagged NLRP3, and then cell lysates were immunoprecipitated with anti-Myc antibody, followed by western blot analysis with anti-HA antibody and anti-Myc Antibody.

(D) The indicated plasmids of HA-tagged full-length NLRP3 and NLRP3 truncation mutants were respectively transfected into HEK293T cells along with Myc-tagged EphA2, and then cell lysates were immunoprecipitated with anti-Myc antibody, followed by western blot analysis with anti-HA antibody and anti-Myc antibody.

(E) Colocalization of endogenous EphA2 and NLRP3 in AEC cells. AEC cells were unstimulated or stimulated with reovirus for 2h before confocal microscopy analysis. EphA2 was stained with rabbit anti-EphA2 (1:500), followed by Alexa Fluor 594 goat anti-rabbit secondary antibody (red), while NLRP3 was stained with mouse anti-NLRP3 (1:500), followed by Alexa Fluor 488 goat anti-mouse secondary antibody (green). DAPI (4′,6-diamidino-2-phenylindole) served as the nuclei marker (blue). Scale bars represent 20μm. The Pearson's correlation coefficient and the Mander's overlap coefficient in the upper red box are 0.5, and 0.95 respectively, and the lower red box 0.78, and 0.94 respectively.

Data information: The position of protein markers (shown in kDa) is indicated on the right-hand side. Data are representative of three independent biological replicates.
EphA2 Phosphorylates NLRP3 and Inhibits Inflammasomes in Airway Epithelial Cells (2020)
Figure 6

Figure 6

EphA2 phosphorylates NLRP3 at Tyr132

(A) The indicated plasmids of Myc-tagged NLRP3 and GFP-tagged EphA2 were transfected into HEK293T cells and then cell lysates were immunoprecipitated with the anti-Myc antibody, followed by western blot analysis with the anti-p-Tyr antibody, anti-GFP and anti-Myc.

(B) Immunoblot analysis of p-Tyr of NLRP3 precipitated with anti-NLRP3 antibody in AECs without (Ctrl) or with LPS stimulation or reovirus (Reo) infection.

(C) The indicated plasmids of Myc-tagged wild-type NLRP3 or its mutants Y132F, Y164F, Y251F, Y570F and Y589F were respectively transfected into HEK293T cells along with GFP-tagged EphA2, and then cell lysates were immunoprecipitated with the anti-Myc antibody, followed by western blot analysis with the anti-p-Tyr antibody, anti-HA antibody and anti-Myc antibody.

(D) Immunoblot analysis of EphA2, ASC, NLRP3, caspase-1 and IL-1β in HEK293T cells were co-transfected with 200ng EphA2, 40ng ASC, 40ng caspase-1 and 200ng IL-1β, vector or wild NLPR3 or Y132F as indicated. The position of protein markers (shown in kDa) is indicated on the right-hand side.

(E) Densitometry analysis of (D). *P<0.05 (unpaired t test). NS means no statistical difference. Shown is mean ± SD. Data represent three independent biological replicates.
EphA2 Phosphorylates NLRP3 and Inhibits Inflammasomes in Airway Epithelial Cells (2020)
Figure 7

Figure 7

NLRP3 Y132 phosphorylation interferes with inflammasome assembly

(A-B) Confocal microscopy analysis of AEC cells uninfected or infected with reovirus at multiplicity of infection (MOI) of 20 for 3h. AECs primed with LPS and stimulated with ATP for 30 min severed as a positive control. ASC was stained with mouse anti-ASC (1:200), followed by Alexa Fluor 488 goat anti-mouse secondary antibody (green). DAPI served as the nuclei marker (blue).

(C-D) Confocal microscopy analysis of HEK293T cells transfected with flag-ASC and Myc-tagged vector or Myc-tagged NLRP3 or Myc-tagged NLRP3 Y132F. Cells were stained with anti-flag (1:200), anti-Myc (1:200), followed by Alexa Fluor 488 goat anti-rabbit secondary antibody (green) or Alexa Fluor 594 goat anti-mouse secondary antibody (red). DAPI served as the nuclei marker (blue).

Data information: Scale bars represent 50μm. Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.

Expanded View Figure legends
Binary organization of epidermal basal domains highlights robustness to environmental exposure (2022)
Figure 1

Figure1: Basal layer cells express different LRC/non-LRC preferred-genes in scales vs interscales of mouse tail skin

(A) Model of tail skin organization in IFE domains.

(B, C) (B) Whole mount low magnification images show marking in interscale (K10+) vs scale regions, quantified in (C). Error bars are SDs. The data shown is average of 4 different timepoints (2 week, 3month, 6month and 1 year) each consisting of 2 mice and 5 images per mice. p-value is calculated for difference between scale vs interscale using Student's t-test (mixed Anova).

(D) Tail skin sections immunofluorescence stained for interscale (K10) or scale (K31) supra-basal markers, and basal markers. Scale bar =20 µm or 50 µm as indicated on the panels. Asterisks indicate that photoshop was used to erase the autoflourescent cornified layer. Dashed lines indicate ROI in basement membrane.

(E) Quantification of images like those in (D) with n=3-4 mice and 6-7 images per mouse showing preferred-gene enrichment. Error bars are SDs. P-values calculated by Student's t-test (mixed effect model)

(F) Relative gene expression/area levels from (D) and (E), normalized to sum to one, and displayed in a barycentric plot obtained from a total of 3-4 biological replicates and 6-7 images per replicate.

Error bars are SEMs. The proportions in Fig. 1F were computed using log ratios (Aitchison, 1982) (G) Cartoon summarizing spatial distribution of markers in the tail IFE domains.
Binary organization of epidermal basal domains highlights robustness to environmental exposure (2022)
Figure 5

Figure 5: Molecular differences in IFE populations suggest domain-specific adaptation to environmental UV exposure

(A) Schematic showing the tamoxifen injection paradigm for tdTomato-labelling of all IFE lineages in tail and back. Co-expression of markers with the lineage-traced cells at 1-month post-tamoxifen. Dlx1-CrER is shown as example in tail; see also Appendix Figure S2A and C (*Back skin). Scale bar =50 µm

(B) Quantification of images like those in (A) and Appendix Figure S2A, showing % tdTomato+ cells expressing indicated markers. Sox6 (black bar) was not analyzed in K14-Cre/EYFP lineage. See also Appendix Figure S2B for scale vs. interscale split. Error bars are SDs. P-values calculated by Student's t-test (mixed effects model) from n=2-3 mice and 5-6 images per replicate.

(C-F) (C,D) Venn diagram showing bulk RNA-seq data in the three sorted IFE basal lineages and (E,F) heatmap for their differentially regulated Hallmark_Pathways.

(G) Immunofluorescence images of Sox6 staining in noUVB (0hr) or UVB-exposed (6hrs- or 24hrs-post) mouse back skin.

(H) Boxplot shows % Sox6 expressing basal cells quantified from images like those in (G). Central band is the mean value, the top of box indicates 25th percentile while the bottom indicates 75th percentile. Whiskers indicate minimum and maximum values in the data, with dots indicating potential outliers. Linear mixed model statistical test was used with data from 18 images from 3 biological replicates.

(I) Model of IFE LRC/non-LRC domains and marked lineages with distinct degree of UV exposure in normal conditions.
Functional coordination of non-myocytes plays a key role in adult zebrafish heart regeneration (2021)
Figure 2

Figure 2. Transcriptome dynamics of macrophages during zebrafish heart regeneration.

A, B, Joint analysis of nonCM scRNA-seq data from uninjured hearts and hearts at 2, 7, and 14 dpi with LIGER. A, All nonCM visualized on tSNE. B, top, NonCM from each time point visualized on the tSNE embedding in (A). Data was down-sampled to the same cell number at each time point. B, bottom, Pie charts showing contribution of different cell types at each time point. C-F, Zoom-in analysis of macrophages identified in (A). C, tSNE plot colored by MC subpopulations. D, Expression levels of representative markers of each MC subpopulation color-coded and mapped to tSNE embeddings in (C) (top), and corresponding representative GO terms for each subpopulation (bottom). E, Dynamics of MC subpopulations during heart regeneration as shown by the proportion of each subpopulation in total nonCM or total MC. F. Expression of tnfa and csf3b in MC at each time point. G, Fluorescent in situ hybridization for cd74a, tnfa, and ctsc at 7 dpi, respectively. mpeg1 was used to label all MC cells. The blue boxed region is highlighted in the zoom-in image to the right. Scale bar = 50 μm. A stands for arium, V stand for ventricle and OFT stands for outflow tract. White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane.