EMBO/soda-nel huggingface dataset:
Integrated Genomics of Crohn's Disease Risk Variant Identifies a Role for CLEC12A in Antibacterial Autophagy. (2015)
Figure 3

Clec12a−/− Mice Are More Susceptible to Salmonella Infection In Vivo

(A) Confocal micrographs of LC3-Listeria colocalization in WT and Clec12a−/− BMDMs at 1 hr post-infection. The scale bars represent 10 μm.

(B) Quantification of LC3-Listeria colocalization in WT and Clec12a−/− BMDMs. Data shown represent mean ± SD of n = 3 independent experiments; unpaired t test. ∗∗p < 0.01.

(C and D) Salmonella cfus quantified per gram of stool (C) and per organ (D) at 4 days post-infection are displayed. Data are shown as mean ± SD; ∗∗p ≤ 0.01. Data are representative of at least two independent experiments (n = 9 for WT; n = 7 or 8 for Clec12a−/−).

(E) Clinical score for infected mice at 4 days post-infection. Data are shown as mean ± SD. Data are representative of at least two independent experiments. ∗∗∗∗p < 0.0001; unpaired t test. (n = 14 for WT; n = 13 for Clec12a−/−).

(F) Survival curve for infected WT and Clec12a−/− mice (n = 9 mice per genotype).

See also Figure S3.
Integrated Genomics of Crohn's Disease Risk Variant Identifies a Role for CLEC12A in Antibacterial Autophagy. (2015)
Figure 4

CLEC12A Associates with Bacteria Early after Entry into Host Cells and Restricts Intracellular Bacterial Replication

(A) Gentamicin protection assay in HeLa cells showing fold replication of Salmonella in cells treated with the indicated siRNAs. Data are shown as mean ± SD; n = 6. Data are representative of four independent experiments.

(B) Quantitation of CLEC12A-bacteria and LC3-bacteria colocalization at indicated time points. Data are shown as mean ± SD; n = 3 independent experiments.

(C) Representative confocal micrographs of HeLa cells showing association of intracellular bacteria with GFP-CLEC12A at 20, 40, 60, and 80 min post-infection. The scale bars represent 10 μm.

See also Figure S4.
Integrated Genomics of Crohn's Disease Risk Variant Identifies a Role for CLEC12A in Antibacterial Autophagy. (2015)
Figure 5

CLEC12A Colocalizes with Autophagosome Proteins and Plays a Role in Pathogen Defense

(A–C) Representative epifluorescent images of HeLa cells showing colocalization of (A) CLEC12A (green) and galectin 8 or galectin 3 (red) around intracellular Salmonella (blue), (B) GFP-CLEC12A (green) and NDP52 (red) around Salmonella (blue), and (C) endogenous CLEC12A (red) and GFP-LC3 (green) around Salmonella (blue).

(D) CLEC12A or GFP-CLEC12A (green) and GABARAPL2 or ATG16L1 (red) around intracellular Salmonella (blue). Images are representative of three or more independent experiments.

(E) Representative epifluorescent images of HeLa cells transfected with a control siRNA or a siRNA against CLEC12A and infected with DsRed-labeled Salmonella for 1 hr.

(F) Quantitation of ubiquitin-bacteria colocalization at 1 hr post-infection in cells shown in (E). Data are shown as means ± SEM from three independent experiments.

(G) Representative epifluorescent images of HeLa cells transfected and infected as in (E).

(H) Quantitation of NDP52-bacteria colocalization at 1 hr post-infection in cells shown in (G). Data are shown as means ± SEM from three independent experiments.

(I) Representative confocal micrographs of HeLa cells expressing GFP-CLEC12A. Cells were untreated or exposed to hypertonic media containing PEG 1000, followed by hypotonic shock.

All scale bars represent 10 μm. See also Figure S5.
Integrated Genomics of Crohn's Disease Risk Variant Identifies a Role for CLEC12A in Antibacterial Autophagy. (2015)
Figure 6

Integrated Systems Analysis Identifies KLHL9, NEDD8, and KLHL13 as CLEC12A-Interacting Antibacterial Autophagy Molecules

(A) Gene expression analysis of WT and Clec12a−/− BMDMs infected with Listeria for the indicated times. Heatmap shows log2 (fold change) relative to uninfected WT BMDMs at indicated time points. Shown are the top ten most-significant upregulated and downregulated genes in Clec12a−/− BMDMs compared to WT BMDMs.

(B) Cluster analysis of 674 differentially expressed genes in Clec12a−/− BMDMs versus WT BMDMs.

(C) Schematic of integrated proteomic and transcriptomic approach to identify functionally relevant CLEC12A-interacting proteins.

(D) Interactome analysis shows network of proteins identified from quantitative iTRAQ proteomics. Red box highlights the KLHL9-KLHL13-CUL3-NEDD8 complex interaction within the CLEC12A interactome.

(E) HeLa GFP-LC3 cells were transfected with indicated siRNAs for 48 hr and then infected with DsRed-labeled Salmonella for 1 hr. The fraction of GFP-positive intracellular Salmonella is shown. Data are mean ± SD and are representative of three independent experiments.

(F) Gentamicin protection assay in HeLa cells showing increased intracellular bacterial replication after 48 hr of knockdown of ATG16L1, KLHL9, NEDD8, or KLHL13. Data shown as mean ± SD; n = 4. Data are representative of three independent experiments.

See also Figure S6 and Tables S1, S2, and S3.
IL-2/IL-7-inducible factors pioneer the path to T cell differentiation in advance of lineage-defining factors (2020)
Figure 2

Figure 2. A subset of pDHSs in previously activated T cells are dependent on IL-2

A. DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB IL-2nil (32694) ordered by increasing fold change of tag count for TB IL-2 compared to TB IL-2nil. The sequence tag density is also shown in parallel at the same sites for the DNase-Seq analyses of CD4 naïve T cells (TN), TB IL-2 DMSO and TB IL-2 Ruxolitinib and ATAC samples from in vivo derived Th2 (Shih et al., 2016), Th1 and Th17 cells. The location of the 1000 IL-2 pDHSs is shown to the right.

B. Average DNase-Seq tag density profile for the 1000 IL-2 pDHSs in both replicates of TB IL-2 and TB IL-2nil samples, and for naïve T cells (TN).

C. Number of IL-2 pDHSs which are detected in in vivo purified CD4 T cell subsets (Chen et al., 2020, Garg et al., 2019, Shih et al., 2016).

D. Overlap between the subsets of IL-2 pDHSs which are present in in vivo derived Th1, Th2 and Th17 cells.

E. UCSC genome browser tracks with examples of IL-2 pDHSs within the Locus Control Region (LCR) within the Rad50 gene which controls the Th2 cytokines Il13, Il4 and Il5, plus IL-2 pDHSs within the Tnfrsf9 and Cish loci. IL-2 pDHSs are highlighted with blue boxes.
IL-2/IL-7-inducible factors pioneer the path to T cell differentiation in advance of lineage-defining factors (2020)
Figure 3

Figure 3. IL-2 pDHSs bind IL-2 regulated factors

A. De novo identification of DNA consensus motifs enriched in the 1000 IL-2 pDHSs determined by HOMER.

B. Jund mRNA levels in TB IL-2 and TB IL-2nil. Standard deviation is shown from the mean of 3 samples. P-values were calculated from the RNA-seq data using Limma with Benjamini-Hochberg correction for multiple testing.

C. Western blot analyses showing JUND and pSTAT5 protein levels in TB IL-2 and TB IL-2nil (left panel) with the signal calculated from 2 replicates relative to TB IL-2 in the right panel.

D. DNA motif density plots (right) showing the location of binding motifs for RUNX, ETS, STAT and AP-1 transcription factors at the 32694 DHSs detected in TB IL-2 and TB IL-2nil, as depicted in Figure 2A, ordered according to fold enrichment of DNase-Seq tag counts in TB IL-2 compared to TB IL-2nil (left).

E. DNA sequence tag density plots from ChIP-Seq assays of RUNX1, ETS1, STAT5 and JUND in TB IL-2, and STAT5 and JUND in TB IL-2nil ordered as in (D).

F. UCSC genome browser tracks at the Il13/Il4/Il5 Th2 cytokine locus LCR (indicated by the grey box within the Rad50 gene), Gzmb and Il24 showing DNase-Seq and ChIP-Seq for STAT5 and JUND in TB IL-2 and TB IL-2nil. Replicate JUND and STAT5 ChIP-Seq tracks are shown for TB IL-2.
IL-2/IL-7-inducible factors pioneer the path to T cell differentiation in advance of lineage-defining factors (2020)
Figure 4

Figure 4: IL-2 signaling supports PMA/I-inducible DHS formation

A,B. DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB+ IL-2 (A, 38645) and in TB+ IL-2 and TB+ IL-2nil (B, 36067), ordered by increasing fold change in tag count as indicated. The position of the 1015 IL-2-dependent inducible DHSs is shown alongside.

C. Average DHS signal at the 1015 IL-2-dependent inducible DHSs in TB IL-2, TB+ IL-2, TB+ IL-2nil and naïve T cells stimulated with PMA/I (TN+).

D. De novo identification of DNA consensus motifs enriched at the 1015 IL-2 iDHSs as determined by HOMER.

E. Bar graph showing the number of IL-2 iDHSs found within 25 kb of an IL2 pDHS.

F. Examples of IL-2 iDHSs which are proximal to IL-2 pDHSs at the Csf2/Il2 and Il10 loci. IL-2 iDHSs are highlighted in red and IL-2 pDHSs in blue.
IL-2/IL-7-inducible factors pioneer the path to T cell differentiation in advance of lineage-defining factors (2020)
Figure 5

Figure 5: Loss of IL-2 priming correlates with reduced gene expression of proximal genes

A. Principle component analysis (PCA) of TB IL-2, TB IL-2nil, TB+ IL-2 and TB+ IL-2nil from 3 replicates.

B. Hierarchical clustering of RNA-Seq data from 3 replicates as described in (A).

C. MA plot showing the genes differentially regulated between TB IL-2nil and TB IL-2. The number of genes with a fold change >3 and adj p-value <0.05 are highlighted. P-values were calculated from the RNA-seq data using Limma with Benjamini-Hochberg correction for multiple testing.

D. log2 fold change in gene expression in TB IL-2nil compared to TB IL-2 for the nearest gene to the DHSs ranked according to the fold change in DNase-Seq tag count for TB IL-2/TB IL-2nil. The DNase-Seq tag density plots are taken from Figure 2A for TB IL-2, TB IL-2nil and TN.

E,F. UCSC genome browser tracks showing DNase-seq and RNA-seq at the Socs1 (E) and Sell loci (F).
IL-2/IL-7-inducible factors pioneer the path to T cell differentiation in advance of lineage-defining factors (2020)
Figure 6

Figure 6: Loss of IL-2 priming correlates with reduced gene expression of some inducible genes

A,B. Relative mRNA expression levels in TB+ IL-2 and TB+ IL-2nil (A) and TB+ DMSO and TB+ Ruxolitinib (B). The expression level in the IL-2/DMSO TB+ sample is set to 1. (n=3, error bars represent standard deviation, P-values were calculated using a paired t-test with *p<0.05, **p<0.005, ***p<0.0005.)

C. UCSC genome browser tracks showing DNase-Seq and RNA-Seq at the IL-2 regulated genes Csf2/Il3 and at the LCR within the Rad50 gene (grey box) which controls the Th2 cytokines Il13, Il4 and Il5. IL-2 pDHSs are highlighted in blue and IL-2 iDHSs in red.
IL-2/IL-7-inducible factors pioneer the path to T cell differentiation in advance of lineage-defining factors (2020)
Figure 7

Figure 7: IL-2 pDHSs can be maintained in vitro by IL-7 in place of IL-2

A. DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB IL‑2nil (data taken from Figure 2A), duplicate DNase-seq samples for TB IL-7 and TB IL-7nil are shown in parallel for the same sites.

B. Average DNase-Seq tag density profile for the 1000 IL-2 pDHSs in both replicates of TB IL-7 and TB IL-7nil.

C, D. UCSC genome browser tracks with DNase-Seq data for TB IL-7 and TB IL-7nil showing examples of IL-2 pDHSs within the Locus Control Region (LCR) within the Rad50 gene which controls the Th2 cytokines Il13, Il4 and Il5, plus IL-2 pDHSs within the Tnfrsf9 and Cish loci (C), and non-primed control regions from the CD3 gene cluster and the Tbp locus (D). IL-2 pDHSs are highlighted with blue boxes.

E. Average DHS signal at the 1015 IL-2 regulated inducible DHSs in TB+ IL-7 and TB+ IL-7nil.

F. Relative mRNA expression levels in TB+ IL-7 and TB+ IL-7nil. The expression level in the TB+ IL-7 is set to 1. (n=3, error bars represent standard deviation, P-values were calculated using a paired t-test with *p<0.05, **p<0.005, ***p<0.0005.)
IL-2/IL-7-inducible factors pioneer the path to T cell differentiation in advance of lineage-defining factors (2020)
Figure 8

Figure 8. IL-2 pDHSs are reduced in antigen specific memory T cells upon deletion the IL-7 receptor

A. Experimental overview of the tamoxifen-inducible disruption of Il7r following generation of 2W1S-specific Th1 memory T cells. CD4creERT2 x Il7rf/f or Il7rf/f (littermate control) mice were injected i.v. with Lm-2W1S, tamoxifen administered for 4 consective days (19-21 dpi) and spleen cells harvested for ATAC-Seq 14-18 days later.

B. Flow cytometric analysis of IL-7Rα expression by 2W1S-specific CD44hi CD4 T cells in the presence or absence of cre recombinase.

C. Average number of 2W1S-specific cells recovered/mouse after sorting 3 replicate samples of Il7rf/f compared to CD4creERT2 x Il7rf/f . Errors bars indicate the standard deviation.

D. Average ATAC-Seq signal at the 411 IL-2 pDHSs which contain a STAT binding motif.

E. UCSC genome browser tracks at loci with IL-2 pDHSs showing replicate ATAC-Seq samples for CD4creERT2 x Il7rf/f memory T cells, Il7rf/f control memory T cells, replicate DNase-Seq samples for TB IL-2 and TB IL-2nil and TB IL-2 and TB IL‑2nil STAT5 ChIP-Seq samples. The grey boxes signify IL-2 pDHSs which diminish after disruption of the Il7r gene.
IL-2/IL-7-inducible factors pioneer the path to T cell differentiation in advance of lineage-defining factors (2020)
Figure 9

Figure 9: IL-2 pDHSs are present in Th1, Th2 and Th17 cells

A. Venn diagram showing the overlaps between subsets of the 1000 IL-2 pDHSs which are also identified as ChIP-Seq peaks for STAT6 (Wei et al., 2010) and GATA3 (Wei et al., 2011) inTh2 cells (161), STAT4 (Wei et al., 2010) and T-BET (Gokmen et al., 2013) in Th1 cells (510), and STAT3 (Durant et al., 2010) and RORγt (Ciofani et al., 2012) in Th17 cells (333). The table below indicates the proportion of these peaks also seen in T blasts generated using CD3/CD28 antibodies instead of ConA.

B. Model demonstrating priming of Th0 cells by IL-2-inducible AP-1 and STAT5 prior to binding of lineage-determining factors to the same regions.

C. UCSC genome browser tracks showing ChIP-Seq and DNase-Seq at the LCR within the Rad50 gene which controls the Il4/Il13/Il5 Th2 cytokine locus, and also Il17a, Il12rb2 and Tbx21. The grey boxes indicate IL-2 pDHSs which bind STAT5 in Th0 cells and other STAT proteins in other T cell lineages.
IL-2/IL-7-inducible factors pioneer the path to T cell differentiation in advance of lineage-defining factors (2020)
Figure 10

Figure 10: JUNB and JUND bind to IL-2 pDHSs in multiple T cell lineages

A. mRNA expression levels of JUN family transcription factors. Normalised TPM is shown for TB IL-2, TB IL2nil, TB+ IL-2 and TB+ IL-2nil. Standard deviation is shown for 3 replicates.

B. Average JUNB ChIP-Seq signal at the 1000 IL-2 pDHSs in TB IL-2 and TB+ IL-2 (Bevington et al., 2016).

C. Overlap of the 411 JUNB bound IL-2 pDHSs in Th17 cells (Carr et al., 2017) with the 376 JUND bound IL-2 pDHSs in T-blast (Th0) cells.

D. Average JUND ChIP-Seq signal at the 1000 IL-2 pDHSs in Junb+/+CD4Cre (Junb WT) and Junbf/fCD4Cre (Junb KO) Th17 cells.

E. Genome browser tracks showing open chromatin (DNase-Seq or ATAC-Seq) and ChIP-Seq in Th0, Th1 and Th17 cells at the Il17a locus (Carr et al., 2017, Ciofani et al., 2012, Vahedi et al., 2012). The grey box indicates a DHS which binds JUND.

F. Fold change in gene expression in TB+ IL-2 cells for Jund -/- compared to Jund+/+ mice. Data from 2 replicates are shown.

G. Genome browser tracks showing open chromatin (DNase-Seq or ATAC-Seq) and ChIP-Seq in Th0, Th1 and Th17 cells at the Irf8 locus. The grey box indicates an IL-2 pDHS which binds JUND.

Expanded View Figure Legends
Systematic analysis of BRAF(V600E) melanomas reveals a role for JNK/c-Jun pathway in adaptive resistance to drug-induced apoptosis. (2015)
Figure 3

c-Jun activity up-regulation by RAF inhibitors causes resistance to apoptosis

A c-Jun and p-c-Jun(Ser63) changes as measured by RPPA in six melanoma cell lines in response to different doses of RAF and MEK inhibitors for 10 h (or 5 h in the case of WM115 cell line) and 48 h.

B, C Synergistic apoptosis induction in four cell lines (WM115, WM1552C, LOXIMVI, and COLO858) treated for 72 h with combinations of vemurafenib and JNK-IN-8. (B) Dose–response profiles for apoptosis induction with vemurafenib and JNK-IN-8 combination. (C) Excess over the predicted Bliss independence (EOBI) calculated for different combined doses of vemurafenib and JNK-IN-8.

D c-Jun expression in WM115 cells transfected with JUN siRNA relative to no RNA and non-targeting controls quantified in triplicate 48 h after transfection.

E, F Apoptosis in WM115 cells with or without 48 h JUN knockdown after 96-h treatment with increasing doses of vemurafenib (E) and selumetinib (F).

Data information: Data are presented as mean ± SD.Source data are available online for this figure.
Systematic analysis of BRAF(V600E) melanomas reveals a role for JNK/c-Jun pathway in adaptive resistance to drug-induced apoptosis. (2015)
Figure 4

A network perspective on adaptive responses

A Left: A consolidated overview of PLSR-derived variable importance in the projection (VIP) scores mapped onto a simple schematic of immediate-early signaling, indicating the fraction of 10 studied cell lines in which early (1–10 h) or late (24–48 h) up- or down-regulation of each of the cell state markers and phospho-proteins is predictive of response to RAF/MEK inhibitors (72-h viability). Up-regulation or down-regulation of signals with VIP score >1 is shown. Right: Correlation between up- and down-regulation of selected pathways in response to RAF/MEK inhibition across the 10 studied cell lines. Correlations between signaling changes in response to RAF/MEK inhibition were evaluated based on pairwise Spearman's correlation between VIP scores for selected phospho-proteins at 24 and 48 h post-treatment across the 10 studied cell lines. P-values <0.05 were considered significant.

B-D Time–dose–response plots indicating changes in RPPA measurements for six selected signals, p27 versus p-histone H3 (B), pERK(Thr202/Tyr204) versus pAKT(Ser473) (C), and pS6(Ser235/236) versus total c-Jun levels (D), for two selected cell lines (C32 and WM115) after exposure to PLX4720. Mean values of four biological replicates are shown. Protein levels represent log2 fold change of each signal (at a specific dose and time) relative to a DMSO-treated control.
Systematic analysis of BRAF(V600E) melanomas reveals a role for JNK/c-Jun pathway in adaptive resistance to drug-induced apoptosis. (2015)
Figure 6

A multifactorial adaptive response, rather than initial target inhibition, determines melanoma response to RAF inhibitors

Pairwise Spearman's correlation between the 1-h changes in pERK(Thr202/Tyr204) levels as measured by immunofluorescence microscopy and 72-h relative viability both represented by the z-score-scaled AUC of the seven dose–response curves for four RAF inhibitors and one MEK inhibitor across 8/10 cell lines investigated in this study.

Comparison of the level of 1-h pERK inhibition for 8 cell lines treated with SB590885 versus PLX4720.

Comparison of the 72-h measured relative viability AUC for 8/10 cell lines treated with SB590885 versus PLX4720.

Comparison of the level of 1-h pERK inhibition between two cell lines (C32 and WM1552C) treated with four RAF inhibitors and one MEK inhibitor.

Comparison of the 72-h measured relative viability AUC between two cell lines (C32 and WM1552C) treated with four RAF inhibitors and one MEK inhibitor.

Pairwise Spearman's correlation between the 24-h changes in pS6(Ser235/236) levels as measured by immunofluorescence microscopy and 72-h relative viability both represented by the z-score-scaled AUC of the dose–response curves for four RAF inhibitors and one MEK inhibitor across 8/10 cell lines investigated in this study.

Pairwise Spearman's correlation between the 24-h p-cJun levels as measured by immunofluorescence microscopy and 72-h relative viability after treatment with PLX4720 for 8 cell lines. p-cJun levels are averaged over five doses (0.1–5 μM), and viability is represented by the AUC of the dose–response curves.

Comparison of the level of 24-h pS6 inhibition for 8 cell lines treated with SB590885 versus PLX4720.

Comparison of the level of 24-h pS6 inhibition between two cell lines (C32 and WM1552C) treated with four RAF inhibitors and one MEK inhibitor.

Data information: P-values in (B–E, H, I) were calculated using one-tailed paired Student's t-test. Source data are available online for this figure.
PLK1-dependent phosphorylation restrains EBNA2 activity and lymphomagenesis in EBV-infected mice (2021)
Figure 1

Figure 1

EBNA2 and PLK1 complexes in EBV infected B cell lines

A, B Co-immunoprecipitation (IP) of EBNA2 (A) and PLK1 (B) using EBNA2-, PLK1-specific antibodies, or isotype-matched control antibodies performed on total cell lysates (L) of lymphoblastoid long-term B cell cultures (LCLs) infected with recombinant EBV strain 2089, which is based on EBV strain B95-8 (Delecluse et al, 1998).
PLK1-dependent phosphorylation restrains EBNA2 activity and lymphomagenesis in EBV-infected mice (2021)
Figure 2

Figure 2

EBNA2/PLK1 phosphorylation-dependent complex formation

(A-C) (A) Schematic outline of EBNA2, its dimerization domains (END, DIM), the region used by CBF1 to recruit EBNA2 to DNA (WW), the C-terminal transactivation domain (TAD), the nuclear localization signal (N) and the EBNA2 fragments used to map the PLK1 docking site (UniProt ID: P12978.1). The panel on the right summarizes the results of the co-immunoprecipitations in (B) and (C). Transfection of DG75 B cells and immunoprecipitation of (B) HA-tagged or for smaller fragments (C) GFP-tagged EBNA2 fragments to co-precipitate endogenous PLK1. Total protein lysates and immunoprecipitates (IP) were analyzed by Western blotting (WB).

(D) Multiple sequence alignment (left) and superposition (right) of several phosphopeptides present in published crystal structures of PLK1 PBD (phosphorylated residues stained red). Crystal structures of the PBD in complex with peptides show that the positively charged groove of PBD docks in a similar mode to the negatively charged phosphopeptides. References for PDB ID: 1Q4K (Cheng et al, 2003), 1UMW (Elia et al., 2003a), 4E9C (Śledź et al, 2012), 3C5L (Yun et al, 2009), 3HIK (Yun et al., 2009), 5X3S (Lee et al, 2018), 3Q1I (Pavlovsky et al, 2012). Potential residues of EBNA2, which might be a PBD docking site, are listed below.

(E) Immunoprecipitation of HA-tagged EBNA2 mutant ST266AV, TSS377VAA and SPSS467APAA using HA-specific antibodies.

(F) ITC thermogram of PLK1 PBD titrated with the peptide PNTSSPS or the phosphopeptide PNTSpSPS of EBNA2.

(G) GST-pulldown of PLK1 from total cellular extracts using Cyclin B/CDK1 phosphorylated GST-EBNA2 region 342-422.

(H) Co-immunoprecipitation of transfected EBNA2 wt or S379A and endogenous PLK1.