EMBO/soda-nel huggingface dataset:
Systematic analysis of IL-17 receptor signalosome reveals a robust regulatory feedback loop (2020)
Figure 3

Figure 3. Kinases TBK1 and IKKε modulate the composition of IL-17RSC

A-B. ST2 wild type or TBK1/IKKε DKO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (A) The heatmap shows the row-normalized Z-Score calculated from log2 transformed iBAQ intensities from five independent experiments. (B) The ratio between iBAQ intensities of selected IL-17RSC components to iBAQ intensity of IL-17RC. Mean + SEM from five independent experiments is shown, statistical significance was determined by two‐tailed Mann-Whitney test.

C. ST2 wild type or TBK1/IKKε DKO cells were stimulated with SF-IL-17 (500 ng/ml) for 15 minutes or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC and samples were analyzed by immunoblotting. ACT1 phosphorylation detected as upper band in WT cells is absent in TBK1/IKKε DKO cells.

D. Cells were pretreated with TAK1 inhibitor 7-oxozeanol (2 µM) and/or TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL-17 (500 ng/ml) for indicated time points and lysates were analyzed by immunoblotting.

E. ST2 wild type or HOIP-deficient cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL-17 (500 ng/ml) as indicated, solubilized, and analyzed by immunoblotting. * indicates unspecific band.

F. ST2 cells pretreated or not with TBK1/IKKε inhibitor MRT67307 were stimulated with SF-IL-17 for 15 minutes or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated and analyzed via immunoblotting.

Data information: Immunoblot results are representative of two (F), three (C, D) or four (E) independent experiments.
Systematic analysis of IL-17 receptor signalosome reveals a robust regulatory feedback loop (2020)
Figure 4

Figure 4. Enzymatic activity of TRAF6 and NEMO is crucial for the recruitment of both TBK1 and IKKε to IL-17RSC

A. ST2 wild type and ACT1-deficient cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. As a control, cells were first solubilized and SF-IL-17 was added only post lysis. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in two independent experiments are shown.

B. ST2 wild type, ACT1 KO or TRAF6 KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC and samples were analyzed by immunoblotting.

C. ST2 cells deficient in TRAF6 reconstituted with TRAF6(WT) or enzymatically inactive TRAF6(C70A) were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in two independent experiments are shown.

D. TRAF6 deficient cells reconstituted with TRAF6(WT), TRAF6(C70A) or empty vector were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated from lysates by anti-Flag immunoprecipitation and analyzed by immunoblotting. Short and long exposure of the same membrane stained for Act1 are shown for lysate and IP samples, respectively.

E-F. TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) and stimulated with IL-17 (500 ng/ml) for indicated time. (E) The activation of signaling pathways analyzed by immunoblotting. (F) The induction of mRNA for selected genes analyzed by real time PCR. Mean + SEM from four independent experiments is shown, statistical significance was determined using unpaired two‐tailed Student's t‐test.

G. ST2 cells wild type or NEMO KO were stimulated with IL-17 (500 ng/ml) for indicated time points, solubilized and analyzed by immunoblotting.

H-I. ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC. Samples were analyzed by immunoblotting (H). In addition, results from four independent experiments were quantified by densitometry and mean + SEM from four independent experiments is shown (I).

Data information: Immunoblot results are representative of two (B, E), three (D), or four (G, H, I) independent experiments.
Systematic analysis of IL-17 receptor signalosome reveals a robust regulatory feedback loop (2020)
Figure 5

Figure 5. NEMO functions as an inhibitor of IL-17RSC assembly

A-B. ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (A) The heatmap shows the row-normalized Z-Score calculated from log2 transformed iBAQ values from two independent experiments. (B) The ratio between iBAQ intensities of selected IL-17RSC components to iBAQ intensity of IL-17RC. Mean + SEM from two independent experiments is shown.

C-D. ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC (C) or tested for activation of signaling pathways (D) and analyzed by immunoblotting.

E. ST2 cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM) or IKKα/IKKβ inhibitor TPCA1 (10 µM), stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.

F. ST2 wild type or NEMO KO cells were stimulated with TNF (50 ng/ml) for indicated time points and lysates were analyzed by immunoblotting.

G. Schematic model of the negative feedback loop mediated by NEMO-recruited TBK1/IKKε.

Immunoblot results are representative of four (C, D), three (E), or two (F) independent experiments.
Systematic analysis of IL-17 receptor signalosome reveals a robust regulatory feedback loop (2020)
Figure 6

Figure 6. Phosphorylation of ACT1 in disordered region leads to the release of TRAF6 from IL-17RSC

A. Schematic representation of ACT1 with indicated TRAF6-binding domain, SEFIR domain and location of phosphorylation sites identified in our MS analysis (based on Table EV6).

B. Analysis of ACT1 structure using three different algorithms predicting disordered regions. Detected phosphorylation sites are all in the disordered region of the protein.

C-D. ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho-sites mutated to alanines (9ST mut). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band.

E. ACT1 KO cells expressing either ACT1(WT) or ACT1(9ST mut) were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated via anti-Flag immunoprecipitation and analyzed by immunoblotting.

F. ACT1 KO cells were reconstituted with either ACT1(WT) or ACT1(Δ20-380). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band.

G. Analysis of iBAQ intensity of ACT1 normalized to iBAQ intensity of IL-17RC. Data are composite of all 14 MS analyses of IL-17RSC from wild type ST2 cells performed in this study. Mean + SEM is shown.

Data information: Immunoblot results are representative of two (C, E) or four (D, F) independent experiments.
CDK8 and CDK19 act redundantly to control the CFTR pathway in the intestinal epithelium (2022)
Figure 1

Fig. 1. CDK8 knockout does not affect adult mouse intestine homeostasis nor chemically-induced carcinogenesis.

A Immunohistochemical staining of CDK8 in mouse small intestines collected two months after tamoxifen treatment. CDK8+/+ depict mice with floxed Cdk8 alleles. 10 mice were used for the experiment (5 males and 5 females). Scale bar, 25μm.

B Analysis of cell differentiation (left) and proliferation (right) in the intestine after CDK8 deletion as in (A). Olfm4, Lysozyme, PAS and Dclk1 staining was used to reveal, respectively, stem, Paneth, goblet and tuft cells. β-Catenin staining allows detection of cancer cells (cytoplasmic vs nuclear localisation). Cell proliferation was assessed by PCNA, Ki-67 and BrdU (2h pulse) staining. Scatter plots represent the percentage of the area stained by each antibody (relative to the area occupied by hematoxylin). For Paneth cells, BrdU, PCNA and Ki67, only crypts were analysed. For goblet cells, crypts and villi were analysed. For Tuft cell quantification, Dclk1-positive cells were counted in 50 villi. Colour code depicts small intestine (green), proximal colon (blue), and distant colon (red). Mean ± SEM is shown. P-value of unpaired two-tailed t-test is indicated (ns, not significant; p > 0.05). Scale bars, 25μm (Olfm4, Lysozyme, Dclk1 and β-Catenin) and 50μm (PAS, BrdU, Ki-67 and PCNA). (n=9 biological replicates)

C Analysis of mouse colon after AOM/DSS treatment. Quantification of the number of neoplastic lesions (n = 10 for Cdk8+/+, and n = 7 for Cdk8 -/- mice). P-value of unpaired t-test is indicated: ns, not significant (p > 0.05). Mean ± SD is shown.

D Quantification of the percentage of the colon surface occupied by tumours. Intestine samples were stained for β-Catenin, and tumour regions with nuclear β-Catenin localisation were quantified using NDP.view software. Two-tailed p-value of unpaired t-test is indicated; ns, not significant (p > 0.05). Mean ± SD is shown. (n=6 biological replicates)

E Example of IHC with β-Catenin staining of tumour-free regions with membrane β-catenin localisation (left) and tumour regions with nuclear β-catenin localisation (right). Scale bars, 50μm.
CDK8 and CDK19 act redundantly to control the CFTR pathway in the intestinal epithelium (2022)
Figure 2

Fig. 2. Double CDK8/CDK19 knockout intestinal organoids show decreased cell proliferation.

A Genotyping confirms the loss of Cdk8 exon 2 in Cdk8 -/- and Cdk8 -/-/Cdk19 -/- organoids after 7 days of OH-tamoxifen treatment. Control plasmids (a, b and c) are described in Fig EV1C.

B WB of organoid samples after 7 days of OH-tamoxifen treatment; β-actin was used as loading control. C Phase contrast images of organoids before and after 6 days of OH-tamoxifen treatment. Scale bars, 150μm.

D Quantification of organoid size at day 6, shown in (C) (mean ± SEM are shown). P-value, one-way ANOVA (Bonferroni test): (*) p ≤ 0.05, (***) p ≤ 0.001; ns, not significant (p > 0.05). (n=47 biological replicates)

E IHC staining of organoids (day 7 of OH-tamoxifen treatment) with Ki-67 antibody. Scale bars, 100μm. F Quantification of Ki-67 positive area (% of the total area of the organoids; mean ± SEM) in the four different genotypes presented in (E). Areas with positive Ki-67 signal were detected and quantified using QuPath and ImageJ programs. Four technical replicates from 2 biological replicates of each genotype were analysed. Adjusted p-values of ordinary one-way ANOVA (Bonferroni test) are indicated: (**) p-values ≤ 0.01; (*) p-values ≤ 0.05; ns, not significant (p > 0.05).

G Quantification of the increase in size over 9 days of control and Senexin B-treated (10μM) organoids (n=10 biological replicates, mean ± SEM are shown). P-value of unpaired two-tailed t-test is indicated: (*) p ≤ 0.05.
CDK8 and CDK19 act redundantly to control the CFTR pathway in the intestinal epithelium (2022)
Figure 3

Fig. 3. CDK8 and CDK19 show functional redundancy in regulation of gene expression, while their knockout does not perturb differentiation in intestinal organoids.

A Dot plot analysis of differentially expressed genes (DEGs). Red dots: DEGs with p-value ≤ 0.05; purple dots: log2 fold change (LFC) >1 or <-1, p-value ≤ 0.05; grey dots: not significant. Numbers inside plots indicate the number of genes deregulated more than 2-fold. 3 biological replicates of each genotype were used for this analysis.

B Venn diagrams indicating intersection of genes with altered expression in the indicated genotypes.

C Left, IHC staining of WT and Cdk8 -/-/Cdk19 -/- organoids with Olfm4 (stem cells), Lysozyme (Paneth cells), Muc2 (goblet cells), and Dclk1 (tuft cells) antibodies. Scale bars, 50μm (Olfm4, Muc2, Dclk1) and 25μm (Lysozyme). Right, quantification of Olfm4, Lysozyme and Muc2 positive area (% of the total organoid area as indicated by hematoxylin staining; mean ± SEM), or the number of Dclk1 positive cells per organoid. P-value, unpaired two-tailed t-test: (***) p-value ≤ 0.001; ns, not significant (p > 0.05). 20 different organoids from 2 biological replicates of each genotype were used for the analysis.

D qRT-PCR analysis of indicated mRNA levels in Wt and Cdk8-/-/Cdk19 -/- (top) or Senexin B-treated (10μM, for 24h; bottom) organoids, relative to WT or vehicle (DMSO)-treated organoids, respectively, showing mean ± SEM. 3 technical replicates were used for this analysis.
CDK8 and CDK19 act redundantly to control the CFTR pathway in the intestinal epithelium (2022)
Figure 4

Figure 4. CDK8 and CDK19 regulate the CFTR pathway in the small intestine.

A Gene set enrichment analysis (using Enrichr database) of highly deregulated genes in Cdk8-/-/Cdk19-/- organoids. Manually curated signatures extracted from RNA-seq studies in GEO where gene expression was measured before and after drug treatment, gene perturbation or disease.

B Venn diagrams indicating intersection of genes with altered expression in the Cftr mouse knockout small intestine (ileum, top, and duodenum, bottom) and Cdk8-/-/Cdk19 -/- organoids. Numbers below give the percentage of each gene set in the intersection.

C qRT-PCR analysis of indicated mRNA levels in Wt, Cdk8-/-, Cdk19-/- and Cdk8-/-/Cdk19 -/- organoids, showing mean ±SEM. 3 technical replicates were used for the analysis.

D WB of indicated proteins extracted from Wt and Cdk8 -/-/Cdk19 -/- organoids. Included are CFTR positive and negative control cell line samples obtained from the Cystic Fibrosis Foundation (CFF). Amidoblack was used as loading control.

E Dot plot analysis of differentially expressed genes (DEGs) in Senexin B (Snx B)-treated (10μM; for 16h, top, and 24h, bottom) organoids. Red dots: DEGs with p-value ≤ 0.05; purple dots: log2 fold change (LFC) >1 or <-1, p-value ≤ 0.05; grey dots: not significant, NS. Numbers inside plots indicate the number of genes deregulated more than 2-fold.

F Gene set enrichment analysis (using Enrichr database) of highly deregulated genes in Senexin B-treated (16h) organoids. The signatures were extracted from RNA-seq studies in GEO where gene expression was measured before and after drug treatment, gene perturbation or disease, and an additional step of manual curation was performed to select those related to the intestine.
CDK8 and CDK19 act redundantly to control the CFTR pathway in the intestinal epithelium (2022)
Figure 5

Fig. 5. Knockout or inhibition of CKD8/19 leads to Cftr mutant-like phenotype.

A Histological PAS staining of organoids treated for 7 days with OH-tamoxifen. Scale bar, 50μm.

B Quantification of PAS signal (% of total organoid area; mean ± SD are shown) in the four different genotypes presented in (A). Areas containing positive PAS staining were detected and quantified using QuPath and ImageJ programs. Adjusted p-values of ordinary one-way Anova followed by Tukey's multiple comparison test are indicated: (***) p-value ≤ 0.001; ns: not significant (p > 0.05). Four technical replicates from 2 biological replicates of each genotype were analysed.

C Representative phase contrast images of organoids at the indicated time points after 7 days of OH-tamoxifen treatment are shown. Scale bar, 100 μm.

D Quantification of the time needed for mucus release (observed as a dark staining in the center of the organoid; mean ± SD are shown). Adjusted p-values of ordinary one-way Anova followed by Tukey's multiple comparison test are indicated: (***) p-value ≤ 0.001; ns: not significant (p > 0.05); (n= 17 for Wt, 11 for Cdk8 -/-, 18 for Cdk19 -/-, and 12 for Cdk8 -/-/ Cdk19 -/- , all biological replicates).

E Fluorescence confocal microscopy images of Calcein green-labeled WT and Cdk8-/-/Cdk19-/- organoids treated with forskolin. Scale bars, 100 μm.

F Quantification of forskolin-induced swelling in WT organoids treated for 1hour or 24 hours with 0.1μM, 1μM or 10μM Senexin B (SenB), as indicated, or double KO organoids; DMSO vehicle was used as control. The surface area of individual organoids at different time points relative to the area at t = 0 (100%) was measured (mean ± SD, n=8 biological replicates). Linear regression lines are shown.

G qRT-PCR analysis of Cftr and Muc3 mRNA levels in WT organoids either not treated (t=0), or treated with 10µM Senexin B for 2 or 24 hrs, showing mean ± SEM. 3 technical replicates were used for the analysis.

H WB of indicated proteins extracted from Senexin B-treated (10μM, 2h and 24h) organoids (left), and WT and Cdk8 -/-/ Cdk19 -/- organoids (right). Amidoblack was used as loading control.
A cAMP signalosome in primary cilia drives gene expression and kidney cyst formation (2022)
Figure 6

Figure 6: Ciliary cAMP signaling evokes a specific gene expression signature controlling cyst development.

(A) Differentially expressed (DE) genes unraveled by RNA-Sequence analysis of the conditions shown Fig. EV4A and B (p value < 0.05). Wild-type (WT), cilia-bPAC, and cyto-bPAC: light-stimulated cells were compared to cells kept in the dark. Forskolin: WT cells stimulated with 10 µM of Forskolin were compared to WT cells incubated with DMSO (control).

(B) Venn diagram of the DE genes; for more details, see Table 1.

(C) Participation of the DE genes identified in cilia-bPAC cells in specific reactomes; for more details, see Table 2.

(D) Venn diagram of the transcription factor binding-sites unraveled by Transcription Factor Enrichment Analysis of the DE genes detected exclusively in cilia-bPAC or cyto-bPAC cells; for more details, see Table 3.

(E) Quantitative RT-PCR analysis. WT, cilia-bPAC, or cyto-bPAC cells were treated as shown in Fig. EV4A and B. WT, cilia-bPAC, and cyto-bPAC: light-stimulated cells were compared to cells kept in the dark. Forskolin: WT cells stimulated with 10 µM of Forskolin were compared to WT cells incubated with DMSO (control). Data are shown as mean ± S.D., p values were calculated by a two-sided, unpaired Student's t-test compared to WT. Data points show individual experiments.

(F) 3D culture of mIMCD-3 cells stably expressing cilia-bPAC, incubated with DMSO (control) or 20 µM Celecoxib, a COX-2-inhibitor, in the dark or during light exposure (1 light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², started 2 d after pre-incubation with DMSO or Celecoxib). Exemplary images are shown (n = 3). Scale bars are indicated.

(G) Quantification of images exemplified in (F) and from 3D cultures incubated with 1 μM celecoxib. Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data points show individual experiments.

(H-I) Quantification of Forskolin- (H) and PGE2-dependent (I) cyst formation in the presence or absence of 20 μM celecoxib. Exemplary images are shown in the Fig. EV4C-D. Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data points show individual experiments (n =3). Forskolin / PGE2 stimulation started 2 d later than incubation with DMSO (0 µM celecoxib) or 20 µM celecoxib.
Anti-tumoral activity of the G-quadruplex ligand pyridostatin against BRCA1/2-deficient tumours (2022)
Figure 1

Figure 1. Pyridostatin inhibits growth of BRCA2-deficient DLD1 xenograft tumours and inflicts repairable DNA damage in BRCA2-deficient cells.

A, B CB17-SCID male mice were injected intramuscularly into the hind leg muscles with (A) BRCA2-proficient (BRCA2+/+) or (B) BRCA2-deficient (BRCA2-/-) DLD1 cells. Pyridostatin (PDS) was administered intravenously (i.v.; 7.5 mg/kg/day) and talazoparib was administered orally (p.o.; 0.33 mg/kg/day), over the indicated periods of time. Vertical dashed line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 5 mice. Error bars represent SEM. P values were calculated between treated and untreated tumours at day 17, using an unpaired two-tailed t-test. ***, P ≤ 0.001; NS, P > 0.5.

C DNA breaks were measured using alkaline comet assay in BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with 2 µM pyridostatin (PDS) for 16 hours and released into fresh medium without pyridostatin. Representative images are shown. Scale bar represents 100 µm.

D Quantification of DNA breaks shown in (C). Graph and error bars represent the mean and SEM of n = 3 independent experiments. A minimum of 50 cells were analysed per condition per experiment. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; NS, P > 0.5.

E Quantification of γH2AX foci visualised using immunofluorescence staining in cells treated as in (C). A minimum of 200 cells were analysed per condition per experiment. Graph and error bars represent the mean and SEM of n = 3 independent experiments. P values were calculated using an unpaired two-tailed t-test. ***, P ≤ 0.001; NS, P > 0.5.

F BRCA2-deficient (-BRCA2) human DLD1 cells were treated with 2 µM pyridostatin (PDS) for 24 hours. Next, pyridostatin was removed and cells were released in a medium containing 250 nM NU-7441 (NU). Whole-cell extracts were prepared at the indicated timepoints after release and immunoblotted as shown. SMC1 was used as a loading control. KAP1 phosphorylation site is indicated in red.

G Dose-dependent viability assays of BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with pyridostatin (PDS) and NU-7441 at the indicated concentrations for six days. Graphs represent average values obtained from of n = 3 independent experiments, each performed in technical triplicates.

Exact P values for (A-B, D-E) are provided in Appendix Table S8.