EMBO/soda-nel huggingface dataset:
Chromatin-bound cGAS inhibits DNA repair hence accelerates genome destabilization and cell death (2019)
Figure 3

Figure 3. cGAS inhibits DNA damage repair and promotes micronuclei generation and cell death independently of STING.

A, B. BMDMos from cGAS-/- mice exhibit enhanced DNA repair efficiency than those from WT and Sting-/- mice. (A) Representative comet tails of WT, cGAS-/- and Sting-/-BMDMos exposed to γ-irradiation (IR: 10 Gy) on ice then incubated at 37 ̊C for indicated duration. (B) Corresponding quantification of the comet tail moments from 20 different fields with n > 200 comets of 3 independent experiments.

C, D. cGAS promotes micronuclei generation in BMDMos independently of STING. (C) Confocal microscopic visualization of micronuclei (indicated by arrow head) and cGAS staining in WT, cGAS-/- and Sting-/- BMDMos exposed to γ-irradiation (10Gy). Scale bar: 10 μm. (D) Average MNs/cell in corresponding representative images. Bar graphs show mean values from eight different microscopic fields with over 200 cells.

E, F. γ-irradiation-induced cell death in WT, cGAS-/- BMDMos (E). γ-irradiation-induced cell death in Sting-/- and cGAS-/- Sting-/- BMDMos (F).

Data information: Data are presented as Mean ± SEM of n=3 independent experiments. Statistical significance was assessed using two-way ANOVA in (B) and one-way ANOVA in (D),(E),(F) followed by Sidak's post-test. *P < 0.05, **P < 0.01, ***P < 0.001 ****P < 0.0001.
Chromatin-bound cGAS inhibits DNA repair hence accelerates genome destabilization and cell death (2019)
Figure 4

Figure 4. cGAS accelerates γ-irradiation-induced depletion of bone marrow cells independently of STING.

A-D. Kinetics of in vivo depletion of indicated bone marrow cells in WT mice (n=3) after γ-irradiation (9 Gy).

E-H. Bone marrow cells in WT (n=3) and cGAS-/- (n=3) mice 10 hours post γ-irradiation.

I-L. Bone marrow cells in Sting-/- (n=3) and cGAS-/- Sting-/- (n=3) mice 10 hours post γ-irradiation.

Data information: Data in this figure are presented as Mean ± SD. One-way ANOVA followed by Sidak's post-test. NS: P >0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ****P ≤ 0.0001.
Chromatin-bound cGAS inhibits DNA repair hence accelerates genome destabilization and cell death (2019)
Figure 5

Figure 5. DNA binding and subsequent oligomerization of cGAS is essential for inhibition of HR-DNA repair.

A, B. Schematics of HR and NHEJ reporter assays.

C, D. Obtained results showing enhanced HR efficiency upon knockdown of endogenous cGAS in U2OS cells. Immunoblot inserts in C and D depict knockdown efficiency of cGAS and Histone H1.2.

E, F. Results showing the effect of hcGAS, hcGAS△cGAMP, hcGAS△DNA or hcGAS△Oligo on HR (E) or NHEJ (F) in HEK293 cells. Corresponding immunoblot inserts in E and F depict cGAS expression.

Data information: Data are means with SEM, n=3. Statistical significance was assessed using one-way ANOVA followed by Sidak's post-test. NS: P >0.05, * P ≤ 0.05, **** P ≤ 0.0001.
Chromatin-bound cGAS inhibits DNA repair hence accelerates genome destabilization and cell death (2019)
Figure 6

Figure 6. Nuclear cGAS is constantly bound to the chromatin and not specifically recruited to DSB sites.

A. cGAS is not recruited to DSB sites: Confocal microscopic images of GFP-NLS- or GFP-hcGAS-expressing U2OS-DSB reporter cells incubated (or not) with Shield-1 and 4-OHT to induce the expression and translocation of mCherry-LacI-FokI (red) to specific DSB sites. Scale bar: 10μm. The arrowheads indicate DSB sites.

B. cGAS does not colocalize with γ-H2AX at DSB sites: GFP-NLS- or GFP-hcGAS- expressing HEK293 cells exposed (or not) to γ-irradiation (IR: 10 Gy) then stained for γ-H2AX. Scale bar: 10 μm.

(C, D) Nuclear cGAS is mainly chromatin-bound and remains unaltered upon γ-irradiation. (C) Cytosolic (cyto) and nuclear fractions of γ-irradiated (10 Gy, 30 min) BMDMos analyzed for cGAS and indicated molecules. (D) Cytosolic, soluble nuclear and chromatin fractions from BMDMos were immunoblotted for cGAS and indicated proteins.

E-G. cGAS co-isolates with DNA repair proteins because of bound chromatin bridges. (E) Nuclease digestion abrogates the co-isolation of cGAS and DNA repair proteins: Lysates of control (-IR) and γ-irradiated (+IR, 10 Gy, 30 min) GFP-hcGAS expressing HEK293 cells were treated (or not) with Benzonase before cGAS immunoprecipitation and analysis for indicated proteins. (F) Agarose gel analysis of DNA in corresponding cell lysates in (E). (G) Co-isolation of cGAS and DNA repair proteins depends on its binding to DNA: cGAS pulldowns along with lysate inputs of control and γ-irradiated HEK293 cells expressing GFP-hcGAS or GFP-hcGASΔDNA probed for indicated proteins.
Chromatin-bound cGAS inhibits DNA repair hence accelerates genome destabilization and cell death (2019)
Figure 7

Figure 7. cGAS inhibits the HR-DNA repair by impeding RAD51-mediated strand invasion.

A. Confocal images of γ-irradiated GFP-NLS-, GFP-hcGAS-expressing HEK293 cells stained for RAD51 (red) with or without γ-irradiation. Scale bar: 10 μm.

B. Schematics of the D-loop formation assay, including pre-incubation of template dsDNA with cGAS-cat (i), or with cGAS-cat being added after RAD51 was bound to dsDNA (ii).

C. Pre-incubation of dsDNA with mcGAS-cat prevents D-loop formation by human RAD51, but does not affect the RAD1 activity once RAD51 filaments are bound to dsDNA. The percentage of D-loop formed in each reaction (left) was graphed as the average of triplicates ± SD.

D. Schematics of the D-loop assay.

E. Pre-incubation of template dsDNA with hcGAScat blocks subsequent D-loop formation. The percentage of D-loop formation (below) was graphed as the average of triplicates±SD.

Data information: unpaired two-tailed Student's t-test was used for statistical analyses. NS P >0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.
Chromatin-bound cGAS inhibits DNA repair hence accelerates genome destabilization and cell death (2019)
Figure 8

Figure 8. cGAS compacts dsDNA and inhibits D-loop formation via oligomerization.

A. Negative-stain electron micrographs of cGAS-dsDNA complexes following incubation of dsDNA with indicated cGAS variants. Scale bar: 100 nm.

B. Effect of indicated hcGAS variants on D-loop formation when pre-incubated with dsDNA.

C. Percentage of D-loop formed in each reaction (left) graphed as the average of triplicates± SD.

D. Overview of a single 1:1 hcGAS-DNA complex depicting the location of the Y215 within the cGAS-dsDNA interface.

E. DR-GFP assay showing that hcGASΔDNA-Y215E is impaired in HR inhibition.

F. hcGASΔDNA-Y215E but not hcGASΔcGAMP has a decreased affinity to dsDNA24.

G. hcGASΔDNA-Y215E and hcGASΔcGAMP are defective in synthase activity.

H. Negative-stain electron micrographs showing that hcGAScat-ΔDNA-Y215E is defective in inducing cGAS-dsDNA complexes. Scale bar: 100 nm.

I. Effect of indicated hcGAS variants on D-loop formation.

Data information: Data are means ±SD, n=3. unpaired-student's t‐test was used for statistical analyses: NS P >0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Expanded View Figure Legends:
A novel phosphocholine-mimetic inhibits a pro-inflammatory conformational change of C-reactive protein (2022)
Figure 1

Figure 1: Binding studies of tool compound C10M to pCRP in vitro.

A Design of the phosphonate compound C10M was guided by the PC:pCRP complex (PDB ID: 1B09), with two n-butyl substituents (nBu) on the tertiary amine exploiting available space in the binding pocket along the denoted vectors R1 and R2. Calcium cations shown as yellow spheres and the location of pCRP residues Asn 61 (N61), Glu 81 (E81) and Gln 150 (Q150) are indicated. pCRP depicted as a light green molecular surface.

B C10M reduces binding of pCRP to immobilized PC. pCRP was incubated with p-aminophenyl phosphoryl choline agarose beads under binding conditions with and without C10M. Porous solid column chromatography was then used to evaluate the binding capacity of pCRP to PC. Biological replicates, n=3, mean ± SEM. P value was calculated with Student's t-test.

C The crystal structure of pCRP (shown as cartoon) in complex with C10M (pink spheres) confirmed that the compound binds to the same pocket as PC/PE. The asymmetric unit consisted of 4 stacked pCRP pentamers; two pairs of pentamers, 1 & 2 and 3 & 4, are stacked A-face to A-face. Pentamers 2 and 4 are colored grey to show their relative location to pentamers 1 and 3 (colored green). C10M was bound to all CRP monomeric subunits (Figure EV1, Table EV1 and EV2). Each CRP monomeric subunit contained two calcium cations (depicted as yellow spheres). Orthogonal views of the asymmetric unit are shown.

D Structure of one pentamer from the asymmetric unit. Interaction of the phosphonate moiety with the bound calcium cations (yellow spheres) and hydrogen bonds with Asn 61 (N61), Glu 81 (E81), and Gln 150 (Q150) (black dashed lines) anchor the compound in the binding pocket. Full list of interacting residues is given in Table EV1.

E Alignment of C10M (pink/blue/red/orange sticks) and PC (cyan/blue/red/orange sticks) in complex with pCRP via the Cα atoms of pCRP. One monomeric subunit of pCRP is depicted as a molecular surface, the location of acidic (red), basic (blue) and hydrophobic (grey) residues around the PC binding pocket is indicated. Calcium cations shown as yellow spheres.
A novel phosphocholine-mimetic inhibits a pro-inflammatory conformational change of C-reactive protein (2022)
Figure 2

Figure 2: Studies on pCRP binding to activated platelets in the presence of C10M.

A Flow cytometry was used to determine binding of pCRP to activated platelets. P-selectin positive platelets were analyzed for CRP-Atto 594 fluorescence. Platelets were incubated without labeled pCRP (control), and with labeled pCRP (50 μg/ml) ± C10M (1:100 molar ratio). Results are displayed as mean ± SEM. P values were calculated with ANOVA and Tukey's post hoc test. Biological replicate, n=3.

B Confocal fluorescence microscopy of ADP-activated human platelets in the presence of pCRP. Depicted are 3D reconstructions of monolayers of ADP (20 μM) activated platelets. Washed platelets were detected by anti-CD62P (green). 50 μg/ml pCRP-Atto 594 (red) was incubated with isolated platelets with (bottom) or without (top) C10M. pCRP localizes on platelets, which can be inhibited by C10M. Scale bar 10 μm.

C Western blot of pCRP binding to activated platelets. Platelets bind less pCRP when incubated with C10M as detected with the anti-CRP antibody, compared to the anti-GAPDH antibody as a control. Washed human platelets were incubated with pCRP and pCRP+C10M, respectively, lysed and separated on SDS-PAGE.

D Densitometric quantification of protein bands of Western blots (biological replicates, n=3) from the experiment described in (C). P value was calculated by Student's t-test. Graph shows mean ± SEM.
A novel phosphocholine-mimetic inhibits a pro-inflammatory conformational change of C-reactive protein (2022)
Figure 3

Figure 3: Studies on CRP-dependent expression of pro-inflammatory cytokines and inhibiting effects of C10M.

A CRP-coated platelets visualized by confocal microscopy bound to whole blood leukocytes. Washed platelets (green) were incubated in serum supplemented with 50 μg/ml pCRP-Atto 594 (red), without (left) and with C10M (right, 1:100 molar ratio). Platelets were then added to whole blood samples and imaged after RBC lysis. Shown are platelet-leukocyte-aggregates with and without CRP attached. Scale bar indicates 5 µm.

B,C CRP induces platelet-leukocyte aggregation in a time-dependent and conformation-specific manner. Whole blood samples stimulated with either 50 μg/ml pCRP (B) or mCRP (C) were analyzed by flow cytometry. Double positive events (CD62P and CD14) were identified as platelet-monocyte complexes. Incubation was stopped at indicated time points. Displayed are mean ± SEM. P values were calculated with ANOVA and Tukey's post hoc test. Biological replicates, n=6.

D Static adhesion of monocytes to 20 μg/ml fibrinogen was tested as described before. Purified monocytes (3 x 106 per ml) were stimulated with 50 μg/ml pCRP and 20 μM ADP and allowed to adhere for 35 min. ADP was used to activate supplemented platelets. Monocyte adhesion was quantified by calorimetric method and absorbance measured after 30 min. Results are shown as mean ± SEM. P values were calculated using ANOVA and Tukey's post hoc test. Biological replicates, n=5.

E Expression of TNF, IL1β and IL6 (from left to right) in monocytes analyzed by flow cytometry. Addition of C10M to the whole blood samples inhibits the CRP-dependent expression of pro-inflammatory cytokines. P values were calculated with ANOVA and Tukey's post-hoc test. Biological replicates, n=6 (IL6), n=5 (TNF) and n=4 (IL1β), respectively, bars indicate mean ± SEM.

F Representative examples of whole blood samples treated with pCRP and C10M visualized by confocal fluorescence microscopy. Depicted are 3D reconstructions multiple focal planes at 20x magnification. The leukocytes were stained with DAPI (blue) and anti-TNF-FITC (green). Scale bar 50 μm.
A novel phosphocholine-mimetic inhibits a pro-inflammatory conformational change of C-reactive protein (2022)
Figure 5

Figure 5: pCRP*/mCRP-driven exacerbation of renal ischemia/reperfusion injury is reduced by C10M.

A Depiction of the experimental protocol used for ischemic acute kidney injury. Male Wistar rats were subjected to IRI and received i.p. DPBS and pCRP twice (green syringe). C10M was i.v.-injected separately every 6 hours (blue syringe).

B Detection of C10M by mass spectrometry in rat urine. Renal excretion of C10M detected by mass spectrometry in three rats (biological replicates, n=3) intravenously injected with C10M. Urine samples were collected at the indicated timepoints. 80% of the applied C10M mass was excreted after 90 min (Kather et al, 2021).

C Immunohistochemistry of rat kidneys subjected to IRI and i.p. pCRP application revealed distinct staining by anti-pCRP*/mCRP-9C9 antibody (green, arrows). C10M reduces the deposition of total CRP in the impaired tissue. No deposits in the non-ischemic tissue (sham). Exemplary stainings out of at least three are shown. Scale bars, 100 µm.

D Tissue lysates of rat kidney were separated on SDS-PAGE and total CRP was identified with anti-CRP antibody. A band at the size of mCRP (~23 kDa) was detected in kidneys subjected to IRI and pCRP, but not in animals treated additionally with C10M. The household gene protein GAPDH served as a control for loading equal amounts of protein. 50 and 100 ng human pCRP, respectively, served as a positive control. Representative results are shown for replicated assays (n=3).

E Renal excretion is impaired by pCRP*/mCRP-driven tissue damage. Blood urea nitrogen (BUN) was utilized as surrogate marker for the excretion function of the kidney. Blood samples were taken before the surgical procedure (preoperative) and 24 hours after the procedure (harvest). Graph shows mean ± SEM.

F Immunohistochemical detection of transmigrated CD68+ cells in IRI kidneys. Quantification of immunohistochemical results is shown as mean ± SEM. pCRP (25 µg/ml) increased the number of CD68+ cells transmigrated into injured renal tissue significantly, while C10M abolished these effects. Presented are mean cell counts per ROI in each animal.

G Periodic acid-Schiff (PAS) stained kidney sections show increased damage after renal IRI in rats when pCRP (25 µg/ml) was injected i.v. The tubulointerstitial injury was quantified by the loss of tubular brush border and by cast formation following an established protocol (Megyesi et al, 1998, 2001). Quantification of immunohistochemical results is shown as mean ± SEM.

H Representative results for the immunohistochemical detection of transmigrated CD68+ cells in IRI kidneys. CD68+ cells are stained with HistoGreen substrate (green). Scale bars indicate 100 µm.

I Representative results for the periodic acid-Schiff (PAS) stained kidney sections quantified in (G). Scale bars indicate 100 µm.

Data information: Statistical analysis (E, F, G) was performed with ANOVA and Tukey's post-hoc test. Biological replicates, n=6, precise p-values are given.
A novel phosphocholine-mimetic inhibits a pro-inflammatory conformational change of C-reactive protein (2022)
Figure 6

Figure 6: Compound C10M delays CRP-driven transplant rejection in a hindlimb transplantation model.

A Experimental protocol of the acute allograft rejection model in rats. Lewis (Lew) rats served as donors, Brown-Norway (BN) rats as recipients. Hindlimb rejection was assessed clinically and histologically. pCRP (green syringe) and C10M (blue syringe) were injected i.p. and i.v., respectively, at the time-points indicated.

B Kaplan-Meier plots for control, pCRP, and pCRP+C10M (above) and C10M vs control (bottom) treatment for total allograft survival with and without CRP ± C10M. Kaplan-Meier curves for different treatments were compared by Mantel-Cox log-rank test. Allograft survival was significantly reduced by pCRP administration (P=0.0005, median survival control vs pCRP, 7.8 vs 4.8 days). C10M masks the CRP-accelerated hindlimb rejection (median survival 7.2 days). Biological replicates, n=4 per group.

C Representative photographic examples of transplanted VCA hindlimb allografts. Shown are Lew hindlimb transplants in orthotopic situ on BN recipients three days after transplantation. Rats receiving i.p. pCRP presented VCA with massive edema (left). C10M treatment inhibits pCRP-aggravated early graft rejection (middle). The depicted C10M control without CRP shows no clinical signs of rejection after five days (right).

D,E Representative immunofluorescence images from transplanted grafts reveal distinct signals (green) using anti-CD68 and anti-CRP antibodies, respectively. In C10M treated animals, less CD68+ cells infiltrate the transplanted tissue. Scale bars, 100 μm. (D) Immunofluorescence of transplanted muscle and (E) skin tissue revealed distinct CRP-deposition (green) in pCRP treated animals, but only minimal staining in rats treated additionally with C10M. Scale bars, 100 µm.

F Exemplary tissue lysates of muscle and skin probes separated on SDS-PAGE are depicted. Total human CRP was identified with an anti-CRP antibody.
MiRNA Exporter HuR Clears  Macrophage Internalized Pathogens by Promoting Pro-inflammatory Response (2020)
Figure 6

Figure 6 Inverse regulation of pro-inflammatory pathways by miRNA regulators HuR (ELAVL1) and PP2A (PPP2CA) in mammalian macrophage cells

A-C Interaction routes between HuR (ELAVL1) and its target gene PP2A (PPP2CA). Alternate mechanism or regulatory steps that could be involved in establishing the regulatory relationship between HuR (ELAVL1) and its target genes have been investigated. A brief outline of possible alternate routes of regulation between HuR (ELAVL1) and PP2A (PPP2CA) via intermediates at the protein-protein interaction, cellular signalling, or by miRNA have been outlined in panel A. In panel B and C, effect of HA-HuR expression on PP2A both at protein and RNA levels were scored. Values are mean+/- s.e.m. and, n=3

D-F Expression profiles of HuR, PP2A and miRNA let-7a are connected to IL-6 expression in RAW 264.7 cells. Data obtained from experiments described in previous figures are summed up to plot the changes in IL-6 mRNA levels against time of LPS treatment along with changes in PP2A, miRNA let-7a and HuR (D). Change in expression of IL-6 is connected to changes in let-7a concentration. The mathematical equation fitting the curve are shown here. Y represents concentration of IL-6 and X represents miRNA let-7a concentration. A1 is the initial IL-6 levels and A2 is the changed level. LogX0 is the mid-point of value 0.34, a concentration of let-7a where Il-6 expression is reduced to half (E).The variations of IL-6 expression with the altered level of PP2A (black) and HuR (red). For both cases, the data were fitted to an equation, which is shown above. The parameter m denotes the expression level of PP2A/HuR which corresponds to the maximum IL-6 expression; w denotes the width of the distributions for both cases. For PP2A and HuR, the values of m were found to be 0.6 and 0.9 respectively and A is a constant (F).

Data information: In all experimental data, ns; non-significant and * and ** represent P value of <0.05 and <0.01respectively calculated by student's t test. Exact P-values against each experimental set are presented in the Appendix Table S2. Positions of molecular weight markers are marked and shown in the western blots used in different panels.
Elucidation of WW domain ligand binding specificities in the Hippo pathway reveals STXBP4 as YAP inhibitor (2019)
Figure 2

Figure 2. Identification of a conserved 9-amino acid sequence that determines the Hippo WW domain binding specificity. (This Figure is related to Figures EV2 and EV3; Appendix Figures S1-S3; Table EV5)

(A) Sequence alignment of the WW domains derived from the WW domain-containing proteins that are known to bind the Hippo PY motif-containing proteins. The two conserved tryptophan restudies were highlighted in purple. Additional conserved amino acid residues were highlighted in yellow.

(B) Summary of the residue difference in the identified 9-amino acid sequence for the control WW domains. The conserved two tryptophan residues are labelled in grey; the changed residues are labelled in orange; and the unchanged residues are labelled in white.

(C-G) Validation of the identified 9-amino acid sequence in determining the Hippo WW domain binding specificity. The requirement of the identified 9-amino acid sequence for AMOT association was respectively examined for TAZ (C), TAZ-WW domain (D), KIBRA (E), YAP (F) and SAV1 (G). HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.
Elucidation of WW domain ligand binding specificities in the Hippo pathway reveals STXBP4 as YAP inhibitor (2019)
Figure 3

Figure 3. STXBP4 is a Hippo pathway regulator, which contains a WW domain that fits the criterion of the Hippo WW domain binding specificity. (This figure is related to Figure EV4, Appendix Figures S4 and S5; Table EV6)

(A) Schematic illustration of STXBP4 protein, where the identified 9-amino acid sequence of STXBP4-WW domain was aligned across the indicated species.

(B) STXBP4 forms a complex with several Hippo PY motif-containing proteins. Immunoprecipitation was performed with STXBP4 antibody.

(C) The identified 9-amino acid sequence is required for the association between STXBP4 and AMOT. HEK293T cells were transfected with the indicated STXBP4 mutants and subjected to the pulldown assay.

(D) Structural comparison between the YAP-WW1/SMAD7-PY and STXBP4-WW/SMAD7-PY complexes. The identified 9 amino acid residues were indicated for both complexes.

(E) The YAP-WW1/SMAD7-PY and STXBP4-WW/SMAD7-PY complexes show similar cumulative average trend and average binding free energy (∆G) within standard deviation (the shaded region) of one another.

(F) Loss of STXBP4 inhibits YAP phosphorylation and LATS activation. Western blotting was performed with the indicated antibodies.

(G and H) Loss of STXBP4 activates YAP. STXBP4 deficiency promotes YAP nuclear translocation (G) and YAP downstream gene transcription (mean ± s.d., n=3 biological replicates) (H). Scale bar, 20 μm.*** p < 0.001 (Student's t-test).

(I) WW domain is required for the STXBP4-mediated YAP cytoplasmic translocation. STXBP4 KO cells were transfected with the indicated STXBP4 constructs and immunofluorescent staining was performed. HA-positive cells (arrows) from ~30 different views (~200 cells in total) were randomly selected and quantified for YAP localization. Percentage of HA-positive cells with nuclear YAP enrichment is shown. Scale bar, 20 μm.

(J) Loss of STXBP4 attenuates YAP phosphorylation as induced by actin cytoskeleton inhibition. The indicated cells were subjected to serum starvation (treatment with no-serum medium for 12 hours), glucose starvation (treatment with no-glucose medium for 6 hours) or actin inhibition (treatment with 0.5 μg/mL latrunculin B or 5 μM blebbistatin for 30 min). YAP phosphorylation was detected using phospho-tag gel, where the YAP phosphorylation level was indicated.