EMBO/soda-nel huggingface dataset:
Cohesin acetylation and Wapl-Pds5 oppositely regulate translocation of cohesin along DNA (2016)
Figure 4

Figure 4. Cohesin preferentially accumulats in nucleosome-poor regions on chromatin in Xenopus egg extracts

(A) Stretched DNA was incubated in the HSS in the presence or absence of geminin. After washing the HSS, endogenous Smc3 (green) and Mcm2 (magenta) were detected by immunofluorescence staining. DNA was counterstained with SYTOX (gray). Scale bar, 2 µm.

(B) Numbers of Smc3 and Mcm2 signals were counted on 10 μm DNA shown in (A). (22 DNAs per condition, n = 3, mean ± s.e.m. *p < 0.0001, two-tailed Mann-Whitney test).

(C) Chromatin formed in the HSS in the presence of geminin, and cohesinHalo488 (green) was loaded onto the chromatin with Scc2-Scc4. Chromatin was detected by immunostaining of histone H3 (magenta). DNA was counterstained with SYTOX (gray). The distributions of cohesin particles and histone H3 are shown as line-scan plots (bottom). Scale bar, 5 μm.

(D) Cohesin particles observed in (C) were categorized as indicated (> 37 particles per condition. n = 6, mean ± s.e.m.).

(E) Cohesin particle numbers for each condition. Red bars denote the median, lower, and upper quartile values (n ≥ 12, *p < 0.0004, unpaired t-test).

(F) Colocalization of Mcm2 with Smc3 was quantified (50 Mcm2 particles per condition, n = 3, mean ± s.e.m.)
Cohesin acetylation and Wapl-Pds5 oppositely regulate translocation of cohesin along DNA (2016)
Figure 5

Figure 5. XEco2 is required for the dynamic translocation of cohesin along unreplicated DNA in Xenopus egg extracts

(A) Stretched DNA was incubated in the HSS. Endogenous Smc3 (green) and Mcm2 (magenta) were detected by immunofluorescence staining. DNA was counterstained with SYTOX (gray). Colocalization of Smc3 and Mcm2 was quantified (bottom, n = 3, mean ± s.e.m.). Scale bar, 2 μm.

(B) Fluorescently labeled Xenopus cohesins (xCohesinHaloTMR) were loaded onto DNA in the HSS, and DNA-bound cohesin particles were observed in the HSS with or without 5 mM AMP-PNP supplementation. On the kymograph, the magenta lines indicate the duration of DNA binding of each cohesin particle. DNA was counterstained with SYTOX. Scale bar, 5 μm.

(C) Kymographs of DNA-bound xCohesinHaloTMR (green) in HSS. After time-lapse imaging of xCohesinHaloTMR, nucleosomes were immunostained by anti-H3 antibody (magenta). Result of H3 immunostaining was merged with the kymograph. DNA was counterstained with SYTOX. Scale bar, 5 μm.

(D) HSS were subjected to mock or XEco2 immunodepletion and human Esco1-His (hEsco1) was added back to XEco2-depleted HSS. Sperm chromatin was incubated in these extracts and chromatin-bound proteins were analyzed by immunoblotting.

(E) Kymographs of DNA-bound xCohesinHaloTMR in mock-, XEco2-depleted, or hEsco1-added XEco2-depleted HSS. Scale bar, 2 μm.

(F) MSD versus time of cohesin particle in the experiment in (E). D indicates the diffusion coefficient (n = 15, mean ± s.e.m.).
Cohesin acetylation and Wapl-Pds5 oppositely regulate translocation of cohesin along DNA (2016)
Figure 6

Figure 6. Cohesin behaviors on replicating chromatin in Xenopus egg extracts

(A) Experimental design for monitoring replication and cohesin in Xenopus egg extracts.

(B) Kymographs of examples for cohesin motion during DNA replication. xCohesinHaloTMR (magenta) was loaded onto DNA in the HSS, and NPE was introduced to allow DNA replication. DNA replication was monitored by xFen1D179A-PAGFP (green). Thirthy-four particles were categorized into five groups. Arrowheads indicate the time points when cohesin was dissociated from chromatin. Scale bar, 2μm.
CXCR4 engagement triggers CD47 internalization and antitumor immunization in a mouse model of mesothelioma (2021)
Figure 3

Figure 3. BoxA induces the release of Damage Associated Molecular Pattern molecules (DAMPs) but not apoptosis in MM cells.

(A) Surface calreticulin on MM cells was evaluated by flow cytometry after exposure to 800 nM BoxA or 1 μM MTX for 4 or 6 hours. Statistics: One-way Anova with Dunnett's post-test; n=3.

(B) Representative immunohistochemical staining for calreticulin in tumor masses of control (PBS) and BoxA-treated mice. Scale bar 50 μm. The distribution of calreticulin positive pixels describes the location of calreticulin (intracellular vs ecto-calreticulin). Statistics: Mann-Whitney test, n=4.

(C) HMGB1 levels in the medium of AB1 cells treated with BoxA or 1 μM MTX for 24 hours. Statistics: One-way Anova with Dunnett's post-test; n=3.

(D) Ratio of phosphorylated eIF2α (p-eIF2α) over eIF2α in MM cells treated with increasing concentrations of BoxA or 1 μM MTX for 24 hours; β-actin is shown as loading control. Statistics: One-way Anova with Dunnett's post-test; n=2.

(E) MM cells (3x105) were grown for 16 hours before adding increasing concentrations of BoxA or 1 μM MTX, and then further grown for 24 hours; t-16h indicates the number of cells at the start of the experiment.

(F) Western blot analysis of cleaved caspase-3 in MM cells exposed or not to BoxA or MTX for 24 hours; α-tubulin was used for normalization. Statistics: One-way Anova with Dunnett's post-test; n=2.

(G) Confocal immunofluorescence microscopy of cleaved caspase 3 (red) in MM cells treated for 24 hours with MTX (1 μM) or increasing concentrations of BoxA. Nuclei were stained with Hoechst (blue) and cytosol with phalloidin (green). Scale bar 50 μm.

(H) Tumor growth detected via BLI. MM cells pretreated or not with 800 nM BoxA overnight were injected in mice (PBS group, n=7; BoxA pretreated, n=6).

(I) Kaplan-Meier survival curve of mice injected with MM cells pretreated with PBS or BoxA shown in panel H (Log rank Gehan Breslow Wilcoxon test, p=0.59).

Data information: In panels (A, C, D, E, F) columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
CXCR4 engagement triggers CD47 internalization and antitumor immunization in a mouse model of mesothelioma (2021)
Figure 4

Figure 4. MM cells exposed to BoxA are phagocytosed by macrophages following CD47 internalization.

(A) Representative frame of a 16 hour time-lapse microscopy experiment; GFP+ MM cells (green) were co-cultured with mouse bone marrow derived macrophages (gray) in the presence of 200 nM BoxA. Arrows indicate GFP+ MM cells engulfed by macrophages. This experiment was repeated twice.

(B) Fraction of phagocytosed GFP+ MM cells after 16 hours. Statistics: χ2 test, phagocytosed vs non phagocytosed MM cells (n= 68 to 158).

(C) Surface CD47 in MM cells, untreated (blue) or treated for 24 hours with 800 nM BoxA (red). Control isotype (grey).

(D) Surface CD47 on MM cells evaluated by flow cytometry after incubation for 24 hours with the indicated concentrations of BoxA. The experiment shown is representative of 3 performed. Error bars indicate standard deviation. Statistics: One-way Anova plus Dunnett's post-test. *p<0.05, **p<0.01, ***p<0.001.

(E) Representative immunofluorescence staining of CD47 (red) in unpermeabilized and permeabilized MM cells treated or not with 800 nM BoxA for 24 hours. Nuclei are counterstained with DAPI. Scale bar 50 μm.
CXCR4 engagement triggers CD47 internalization and antitumor immunization in a mouse model of mesothelioma (2021)
Figure 5

Figure 5. BoxA exerts therapeutic effects in a model of colon cancer.

(A) Ecto-calreticulin exposure (expressed as MFI) in CT26 cells treated with 800 nM BoxA or 1 μM MTX for 2 and 4 hours. Error bars indicate standard deviation. Data were pooled from two different experiments.

(B) HMGB1 levels in the medium of CT26 cells treated with BoxA or 1μM MTX for 24 hours (n=3).

(C) Ratio of phosphorylated eIF2α (p-eIF2α) over eIF2α in CT26 cells treated with increasing concentrations of BoxA or 1μM MTX for 24 hours. β-actin is shown as loading control (n=3).

(D) Surface CD47 on CT26 cells after incubation for 24 hours with the indicated concentrations of BoxA. The experiment shown is representative of 2 performed, in biological triplicate.

(E) Cell proliferation. CT26 cells (3x105) were seeded at t-16 hours, then exposed to increasing concentrations of BoxA or 1 μM MTX and counted after further 24 hours (n=3).

(F) Western blot analysis of cleaved caspase-3 in CT26 cells exposed or not to BoxA or MTX for 24 hours. α-tubulin was used for normalization (n=2).

(G) Scheme of the experiment. Twenty-two BALB/c mice were inoculated i.p. with 3.5x104 CT26-LUC cells and treated with either 800 μg BoxA or PBS 3 times a week, 10 times in total. Yellow arrows represent BLI imaging.

(H) Tumor growth was detected by BLI. Lines that do not reach day 20 correspond to mice that were sacrificed for ethical reasons. One BoxA treated mouse survived without any BLI signal and was re-challenged with CT26-LUC cells at day 65; at day 79 it was sacrificed and no tumor masses were detected. (I) Kaplan-Meier survival curves of mice shown in panel H. Statistics: Gehan-Breslow-Wilcoxon test.

(J) BLI was measured two hours after re-challenge (upper images) and one week later (lower images). The survivor was imaged also at much higher sensitivity (note the different scales for radiance).

Data information: In panels A-F, bars and error bars represent mean ± SD; statistics: One-way Anova with Dunnett's post-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
CXCR4 engagement triggers CD47 internalization and antitumor immunization in a mouse model of mesothelioma (2021)
Figure 6

Figure 6. Engagement of CXCR4 by BoxA mediates CD47 internalization.

(A) Confocal images of Proximity Ligation Assays performed on CD47 and CXCR4, CD47 and RAGE, CD47 and TLR4. Representative images from one experiment out of 3 performed are shown. MM cells were incubated overnight with 400 nM BoxA or PBS. Nuclei are in blue (DAPI), phalloidin is in green. Red dots represent physical contact of CD47 with CXCR4 or RAGE or TLR4. Scale bar, 20 μm. The intensity of red signal in PLA was quantified as described in Materials and Methods in individual cells (n as indicated). Insets are enlargements of the y axis. Mean and SD are indicated. Statistics: Kolmogorov-Smirnov test.

(B) Cxcr4 mRNA expression in MM cells transfected with shCxcr4 (pools 1 and 2) relative to MM cells transfected with shScramble (AB1 shCTR), one biological replicate, in technical triplicate.

(C) Cell proliferation. shCTR (n=3) and shCxcr4 MM cells (pools1+2, n=6)) were plated (4.4x104 at the start of the experiment, t-16h), treated or not with 800 nM BoxA for 24 hours and counted. Statistics: t-test.

(D) Flow cytometry analysis of surface CD47 in shCTR and shCxcr4 MM cells (pools 1+2) exposed or not to 800 nM BoxA for 24 hours.

(E) Flow cytometry analysis of surface CD47 on MM cells after exposure to 800 nM BoxA and 100 nM AMD3100 for 24 hours.

Data information: In panels D, E bars and error bars represent mean ± SD; statistics: One-way Anova plus Tukey's post-test. In all panels, error bars indicate standard deviation; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
CXCR4 engagement triggers CD47 internalization and antitumor immunization in a mouse model of mesothelioma (2021)
Figure 7

Figure 7. CXCL12 induces CD47 internalization and macrophage phagocytosis.

(A) Ratio of phosphorylated eIF2α (p-eIF2α) over eIF2α in MM cells exposed to 800 nM BoxA or increasing concentrations of CXCL12 for 24 hours (n=2). β-actin is shown as loading control.

(B) Western blot of HMGB1 released from MM cells exposed to BoxA and CXCL12 for 24 hours. HMGB1 is expressed as relative units compared to untreated cells. The samples (n=2) were loaded on the same gel, but intervening non-relevant samples were cropped from the image. Ponceau S staining was used as a loading control.

(C) Surface CD47 on MM cells after incubation for 24 hours with the indicated concentrations of CXCL12. The experiment shown is representative of 2 performed, in biological triplicate.

(D) PLA intensity was quantified as described in Materials and Methods in individual cells (n as indicated). Mean and SD are indicated. Statistics: Kolmogorov-Smirnov test.

(E) Fraction of phagocytosed GFP+ MM cells after 16 hours treatment with 10 nM CXCL12. The numbers above the columns indicate the phagocytosed/non-phagocytosed MM. Statistics: χ2 test, phagocytosed vs non phagocytosed tumor cells.

Data information: In panels A-C bars and error bars represent mean ± SD; statistics: one-way Anova plus Dunnett's post-test. In all panels, *p<0.05, **p<0.01, ****p<0.0001.
Allosteric gate modulation confers K+ coupling in glutamate transporters (2019)
Figure 4

Figure 4. K1 site mutations abolish K+ translocation in EAAT2 and K+ binding to GltPh.

(A) Simulated changes in the free energy of K+ binding to GltPh induced by different mutations. Values are calculated as differences of free energy changes during alchemical transformations of amino acid side-chains with the protein either in the K1 bound or in the apo state (Mean ± SD of 1000 bootstrap samples).

(B) Representative WT and mutant whole-cell current responses to voltage jumps before and after removal of external K+. Cells were dialyzed with a KNO3-based pipette solution and the external solution contained either 140 mM CholineNO3 or 140 mM KNO3.

(C) Relative changes in fluorescence for WT, D312N and D405N GltPh upon a temperature jump in MST experiments as a function of [K+]. Solid lines represent non-parametric fits, and shaded area show bootstrapped 99% confidence intervals for those fits. Non-overlapping confidence intervals indicate significant difference between WT and mutant binding curves. Values are given as mean ± SD (n≥3).

(D) Representative whole-cell current recordings from cells internally dialyzed with NaNO3 and glutamate in choline-NO3-based external solutions or in NaNO3-based solutions supplemented with 1 mM L-glutamate. Glutamate-induced changes in anion current amplitudes indicate that D399N and D486N EAAT2 (GltPh D312 and D405) are K+-independent, but are functionally expressed in the plasma membrane.

(E) Current-voltage relationships of steady-state currents from cells dialyzed with a KNO3-based pipette solution. Currents were measured at external 140 mM KNO3 and normalized to the current at -170 mV from a consecutive recording of the same cell in K+-free external solutions. Values are given as mean ± SD (n≥3)

Data information: See also Appendix Figure S6
Allosteric gate modulation confers K+ coupling in glutamate transporters (2019)
Figure 6

Figure 6. Mutations that stabilize the closed gate generate K+ independent EAATs.

(A) Root-mean-squared fluctuation (RMSF) of the HP2 backbone in the open and closed state for WT and L448A EAAT1 (GltPh A360). Values are given as mean ± SD of 10 blocks. Amino acid conservation in the indicated stretch (residue range 444-452 (GltPh 356-368) is shown as sequence logo for all K+-independent (GltX and ASCT1/2, upper row) and all K+-dependent (EAAT1-5, lower row) transporters.

(B) HP2 closure is accompanied by deformation of the backbone structure around residue L448 (GltPh A360).

(C) Probability density distribution for HP2 opening in WT (from Figure S7C), L448A or L448T EAAT1 (GltPh A360); the shaded area indicates closed states.

(D) Probabilities of HP2 closure from the integrated densities in (B) (Mean ± SD from 1000 bootstrap samples). Statistical significance was tested by comparing bootstrapped confidence intervals (*, 95% confidence intervals do not overlap).

(E) Representative transport current recordings from cells expressing L448A or L448T (GltPh A360) or WT EAAT1 upon voltage jumps to ˗140 mV before and after superfusion with 1 mM l-glutamate. Cells were intracellularly dialyzed with a K+-free solution without permeating anions to isolate K+-independent transport currents. Transient capacitive currents during the first 5 ms after a voltage jump were blanked.

(F) Current-voltage relationship for net transport current amplitudes for WT and L448A/T EAAT1 (GltPh A360) for choline-based pipette solutions. Values are given as mean ± SD with indicated numbers of experiments.

(G) Time-dependent changes in L448A EAAT1 (GltPh A360) uptake currents during two repetitive glutamate applications and subsequent removals. Current amplitudes were measured at the end of voltage jumps to -140mV and plotted versus the time. Glutamate application is indicated by green bars. Values are given as mean ± SD of the last 100 data points in the current trace.

(H) Net transport current amplitudes for WT and L448A/T EAAT1 (GltPh A360) for different pipette solutions, values for internal KGluc are taken from current-voltage relationships in Appendix Figure S9. Values are given as mean ± SD with indicated numbers of experiments (***, p<0.001 Student's t-test).

Data information: See also Appendix Figure S9.
Allosteric gate modulation confers K+ coupling in glutamate transporters (2019)
Figure 7

Figure 7. A conserved salt bridge prevents HP2 closure in apo EAATs.

(A) The E406-R479 (GltPh Q318, R397) salt bridge locks HP2 in the open state in EAAT1. Image shows the region around residues 406 and 479 (shown as spheres) with HP2 open (gray cartoon). Both residues sterically prevent HP2 closure by forming a salt bridge.

(B) Contour plot of the HP distance vs. the R479-E406 (GltPh Q318, R397) distance distribution. Gray lines indicate thresholds for the HP2-closed and salt bridge-formed states.

(C) Probability density distribution for HP2 opening in WT (from Figure S7C) and R479A EAAT1 (GltPh R397).

(D) Probability of HP2 closure calculated by integrating the densities in (C) (Mean ± SD from 1000 bootstrap samples). Statistical significance was tested by comparing bootstrapped confidence intervals (*, 95% confidence intervals do not overlap).

(E) Representative transport current recordings from a cell expressing R478A EAAT2 (GltPh R397) in the absence of internal K+. Transport currents were elicited by voltage jumps to -140mV before and after superfusion with 5 mM l-serine. Transient capacitive currents during the first 5 ms after a voltage jump were blanked.

(F) Time-dependent changes in steady-state R478A EAAT2 (GltPh R397) uptake currents during two consecutive serine applications and subsequent removals. Current amplitudes were measured at the end of voltage jumps to -140mV and plotted versus time. Serine application is indicated by green bars. Values are given as mean ± SD of the last 100 data points in the current trace.

(G) Current-voltage relationship for mean net transport currents for R478A EAAT2 induced by 5 mM l-serine for different intracellular solutions. Values are given as mean ± SD for the indicated number of experiments.

(H) Mean net transport currents for WT and R478A EAAT2 (GltPh R397) induced by 5 mM l-serine for different intracellular solutions. Values are given as mean ± SD for the indicated number of experiments.
Viral mediated redirection of NEMO/IKKγ to autophagosomes curtails the inflammatory cascade. (2012)
Figure 1

M45 inhibits TLR- and IL-1R-mediated NF-κB but not p38 activation.

(A) NIH-3T3 cells were transduced with retroviral vectors expressing M45 or GFP. Two days after transduction cells were stimulated with the TLR4 agonist LPS (10 µg/ml), the TLR2 agonist LTA-SA (10 µg/ml), or IL-1β (20 ng/ml). IκBα levels were determined by immunoblotting. (B) NIH-3T3 cells were transduced with an M45-expressing or an empty retroviral vector. 30 min after stimulation with IL-1β (10 ng/ml) cells were fixed, and subcellular localization of the NF-κB p65 subunit was analyzed by immunofluorescence. (C) NIH-3T3 cells expressing an NF-κB-dependent secreted alkaline phosphatase (SEAP) reporter were transduced with retroviral vectors expressing M45 or GFP and stimulated 78 h later with the TLR2 agonist Pam3CSK4 (Pam.,1 µg/ml), the TLR4 agonist LPS (0.1 µg/ml), or IL-1β (5 ng/ml). SEAP activity in the supernatant was quantified 15 h after stimulation and is shown as fold induction of SEAP activity of stimulated cells compared to non-stimulated cells (mean ± SD) (D) NIH-3T3 cells transduced with retroviral vectors expressing M45 or GFP were stimulated with IL-1β (20 ng/ml, 15 min) or TNFα (10 ng/ml, 5 min). Phosphorylated and total p38 in cell lysates was detected by immunoblotting.
Viral mediated redirection of NEMO/IKKγ to autophagosomes curtails the inflammatory cascade. (2012)
Figure 2

M45 inhibits IκBα degradation, TNFα and IL-6 production during MCMV infection.

(A) NIH-3T3 cells were infected with wt MCMV-GFP, an M45 deletion mutant (ΔM45) or a revertant virus (RM45) at an MOI of 10 and treated 5 h postinfection with the TLR2 agonist Pam3CSK4 (Pam., 0.1 µg/ml), the TLR4 agonist LPS (10 µg/ml), or IL-1β (20 ng/ml) for the indicated times. Levels of the indicated proteins were analyzed by immunoblotting. (B) BMDMs were infected with wt MCMV-GFP, ΔM45, or RM45 at an MOI of 3 and stimulated 8 h postinfection for 16 h with the TLR7 agonist R848 (0.1 µM). TNFα and IL-6 levels in the supernatant were determined by ELISA (mean ± SD). (C) RAW264.7 macrophages were infected with wt MCMV-GFP or ΔM45 at an MOI of 0.1, stimulated 24 h postinfection for 4 hours with TLR agonists Pam3CSK4 (Pam.) or Malp-2 (TLR2), LPS (TLR4), R837 (TLR7), or CpG (TLR9), in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SD).
Viral mediated redirection of NEMO/IKKγ to autophagosomes curtails the inflammatory cascade. (2012)
Figure 3

M45 interacts with the IKK complex.

(A) 293A cells were transfected with plasmids expressing HA-tagged M45 or an unrelated MCMV control protein (m143) and Flag-tagged IKK subunits. Lysates were subjected to immunoprecipitation (IP) using an anti-HA antibody. Immunoprecipitates were analyzed by immunoblotting (IB) using anti-Flag antibody. Whole cell lysates (WCL) were immunoblotted using Flag-, HA-, and actin-specific antibodies, respectively. (B) Nemo−/− MEFs were transfected with Flag-tagged IKK subunits and HA-tagged M45 or an unrelated MCMV control protein (m142), respectively. IP and IB were performed using anti-HA and anti-Flag antibodies, respectively. WCL were immunoblotted with anti-NEMO antibody and as described for panel A. (C) rip1−/− MEFs were transfected with HA-tagged M45 or an unrelated MCMV control protein (m142) and Flag-tagged NEMO. IP was done with an anti-HA antibody. Immunoprecipitates and WCL were analyzed by immunoblotting using the indicated antibodies. (D) 293A cells were transfected with plasmids expressing HA-tagged M45 or m143 (as a control). Lysates were subjected to anti-HA immunoprecipitation. Immunoblotting of precipitates and the WCL was done with antibodies as indicated. (E) NIH-3T3 cells were infected for 4 hours with RM45 (MCMV expressing HA-tagged M45), ΔM45, or Rm142 (MCMV expressing HA-tagged m142) at an MOI of 5. Cell lysates were subjected to IP and IB with antibodies as indicated.