EMBO/soda-nel huggingface dataset:
Actin filaments regulate microtubule growth at the centrosome. (2019)
Figure 4

Figure 4: Blockage of microtubule growth by actin filaments on isolated centrosomes.

Schematic illustration of the first dynamic assay: sequential addition of tubulin followed by tubulin and actin on isolated centrosomes.

Representative images showing microtubules (top line) and the merged images of actin and microtubules (bottom line) for the two steps of the assay; in the presence of tubulin only (left column) and in the presence of tubulin and actin (right column). Scale bar: 10 µm.

Quantification of the differences in the number of microtubules per centrosome between the two stages of the experiment described above on centrosomes capable (first condition), or not (second condition), to grow actin filaments. Data were collected from a single experiment; asters without actin filaments: n= 29; asters with actin filaments: n= 13. Data were analyzed using Mann-Whitney test.

Schematic illustration of the second dynamic assay: tubulin is added to measure centrosome nucleation capacity and washed out. Then actin is added followed by actin and tubulin.

Representative images showing microtubules (top line) and the merged images of actin and microtubule (bottom line) during the three steps of the assay; in the presence of tubulin only (left column), in the absence of tubulin and presence of actin (middle column) and in the presence of tubulin and actin (right column). Scale bar:10 µm. Arrowheads indicate microtubules unable to re-grow after assembly of actin filaments.

Quantification of the differences in the number of microtubules per centrosome between the first and last steps of the experiment described above (panels D and E) on centrosomes capable (first condition), or not (second condition), to grow actin filaments. Data were pooled from 2 independent experiments; asters without actin filaments: n= 24; asters with actin filaments: n= 13. ****:P<0.001 Student t-test.
Actin filaments regulate microtubule growth at the centrosome. (2019)
Figure 5

Figure 5: Reconstitution of the interplay between actin and microtubules on micropatterns.

Schematic illustration of the micropatterning method to graft microtubules seeds (green) via neutravidin (yellow) and actin-nucleation-promoting complexes (orange) on 8-micron-wide discoidal micropatterns. A glass coverslip (deep blue) coated with polyethyleneglycol (PEG) (light blue) was placed in contact with a transparency mask and exposed to deep UV light. The exposed coverslip was then immersed with neutravidin to fix biotinylated microtubule seeds (green) on exposed regions. Streptavidin-WA was immobilized on microtubule seeds via their interaction with biotin. Tubulin dimers and actin monomers were then added to allow filaments elongation.

Representative images of microtubules (top) and actin filaments (bottom) growth from micropatterns. Scale bars: 20 μm.

Schematic illustration of the assay on micropatterned substrate. Tubulin was first added alone to measure the nucleation capacity of each micropattern, and then washed out. Later on, actin was added followed by actin and tubulin. Finally, actin was rinsed out and gelsolin was added to fully disassemble actin filaments. Representative images showing microtubules (top line) and the merged images of actin and microtubules (bottom line) during the four steps of the assay; in the presence of tubulin only, in the absence of tubulin and presence of actin, in the presence of tubulin and actin, and finally in the presence of tubulin and gelsolin but in the absence of actin filaments (from left to right). Scale bar: 10 μm.

Quantification of the number of microtubules per micropattern in the presence of tubulin only (left), actin and tubulin (middle) and tubulin only after actin filaments disassembly (right). Data were pooled from 2 independent experiments; n= 133. ****:P<0.001 Student t-test.

The graph shows the same measurements as in panel (E) in an XY representation of individual measurements. It illustrates the differences in the number of microtubules per micropattern between the first to the second step (tubulin only versus actin and tubulin together) with respect to the density of actin filaments per micropattern.
Actin filaments regulate microtubule growth at the centrosome. (2019)
Figure 6

Figure 6: Modulation of microtubule growth by cell spreading and centrosomal actin.

Schematic illustration of our model according to which cell spreading sequesters monomeric actin to the cortex and thereby enables the centrosome to grow more microtubules. Drawings show top (top line) and side views (bottom line) of cells with increased spreading from left to right. Actin filaments are in green, microtubules in red.

RPE1 cells stably expressing centrin1-GFP were plated for 3 h on coverslips coated with different ratios (100:0; 50:50 or 1:99) of fibronectin and PLL-PEG prior to fixation and staining for F-actin (top line and magnified views around centrosome below. Scale bars: 10 µm and 2 µm, respectively) and α-tubulin (bottom line. Scale bar: 10 µm).

IIA1.6 B lymphoma cells were plated for 60 min on poly-L-lysine, fibronectin or ICAM-1 coated cover slides prior to be fixed and stained for F-actin (top line) and α-tubulin (bottom line). Scale bar: 3 µm.

Quantification of the area occupied by RPE1 cells on the substrate (top left), F-actin content at the centrosome (top right) polymerized tubulin at the centrosome (bottom left) and in the entire cell (bottom right) for the three conditions of cell adhesion described in (B). Measurements came from 3 independent experiments with more than 60 analyzed cells in each. Errors bars represent standard deviations. F-actin and microtubule contents were compared using Mann-Whitney test, and variations of the cell area were compared using unpaired t-test.

Quantification of the area occupied by B lymphoma cells on the substrate (top left), F-actin content at the centrosome (top right) polymerized tubulin at the centrosome (bottom left) and in the entire cell (bottom right) for the three conditions of cell adhesion described in (D). Measurements came from 3 independent experiments with more than 80 analyzed cells in each. Errors bars represent standard deviations. F-actin and microtubule contents were compared using Mann-Whitney test, and variations of the cell area were compared using unpaired t-test.

Expanded View Figure legends
CDC20 promotes bone formation via APC/C dependent ubiquitination and degradation of p65 (2021)
Figure 1

Figure 1. Conditional knockout of Cdc20 impairs bone formation and blunts bone regeneration.

(A) Representative micro-CT images and H&E staining of trabecular bone from the femoral metaphysis of 6-week-old male Sp7-Cre;Cdc20f/f and littermate control mice. Scale bar, 500 μm.

(B) Histomorphometric analyses of 6-week-old male femurs. (n=6)

(C) Representative micro-CT images and H&E staining of trabecular bone from the femoral metaphysis of 6-week-old female Sp7-Cre;Cdc20f/f and littermate control mice. Scale bar, 500 μm.

(D) Histomorphometric analyses of 6-week-old female femurs. (n=6)

(E) Representative micro-CT images of tibial cortical bone defects in Sp7-Cre;Cdc20f/f and littermate control mice. The green lines show the position of the original defect position. The green circle represents the regenerated bone. Scale bar, 500 μm.

(F) Histomorphometric analysis of the regenerated bone in tibial cortical gaps. (n=5)

Data information: Data are displayed as mean ± SD and show one representative of n ≥ 3 independent experiments with three biological replicates. Statistical significance was calculated by a two-tailed unpaired Student's t-test defined as *p<0.05; **p<0.01; ***p<0.001.
CDC20 promotes bone formation via APC/C dependent ubiquitination and degradation of p65 (2021)
Figure 2

Figure 2. CDC20 modulates osteogenic differentiation of BMSCs.

(A) Western blot analyses of the knockout efficiency of Cdc20 in Cdc20f/f and Sp7-Cre;Cdc20f/f BMSCs.

(B, C) The ALP staining (B) and ALP quantification (C) of Cdc20f/f and Sp7-Cre;Cdc20f/f BMSCs after 7 days osteogenic differentiation. (n=6)

(D) The efficiency of CDC20 knockdown determined by western blot in hBMSCs.

(E, F) The ALP staining (E) and ALP quantification (F) of NC and CDC20sh hBMSCs after 7 days osteogenic differentiation. (n=5)

(G) Schematic representation of the CDC20 truncated mutant fragments. The boundaries of its WD40 domain (amino acids 171-471) were shown.

(H, I) The ALP staining (H) and ALP quantification (I) of Vector and truncated fragments overexpression of CDC20 in CDC20sh hBMSCs after 7 days osteogenic differentiation. (n=5)

(J, K) The H&E staining (J) and Masson's trichrome (K) of the histological sections from implanted NC and CDC20sh hBMSCs scaffold hybrids. Scale bar, 100 μm.

Data information: Data are displayed as mean ± SD and show one representative of n ≥ 3 independent experiments with three biological replicates. Statistical significance was calculated by one-way ANOVA followed by a Tukey's post hoc test and defined as *p<0.05; **p<0.01; ***p<0.001.
CDC20 promotes bone formation via APC/C dependent ubiquitination and degradation of p65 (2021)
Figure 3

Figure3. CDC20 induces proteasome-dependent degradation of p65.

(A) Western blot analyses of the degradation of endogenous p65 protein in BMSCs from Sp7-Cre;Cdc20f/f and Cdc20f/f mice.

(B) Western blot analyses of the degradation of endogenous p65 protein in NC and CDC20si hBMSCs.

(C) Western blot analyses of the degradation of nuclear p65 protein after the knockdown of CDC20. The cytoplasmic and nuclear protein of NC and CDC20si HEK293T cells were extracted after TNF-α treatment for 30 minutes.

(D) The graphically presented amount of p65 of NC and CDC20si HEK293T cells was normalized with α-tublin and H3 expression. The percent of p65 protein was calculated accordingly. (n=6)

(E) Western blot analyses of the degradation of nuclear p65 protein after the overexpression of CDC20. The cytoplasmic and nuclear protein of overexpressed Myc-CDC20 and Vector HEK293T cells were extracted after TNF-α treatment for 30 minutes.

(F) The graphically presented amount of p65 of overexpressed Myc-CDC20 and Vector HEK293T cells was normalized with α-tublin and H3 expression. The percent of p65 protein was calculated accordingly. (n=5)

(G) Western blot analyses of the degradation of p65 protein after the overexpression of Myc-CDC20. hBMSCs were transfected with Vector and Myc-CDC20 plasmids for 36 h, cells were treated with or without 10 μM MG132 for 6 h before collected.

Data information: Data are displayed as mean ± SD and show one representative of n ≥ 3 independent experiments with three biological replicates. Statistical significance was calculated by one-way ANOVA followed by a Tukey's post hoc test and defined as *p<0.05; ***p<0.001.
CDC20 promotes bone formation via APC/C dependent ubiquitination and degradation of p65 (2021)
Figure 4

Figure 4. CDC20 interacts with p65.

(A, B) Co-immunoprecipitation of ectopically expressed Myc-CDC20 with ectopically expressed Flag-p65 in HEK293T cells.

(C, D) Co-immunoprecipitation of ectopically expressed Myc-CDC20 with endogenous p65 and ectopically expressed Flag-p65 with endogenous CDC20 in HEK293T cells.

(E, F) Co-immunoprecipitation of endogenous CDC20 with endogenous p65 in HEK293T cells.

(G) Co-immunoprecipitation of purified GST-CDC20 protein with endogenous p65 in HEK293T cells and the construct of GST-CDC20. Whole cell lysates (WCL) were subjected to perform GST pull-down analyses with purified GST-CDC20 and GST as a negative control.

(H) Co-immunoprecipitation of purified GST-p65 protein with endogenous CDC20 in HEK293T cells and the construct of GST-p65. Whole cell lysates (WCL) were subjected to perform GST pull-down analyses with purified GST-p65 and GST as a negative control.
CDC20 promotes bone formation via APC/C dependent ubiquitination and degradation of p65 (2021)
Figure 5

Figure 5. The functional domain of CDC20 and p65.

(A, B) Co-immunoprecipitation of ectopically expressed Myc-CDC20 171-499 fragment (containing WD40 domain) (A) rather than Myc-CDC20 1-170 fragment (lacking WD40 domain) (B) with endogenous p65 in HEK293T cells.

(C) Schematic representation of RHD domain location in p65 peptides (amino acids 19-306).

(D) Western blot analyses of Flag-p65, Flag-p65 1-310 fragment (containing RHD domain), Flag-p65 311-551 fragment (lacking RHD domain) plasmids expression in HEK293T cells.

(E, F) Co-immunoprecipitation of ectopically expressed Flag-p65 1-310 fragment (containing RHD domain) (E) rather than Flag-p65 311-551 fragment (lacking RHD domain) (F) with endogenous CDC20 in HEK293T cells.

(G, H) Co-immunoprecipitation of ectopically expressed Myc-CDC20 171-499 with ectopically expressed Flag-p65 1-310 fragment in HEK293T cells.
CDC20 promotes bone formation via APC/C dependent ubiquitination and degradation of p65 (2021)
Figure 6

Figure 6. CDC20 regulates p65 degradation in an APC11-dependent manner.

(A, B) Co-immunoprecipitation of endogenous APC11 with endogenous p65 in HEK293T cells.

(C) Western blot analyses of the degradation of p65 protein in NC and APC11si HEK293T cells.

(D) Western blot analyses of the degradation of p65 protein by APC11. HEK293T cells were transfected with Vector and HA-APC11 plasmids for 36 h, cells were treated with or without 10 μM MG132 for 6 h before collection.

(E) Co-immunoprecipitation of APC11 with endogenous p65 in NC and CDC20sh HEK293T cells.

(F) Western blot analyses of p65 expression in NC or CDC20sh HEK293T cells after the overexpression of Vector or HA-APC11 plasmids.

(G) The efficiency of APC11 knockdown determined by western blot in hBMSCs.

(H, I) The ALP staining (H) and ALP quantification (I) of NC and APC11si hBMSCs after 7 days osteogenic differentiation. (n=6)

Data information: Data are displayed as mean ± SD and show one representative of n ≥ 3 independent experiments with three biological replicates. Statistical significance was calculated by one-way ANOVA followed by a Tukey's post hoc test and defined as **p<0.01.
CDC20 promotes bone formation via APC/C dependent ubiquitination and degradation of p65 (2021)
Figure 7

Figure 7. The ubiquitination of p65 by APC11CDC20.

(A) Immunoblot of Flag-p65-linked ubiquitination promoted by Myc-CDC20. HEK293T cells were co-transfected with Flag-p65, HA-Ubiquitin, with or without Myc-CDC20 or Vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection.

(B) Immunoblot of Flag-p65-linked ubiquitination promoted by HA-APC11. HEK293T cells were transfected with Flag-p65, His-Ubiquitin, with or without HA-APC11 or Vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection.

(C, D) Immunoblot of Flag-p65-linked in vitro ubiquitination promoted by GST-CDC20 (C) or GST-APC11 (D). Bacterially expressed and purified GST-CDC20 or GST-APC11 were incubated with purified proteins, including HA-ubiquitin, E1, E2, and Flag-p65 as indicated at 32 ℃ for 1 h.

(E) Immunoblot of Flag-p65-linked ubiquitination promoted by truncated Myc-CDC20 fragments. HEK293T cells were transfected with Flag-p65, HA-Ubiquitin, with or without Myc-CDC20 171-499 fragment (containing WD40 domain), Myc-CDC20 1-170 fragment (WD40 domain deficient) and Vector plasmids. Transfected cells were then treated with 10 μM MG132 for 6 h before collection.

(F) Immunoblot of Flag-p65-linked ubiquitination promoted by HA-APC11 in NC and CDC20sh cells. NC or CDC20sh HEK293T cells were co-transfected with Flag-p65, His-Ubiquitin, with or without HA-APC11 and Vector plasmids. Transfected cells were treated with 10 μM MG132 for 6 h before collection.
A DNA/HDAC dual-targeting drug CY190602 with significantly enhanced anticancer potency. (2015)
Figure 1

The side chain structure of CY enhances nitrogen mustard activity in cancer cells

Chemical structures of bendamustine, CY, compound A, and compound B (referred to as Cpd A and Cpd B hereafter).

Growth inhibitory concentration (GI50) of CY and bendamustine in the NCI60 cell line panel.

Nontoxic dose of Cpd A enhances bendamustine cytotoxicity in cancer cells. Images were taken at 48 h posttreatment.

Cpd A augments DNA damage caused by bendamustine. H1650 cells were treated with 100 μM Bend, 50 μM Cpd A, 100 μM Bend plus 50 μM Cpd A, or 10 μM CY for 12 h, and cell lysates were blotted for γ-H2AX. Actin was used as a loading control.

Source data are available online for this figure.
A DNA/HDAC dual-targeting drug CY190602 with significantly enhanced anticancer potency. (2015)
Figure 2

CY harbors HDAC inhibitory activity that enhances nitrogen mustard's anticancer efficacy

Upper panel, in vitro HDAC1 inhibition assay using bendamustine and CY. Lower panel, summary of CY IC50 against other HDACs.

CY and CpdA, but not bendamustine or CpdB, inhibited HDAC activities in cancer cells. H1650 cells were treated with 20 μM CY, 20 μM CpdA, 200 μM CpdB, 200 μM Bend, or 10 μM SAHA for 12 h, and cell lysates were blotted for histone acetylation markers. Actin was used as a loading control.

Molecular docking of CY with the structure of HDAC1/2 enzyme pocket.

Alteration of CY side chain (Cpd B) caused loss of HDAC inhibitory activity and resulted in reduced ability to induce DNA damage. Cells were treated for 12 h. Cells treated with 200 μM Cpd B and 20 μM CY exhibited similar levels of γ-H2AX. Actin was used as a loading control.

Summary of lethal dose 50 (LD50) of bendamustine, CY, Cpd A (HDAC inhibition only), and Cpd B (nitrogen mustard only) against 11 human cancer cell lines.

Cpd A enhances the anticancer activity of bendamustine. H1650 cells were treated with various doses of bendamustine with or without 40 μM Cpd A (nontoxic dose, see Fig1C and Supplementary Fig S2) for 48 h and subjected to MTT viability assays.

Source data are available online for this figure.
A DNA/HDAC dual-targeting drug CY190602 with significantly enhanced anticancer potency. (2015)
Figure 3

HDAC inhibition by CY leads to downregulation of several histone acetyltransferases involved in DNA repair

CY, but not bendamustine treatment, led to suppression of CBP, TIP60, MORF, and MSL1 in H1650 cells. Cells were treated with DMSO, bendamustine (350 μM), CY (15 μM), or SAHA (10 μM) for 6 h, and mRNA was extracted for qPCR analysis. Data represent mean ± SEM from three independent experiments, and statistical significance was determined by unpaired two-tailed t-test. ***P < 0.01.

Drug treatment up to 10 h at the concentrations indicated in (A) did not cause apoptosis as judged by PARP cleavage. Actin was used as a loading control.

ShRNA-mediated suppression of CBP, TIP60, MORF, and MSL1 at mRNA level. Data represent mean ± SEM from three independent experiments, and statistical significance was determined by unpaired two-tailed t-test. *P < 0.1, **P < 0.05, ***P < 0.01.

Suppression of CBP, TIP60, MORF, and MSL1 sensitized cells to bendamustine. Y-axis represents relative resistance calculated from results of GFP competition assays, and statistical significance was determined by unpaired two-tailed t-test. Data represent mean ± SEM from two independent experiments. **P < 0.05, ***P < 0.01.

Source data are available online for this figure.
Monomeric cohesin state revealed by live cell single molecule spectroscopy (2019)
Figure 2

Figure 2. Diffusion of cohesin subunits throughout the cell cycle.

A. Typical fluorescence intensity trace collected over 10 seconds.

B. Typical normalized FCS curves of Smc3-GFP at different cell cycle phases. Dotted lines indicate fitting of the one-component anomalous diffusion model to the respective FCS curve.

C-F. Diffusion times of Smc3-GFP, Scc3-GFP, and Scc1-GFP at the G1 phase (C), S phase (D), G2 phase (E), and mitosis (F). Data shown as mean ± s.e.m. from at least three independent experiments. *: p<0.01, #: p<10-13 with the Mann-Whitney test.
Monomeric cohesin state revealed by live cell single molecule spectroscopy (2019)
Figure 3

Figure 3. The effect of subunit composition on cohesin diffusion time throughout the cell cycle.

Representative normalized FCS curve of Scc1-GFP at different cell cycle phases. Dotted lines indicate fitting of one-component anomalous diffusion models to the respective FCS curve.

B-D. The diffusion times of Scc1-GFP at S phase (B) G2 phase (C) and M phase (D) were measured in strains yIO664 (wild-type) and yEB005 (WPL1∆), and in auxin-depleted yEB002 (PDS5-AID), yAM085 (SCC3-AID) and yAM946 (ECO1-AID) cells. Data are shown as mean ± s.e.m. from at least three independent experiments. *: p< 10-5, **: p< 10-13.
Monomeric cohesin state revealed by live cell single molecule spectroscopy (2019)
Figure 4

Figure 4. Photon counting histogram (PCH) reveals a monomer form of cohesin.

A. Typical fitted photon counting histograms of GFP and GFP-GFP in live yeast cells.

B. The molecular brightness of different GFP tagged constructs was obtained with GFP monomer and GFP-GFP dimer as the brightness standard. Cells of strain yIO664 (SCC1-GFP) were analyzed throughout the cell cycle by PCH. To examine the effects of Wpl1, Pds5, and Eco1 on cohesin dimerization, the PCH analysis was repeated in strain yEB005 cells (SCC1-GFP WPL1∆), strain yEB002 cells (SCC1-GFP PDS5-AID), and yAM946 (SCC1-GFP ECO1-AID) in which Pds5-AID/Eco1-AID was depleted with auxin.

C. PCH analysis of strain yAM085 (SMC3-GFP) throughout the cell cycle.

D. PCH analysis of strains yAM953 (SCC1-GFP SMC3-GFP) and yAP941 (SCC1-GFP SCC3-GFP) containing two GFP tagged subunits. Data shown as boxplot (+data overlap) with values of minimum, 5% percentile, 25% percentile, median, 75% percentile, maximum, and mean (center square marks) from at least three independent experiments.

Tables

Table 1. Summary of cohesin holocomplex and subunit depleted complexes on diffusion times throughout the cell cycle. Data is shown as mean ± s.e.m from at least three independent experiments.

Table 2. Cellular concentration of cohesin subunits throughout the cell cycle. Protein concentrations are in nM.

Expanded View Figure Legends
A preclinical model of peripheral T-cell lymphoma GATA3 reveals DNA damage response pathway vulnerability (2022)
Figure 1

Figure 1. In vivo expansion of a novel transplantable disseminated lymphoma. A. A schematic showing the procedure by which a transplantable lymphoma line was passaged from a Cγ1-Cre Blimp1fl/fl mouse into C57bl/6 mice. Total splenocytes were inoculated intravenously (IV) and a lymphoma serially expanded in vivo. Each black dot represents a single mouse passage.

B. Time-to-welfare endpoint in C57bl/6J mice intravenously injected with a varying inoculum of splenocytes (Passage (P) 5). Log-rank test results per inoculum: 1x104 vs. 2x105 cells= p=0.008. 5x104 vs. 2x105 cells= p=0.0179.

C, D (C) Mass of lymphoma-infiltrated vs. tumor-free C57bl/6J tissues (n=6 biological replicates) and (D), gross morphology of spleens and mesenteric lymph nodes (MLN) at welfare endpoint in representative mice. T-test result was corrected for multiple comparisons by the Holm-Sidak method. Adjusted p<0.000001 (spleen) and p=0.000629 (liver). Error bars represent SEM.

E. H&E stain of infiltrated secondary lymphoid tissues with follicle structures (fo), red pulp (rp) and white pulp (wp) indicated in spleens.

F. Immunostain for CD21 in MLN labels B-cells and at higher staining intensity, follicular dendritic cell (FDC) network. IHC data in E and F is representative of 6 mice. Dotted lines represent the region magnified in the lower images. Dashed lines show the outline of the FDC network.

G. Immunostain for Ki67 in wildtype and lymphoma-bearing spleen. Representative images are shown at left. The proportion of nuclei positive for Ki67 (proliferation index) is quantified at right for n=3 spleens(T-test p=0.00002331).

H. Sequential sections of spleen stained by H&E, for B-cells (B220) and T-cells (CD3). The box shows an expanded region at right. wp=white pulp and rp=red pulp. *p<0.05, **p<0.01, ***p<0.001.