EMBO/soda-nel huggingface dataset:
The novel BET-CBP/p300 dual inhibitor NEO2734 is active in SPOP mutant and wild-type prostate cancer (2019)
Figure 3

Figure 3. Q165P mutation affects SPOP dimerization

A. Root mean square deviation (RMSD) calculations of both SPOP WT and Q165P mutant on CA atoms with reference to the crystal structure (3HQI.pdb).

B. Overall architecture of SPOP dimer. Superposition of all Cα atoms of WT structure (a) and Q165P structure (b) from simulation at 80 ns over the crystal structure. The two SPOP molecules in the crystal structure are colored cyan and gray, respectively; the WT structure from simulation are red and warm pink, and Q165P are green and pale green, respectively.

C, D. Close-up views of the MATH domain from top of (B). The Cα atoms of Q165 or P165 are represented by spheres.

E. Several amino acids are predictively important for SPOP dimerization. Mutations on the following residues might destroy the dimer interface for MATH domain: any residue in blue color to Pro at the strand where Q165 locates or any residue in green color to Pro at the strand on the dimer interface. Residues mutated in PCa patients F102, F133 and Q165 are in red.

F, G. Co-IP assay was performed to detect the efficiency of dimerization between SPOP WT and Q165P mutant (F) and the interaction between CULLIN3 with SPOP WT or Q165P (G).
The novel BET-CBP/p300 dual inhibitor NEO2734 is active in SPOP mutant and wild-type prostate cancer (2019)
Figure 4

Figure 4. BET and CBP/p300 dual inhibitor NEO2734 overcomes JQ1 resistance in SPOP mutated PCa cells in vitro

A. DU145 PCa cells infected with virus expressing EV or SPOP Q165P were harvested for western blot analysis.

B. MTS was performed to compare the growth between EV and Q165P DU145 cells after the treatment with indicated inhibitors (2 µM of JQ1, 2 µM of CPI-637, 2 µM NEO2734) for 72 h. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001. See Appendix Table S4 for the detailed comparison, P values and sample number (n).

C - E. Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP Q165P DU145 cells. The survival curve showed IC50 for SPOP Q165P (0.69 µM) and EV cells (1.08 µM) (C). DU145 cells expressing lenti-EV or lenti-HA-SPOP-Q165 constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvest. The number of colonies with more than 50 cells was counted. Representative images of colonies are shown in (D) with quantitative data shown in (E). All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, ** P < 0.01. See Appendix Table S4 for the detailed comparison, P values and sample number (n).

F. DU145 cells were treated with the indicated inhibitors and harvested for ChIP with BRD4 antibody. The enrichment of BRD4 at c-MYC enhancer and RAC1 promoter was analyzed using qPCR. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01. See Appendix Table S4 for the detailed comparison, P values and sample number (n).

G. DU145 cells were treated with the indicated inhibitors and harvested for ChIP with Ac-H3 antibody. The level of Ac-H3 at c-MYC enhancer and RAC1 promoter is analyzed using qPCR. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; ** P < 0.01. See Appendix Table S4 for the detailed comparison, P values and sample number (n).
The novel BET-CBP/p300 dual inhibitor NEO2734 is active in SPOP mutant and wild-type prostate cancer (2019)
Figure 6

Figure 6. JQ1-resistant SPOP hotspot mutant organoids are sensitive to NEO2734

A, B. Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were harvested for western blot analysis with the indicated antibodies (A) and IFC with p-AKT-S473 antibody (B). Asterisk, SRC3 at expected molecular mass. E-cadherin antibody was used to indicate the cell membrane (red) and DAPI for nucleus (blue). Scale bars: 25 µm.

C, D. Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were cultured for five days, followed by the treatment with JQ1 (2 µM), CPI-637 (2 µM), JQ1 (2 µM) + CPI-637 (2 µM) or NEO2734 (2 µM) for five more days. The representative images of organoids after the treatment are shown in (C) and the quantified data of the organoid diameter are shown in (D). Scale bars: 25 µm. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. See Appendix Table S4 for the detailed comparison, P values and sample number (n).

E, F. 313HR (SPOP WT) and 573R (Q165P) organoids were cultured for 5 days, followed by the treatment with the indicated inhibitors for 5 days. The organoids were stained with Caspase-3 antibody (green) to detect apoptotic cells. E-cadherin antibody staining was used to define the cell membrane (red) and the nucleus was counterstained with DAPI (blue). The representative images of cleaved Caspase-3 IFC staining are shown in (E). Scale bars: 25 µm. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001. See Appendix Table S4 for the detailed comparison, P values and sample number (n).
The novel BET-CBP/p300 dual inhibitor NEO2734 is active in SPOP mutant and wild-type prostate cancer (2019)
Figure 7

Figure 7. JQ1-resistant SPOP hotspot mutant xenografts are sensitive to NEO2734

A. C4-2 PCa cells infected with virus expressing EV, SPOP Q165P or F133V were harvested for western blot analysis.

B MTS was performed to compare the cell growth between EV, Q165P, F133V C4-2 groups after the treatment with indicated inhibitors (2 µM of JQ1, 2 µM of CPI-637, 2 µM NEO2734) for 72 h. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. See Appendix Table S4 for the detailed comparison, P values and sample number (n).

C. Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP mutant C4-2 cells. The survival curve showed IC50 for EV (1.01 µM), SPOP Q165P (0.621 µM) and SPOP F133V cells (0.395 µM).

D, E. C4-2 cells expressing EV, Q165 or F133V constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvesting. The number of colonies with more than 50 cells was counted. Representative images of colonies are shown in (D) with quantification data in (E). All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001. See Appendix Table S4 for the detailed comparison, P values and sample number (n).

F - H. A schematic depicts the procedure of the establishment of SPOP mutant xenograft models and inhibitor administration (F). When the tumor reached 100 mm3, mice were treated with vehicle (40% polyethylene glycol), JQ1 (50 mg/kg) or NEO2734 (30 mg/kg) five days a week for three consecutive weeks. Tumors isolated from mice at day 21 of treatment were photographed (G) and tumor growth are shown in (H). All data shown are means ± SEM. The P value comparing the tumor volume at day 21 post- treatment was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, ** P < 0.01; *** P < 0.001. See Appendix Table S4 for the detailed comparison, P values and sample number (n).

Expanded View Figure Legends
A microtubule polymerase is required for microtubule orientation and dendrite pruning in Drosophila (2020)
Figure 1

Figure 1. Msps is required for dendrite pruning in ddaC neurons.

A A schematic illustration of the dendrite pruning process in ddaC neurons. The time windows for three subdivided phases including severing, fragmentation, and clearance are listed. Red arrowheads point to the ddaC somas.

B-F Live confocal images of ddaC clones expressing mCD8::GFP driven by ppk-Gal4 at WP stage or 16 h APF. l(3)810 and l(3)924 mutant ddaC clones (C, E) displayed dendrite arborization defects at WP stage and pruning defects at 16 h APF. (D, F) Introduction of one genomic construct of msps, HN267/g-msps, rescued dendrite pruning defects in msps810 and msps924 clones. Red arrowheads point to the ddaC somas.

G Percentages of ddaC clones showing severing defects at 16 h APF.

H Quantitative analysis of unpruned dendrite lengths at 16 h APF.

Data information: In (G), the numbers above the bars represent the sample sizes. Error bars represent S.E.M. from three independent experiments. Statistical significances were determined by One-way ANOVA with Bonferroni test, ***p<0.001, ns indicates statistically not significant. Scale bars represent 50 μm. Source data are available online for this figure.
A microtubule polymerase is required for microtubule orientation and dendrite pruning in Drosophila (2020)
Figure 2

Figure 2. Msps and TACC associate and stabilize each other in postmitotic neurons.

A Pan-neuronal driver elav-Gal4 was used to co-express GFP-Msps and TACC in postmitotic neurons. elav-Gal4 driven mCD8::GFP expression was used as a control. TACC was pulled down by GFP-Msps but not by mCD8::GFP. Alpha-tubulin was used as a loading and probing control. Neither mCD8::GFP nor GFP-Msps could pull down alpha-tubulin.

B Endogenous Msps proteins were co-immunoprecipitated with Venus-TACC by GFP beads but not by control IgG beads. Alpha-tubulin was used as loading and probing control.

C Endogenous Msps or TACC was pulled down by their respective antibodies, and the other protein was simultaneously detected in the immunoprecipitated contents. Alpha-tubulin was used as loading and probing control.

D-G Confocal images of ddaC neurons expressing control RNAi (D), msps RNAi (E), tacc RNAi (F) and αTub84B RNAi (G) that were immunostained for TACC at wL3 stage. ddaC somas are labeled by dashed lines. ddaC neurons are identified by ppk-Gal4 driven mCD8::GFP (green channel) expression, as shown at the top right corner.

H Quantitative analysis of normalized TACC fluorescence intensity in ddaC somas.

I-L Confocal images of ddaC neurons expressing control RNAi (I), msps RNAi (J), tacc RNAi (K) and αTub84B RNAi (L) that were immunostained for Msps at wL3 stage. ddaC somas are labeled by dashed lines. ddaC neurons are identified by ppk-Gal4 driven mCD8::GFP (green channel) expression, as shown at the top right corner.

M Quantitative analysis of normalized Msps fluorescence intensity in ddaC somas.

N, O Protein expression of Msps and TACC in brain tissues. (N) Larval brains of msps810/mspsP transheterozygotes at early wL3 stage were immunoblotted and probed with α-Msps and α-TACC antibody. Actin blotting was used as loading control. The dot plot on the right depicts the quantitative analysis of total TACC levels in control and msps transheterozygous larvae. (O) Adult brains of tacc59/ tacc74 transheterozygotes were immunoblotted and probed with α-Msps and α-TACC antibody. Actin blotting was used as loading and probing control. The dot plot on the right displays the quantitative analysis of total Msps level in control and tacc transheterozygotes.

Data information: The numbers above the x-axis represent the sample sizes. Error bars represent S.E.M. from three independent experiments. In (H, M), statistical significances were determined by One-way ANOVA with Bonferroni test, ***p<0.001, ns indicates statistically not significant. In (N, O), statistical significance was determined by two-tailed Student's T test, ***p<0.001. Scale bars in (D-G, I-L) represents 10 μm. Source data are available online for this figure.
A microtubule polymerase is required for microtubule orientation and dendrite pruning in Drosophila (2020)
Figure 3

Figure 3. tacc is required for dendrite pruning in ddaC neurons.

A-C Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. While expression of control RNAi in ddaC neurons (A) did not affect the dendrite morphogenesis nor the pruning processes, knockdown of tacc using RNAi #1 (B) or tacc59/tacc74 transheterozygous mutant (C) severely perturbed the dendrite arborization as well as pruning. Red arrowheads point to the ddaC somas.

D Percentages of ddaC neurons showing severing defects in (A-C) at 16 h APF.

E Quantitative analysis of unpruned dendrite lengths in (A-C) at 16 h APF.

F-H Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. Simultaneous knockdown of tacc and msps via co-expressing tacc RNAi and msps RNAi (H) in the ddaC neurons did not enhance the pruning defects, compared to those neurons co-expressing control RNAi and msps RNAi (F). Red arrowheads point to the ddaC somas.

I Percentages of ddaC neurons showing severing defects in (F-H) at 16 h APF.

J Quantitative analysis of unpruned dendrite lengths in (F-H) at 16 h APF.

Data information: In (D, I), the numbers above the bars represent the sample sizes. Error bars represent S.E.M. from three independent experiments. Statistical significances were determined by One-way ANOVA with Bonferroni test, ***p<0.001, ns indicates statistically not significant. Scale bars represent 50 μm. Source data are available online for this figure.
A microtubule polymerase is required for microtubule orientation and dendrite pruning in Drosophila (2020)
Figure 4

Figure 4. Msps and TACC are required for proper distribution of dendritic and axonal MT markers.

A-F Confocal images of ddaC neurons expressing UAS-mCD8::GFP, UAS-Nod-lacZ or UAS-Kin-lacZ and immunostained for β-galactosidase at wL3 stages. (A-C) Wild-type ddaC neurons (A) displayed normal distribution of Nod-lacZ signals in dendrites. In contrast, in msps810 mutants (B) and tacc59/tacc74 mutants (C), ddaC neurons exhibited altered Nod-lacZ distribution patterns, with highly enriched stainings in the somas and decreased signals in the dendrites. (D-F) Kin-lacZ signals were present in axons but not detectable in dendrites of the control ddaC neurons (D), whereas part of the Kin-lacZ were mislocalized to dendrites in msps RNAi (E) and tacc RNAi (F) ddaC neurons. Asterisks indicate the location of ddaC somas. White arrows indicate the location of axons. Curly brackets mark the dendritic regions where fluorescence intensity of Nod-lacZ was measured. White arrowheads point to dendritically localized Kin-lacZ signals.

G Percentages of ddaC neurons showing defective Nod-lacZ distribution in mutants or RNAi-expressing neurons.

H Quantitative analysis of normalized Nod-lacZ intensity for 20 μm of major dendrite located 40 μm away from the soma.

I Percentages of ddaC neurons showing defective Kin-lacZ distribution.

Data information: In (G, H, I), the numbers above the bars represent the sample sizes. Error bars represent S.E.M. from three independent experiments. Statistical significances were determined by One-way ANOVA with Bonferroni test, ***p<0.001. Scale bars represent 20 μm. Source data are available online for this figure.
A microtubule polymerase is required for microtubule orientation and dendrite pruning in Drosophila (2020)
Figure 5

Figure 5. msps and tacc are required for minus-end-out MT orientation in dendrites.

A-F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL. Horizontal arrow indicates the direction towards the somas, and vertical arrow indicates the time. EB1::GFP expression was driven under Gal44-77. In the control ddaC neurons (A, D), EB1 comets move predominantly in retrograde direction. EB1 comets were largely diminished in msps810/mspsP mutant neurons (B). Interestingly, in msps810/mspsP18 mutants (C), tacc RNAi (E) and tacc59/tacc74 mutant (F), increased anterograde comets were detected in ddaC dendrites.

G-J Quantitative analysis of the percentages of anterograde EB1::GFP comets, the track length, the number (per 30 µm) and the velocity of the comets in each neuron imaged.

Data information: In (G-J), the numbers above the x-axis represent the sample sizes. Error bars represent S.E.M. from three independent experiments. Statistical significances were determined by two-tailed Student's T test (pairwise comparisons) or One-way ANOVA with Bonferroni test (multiple-group comparisons), **p<0.01, ***p<0.001, ns indicates statistically not significant. Scale bar in (A-F) represents 10 μm. Each kymograph was generated by analyzing a 3-minute movie. Source data are available online for this figure.
A microtubule polymerase is required for microtubule orientation and dendrite pruning in Drosophila (2020)
Figure 6

Figure 6. Excessive MT depolymerization perturbs dendrite pruning.

A-F Confocal images of ddaC neurons expressing UAS-mCD8::GFP (Green channel), UAS-Nod-lacZ and immunostained for β-galactosidase at wL3 stages. Simultaneous knockdown of klp10A and msps (B) significantly restored Nod-lacZ localization in the dendrites, compared to msps and control RNAi (A). ddaC neurons with colchicine treatment (D) or with Kat-60 overexpression (F) exhibited perturbed Nod-lacZ distribution with highly enriched staining in the soma and decreased signals in the dendrites. Asterisks indicate the location of ddaC somas. White arrows indicate the location of axons. Curly brackets mark the dendritic regions where fluorescence intensity of Nod-lacZ was measured.

G-L Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. Simultaneous knockdown klp10A and msps (H) almost fully rescued the dendrite morphological defects and the pruning defects compared with the msps, control RNAi neurons. Wild type larvae fed with colchicine (J) showed pruning defects in comparison with those feed with DMSO (I). ddaC neurons with Kat-60 overexpression (L) exhibited abnormal dendrite arborization and pruning defects. Red arrowheads point to the ddaC somas.

M, N Quantitative analysis of normalized Nod-lacZ intensity for 20 μm of major dendrite located 55 μm or 40 μm away from the soma respectively.

O Percentages of ddaC neurons showing severing defects at 16 h APF.

P Quantitative analysis of unpruned dendrite lengths at 16 h APF.

Data information: In (M-O), the numbers above the x-axis represent the sample sizes. Error bars represent S.E.M. from three independent experiments. In (M, N, P), statistical significance were determined by two-tailed Student's T test (pairwise comparisons in M, N, P) or One-way ANOVA with Bonferroni test (multiple-group comparison in P), **p<0.01, ***p<0.001. Scale bars in (A-F) and (G-L) represent 20 μm and 50 μm, respectively. Source data are available online for this figure.
A microtubule polymerase is required for microtubule orientation and dendrite pruning in Drosophila (2020)
Figure 7

Figure 7. Excessive MT depolymerization perturbs dendritic MT orientation.

A-F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL. Horizontal arrow indicates the direction towards the somas, and vertical arrow indicates the time. (B) RNAi knockdown of klp10A in msps810/mspsP18 mutants restored the retrograde movement pattern of EB1 comets. MT depolymerization or severing induced by Colchicine treatment (D) or overexpression of Kat-60 (F) led to mixed MT orientation in ddaC neurons.

G-J Quantitative analysis of the percentages of anterograde EB1 comets, the track length, number (per 30 µm) and the velocity of EB1 comets in each neuron imaged.

Data information: In (G-J), the numbers above the x-axis represent the sample sizes. Error bars represent S.E.M. from three independent experiments. Statistical significances were determined by two-tailed Student's T test, *p<0.05, ***p<0.001, ns indicates statistically not significant. Scale bar in (A-F) represents 10 μm. Source data are available online for this figure.
Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding (2022)
Figure 1

Figure 1

Clustering of PS by eVP40 in vitro and in HEK 293 cells.

A Representative 3D reconstructed confocal images of immobilized GUVs (DPPC:Cholesterol:DPPS:PI(4,5)P2:TopFluor® TMR-PS(red)). Left panel: GUVs incubated without eVP40-Alexa488. Right three panels: GUVs incubated with 1.25 µM eVP40-Alexa488 (green). Scale bar = 5 μm.

B Index of correlation (Mander's coefficient) between TopFluor® TMR-PS and eVP40-Alexa488 of different GUVs compositions incubated with 1.25 µM eVP40-Alexa488. Values are reported as mean ± s.d. A one-way ANOVA with multiple comparisons was performed; n=3 for 3 technical replicates ****p<0.0001.

C % TopFluor® TMR-PS quenching by eVP40 using GUVs (DPPC:Cholesterol:TopFluor®TMR-PS + increasing mol% of PS), 2.5% PI(4,5)P2 was added to GUVs with 60% PS. Fluorescence spectra were recorded (Ex: 547 nm; Em: 550-600 nm); n=2 technical replicates.

D Representative confocal images of HEK293 cells expressing various GFP-fused proteins (green) and supplemented with TopFluor® TMR-PS (red); scale bar = 10 µm. Yellow arrows indicate high intensity PS fluorescent regions.

E %PM with PS clusters = area of high intensity fluorescent PS clusters over total plasma membrane area from images in panel D. Black bars are control proteins and blue bars are eVP40 proteins. Values are reported as mean ± s.d.; N>18 cells per biological replicate, n=3 for 3 biological replicates; A one-way ANOVA was performed with multiple comparisons compared to the control GFP %PS clustering (***p=0.0007, **p=0.004).

Data information: DPPC: dipalmitoyl-phosphatidylcholine; DPPS: dipalmitoyl-phosphatidylserine; GUVs: giant unilamellar vesicles; PS: phosphatidylserine; PM: plasma membrane.
Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding (2022)
Figure 2

Figure 2

Effect of PS concentration on eVP40 binding affinity to and oligomerization on membranes.

A-C SPR demonstrates that eVP40 affinity to LUVs increases in relation to PS concentration. A Representative normalized sensorgram of His6-eVP40 binding to LUVs containing 1% PS indicating an apparent affinity of 2.5 µM. B Representative normalized sensorgram of His6-eVP40 binding to LUVs containing 11% PS indicating an apparent affinity of 0.65 µM. C Representative normalized sensorgram of His6-eVP40 binding to LUVs containing 22% PS indicating an apparent affinity of 0.18 µM.

D, E PS concentration in LUVs enhances the ability of His6-eVP40 to oligomerize on membranes. D Representative western blot of chemical crosslinking performed on His6-WT-eVP40 following incubation with LUVs of varying PS content (detected by Mouse α-His antibody & HRP-Sheep α-Mouse). E Oligomerization capacity was determined from the western blot band density ratio of oligomers/(monomer + dimer) from chemical crosslinking experiments. A one-way ANOVA was performed with multiple comparisons compared to the control 0% PS LUVs control (30% PS *p= 0.02; 60% PS *p=0.017). n=3 biological replicates. Values are reported as mean ± s.d.;

Data information: SPR: surface plasmon resonance; LUVs: large unilamellar vesicles; PS: phosphatidylserine; HRP: horseradish peroxidase.