EMBO/soda-nel huggingface dataset:
The NFIB-ERO1A axis promotes breast cancer metastatic colonization of
disseminated tumour cells (2021)
Figure 2

Figure 2. Nfib ablation delays mammary tumour formation and abrogates metastasis.

A The kinetics of LM1 (n = 10), LM9 (n = 4), LM1 Nfib KO (n = 4), and LM9 Nfib KO (n = 4) tumour growth upon orthotopic injection of 250 x 103 cells into NOD/SCID mice. Curves show means of tumour volume ± s.d., two-way ANOVA group analysis of the times to reach 250 mm3 (dashed line).

B Kaplan-Meier plot depicting metastatic onset after tumour removal in mice injected orthotopically with LM1 (n = 5), LM9 (n = 4), LM1 Nfib KO (n = 3), or LM9 Nfib KO (n = 4) cells, two-tailed log-rank test.

C Nfib knockout abrogates metastasis in the orthotopic LM1 model. Representative bioluminescence images (left panel) and bar plot quantification (right panel) of lung metastases at 25 (LM1) and 40 (KO) days after primary tumour removal; n = 3 mice, means ± s.d., two-tailed Student's t-test.

D Nfib knockout impairs experimental metastases formation in the LM models. Kaplan-Meier plot showing metastatic incidence of animals inoculated i.v. with LM1 (n = 4), LM9 (n = 4), LM1 Nfib KO (n = 3), or LM9 Nfib KO (n = 5) cells, two-tailed log-rank test.

E Representative bioluminescence images (left panel) and bar plot quantification (right panel) of lung metastases 16 days after i.v. injection of LM1 or LM1 Nfib KO cells, n = 3 mice, means ± s.d., two-tailed Student's t-test.
The NFIB-ERO1A axis promotes breast cancer metastatic colonization of
disseminated tumour cells (2021)
Figure 3

Figure 3. Nfib is sufficient to induce metastasis.

A Kaplan-Meier plot depicting metastasis onset after tumour removal in mice injected orthotopically with HR1 gCtrl (n = 13) or HR1 g4Nfib (n = 14), two-tailed log-rank test.

B Nfib overexpression in HR1 parental cells enhances metastasis in the orthotopic model. Representative bioluminescence images (left panel) and bar plot quantification (right panel) of lung metastases at day 20 (HR1 gCtrl) and day 2 (HR1 g4Nfib) after primary tumour removal. n = 4 mice, means ± s.d., two-tailed Student's t-test.

C Nfib overexpression in the HR1 parental cells enhances experimental metastases. Kaplan-Meier showing metastatic incidence of animals inoculated i.v. with HR1 gCtrl (n = 11) or HR1 g4Nfib (n = 11) cells, two-tailed log-rank test.

D Representative bioluminescence images (left panel) and bar plot quantification (right panel) of lung metastases 16 days after i.v. injection of HR1 gCtrl or HR1 g4Nfib cells. n = 4, means ± s.d., two tailed Student's t-test.

E NFIB overexpression enhances experimental metastases formation in the SUM159PT model. Kaplan-Meier survival analysis of animals inoculated i.v. with SUM159PT gCTRL (n = 5) or SUM159PT gNFIB (n = 6) cells, two-tailed log-rank test.

F Representative bioluminescence images (left panel) and bar plot quantification (right panel) of lung metastases 20 days after i.v. injection of SUM159PT gCTRL or SUM159PT gNFIB (n = 5), means ± s.d., two-tailed Student's t-test.
The NFIB-ERO1A axis promotes breast cancer metastatic colonization of
disseminated tumour cells (2021)
Figure 4

Figure 4. The NFIB-ERO1A axis enhances lung metastatic colonization.

A, B Intersection of the top 200 differentially expressed genes (sorted by statistical significance) in tumourspheres and tumours (Nfib high-expression vs. the respective controls) and putative transcriptional targets of Nfib determined by ChIP-seq in mammary gland (Shin et al, 2016) (A) and epithelial-melanocyte stem cells (Chang et al, 2013) (B) (association rule: Basal+extension: 5000 bp upstream, 1000 bp downstream, 10000 bp max extension, curated regulatory domains included).

C ChIP was performed in all murine cell lines (Nfib high-expression and the respective Nfib low-expression or KO controls) against Nfib followed by qPCR with primers specific for Ero1l promoter region proximal to transcription starting site (Ero1l_492 and Ero1l_493). Meg3 was used as control. Graph shows qPCR results with % input method as indicated (Nfib high-expression vs. the respective Nfib low-expression or KO controls). n = 2 biological replicates and n = 2 technical replicates, means ± s.d., two-tailed Student's t-test, n.s. = not significant.

D Kaplan-Meier survival analysis of NSG mice inoculated i.v. with SUM159PT gNFIB shCTRL (n = 9), SUM159PT gNFIB sh1 ERO1A (n = 7), or SUM159PT gNFIB sh2 ERO1A (n = 7) cells. Two-tailed log-rank test.

E Representative bioluminescence images (left panel) and bar plot quantification (right panel) of lung metastases 8 (SUM159PT gNFIB shCTRL) and 20 (SUM159PT gNFIB sh1 ERO1A or SUM159PT gNFIB sh2 ERO1A) days after i.v. injection of the respective cells. n = 4, means ± s.d., two-tailed Student's t-test.

F Kaplan-Meier survival analysis of overall survival of mice injected i.v. with SUM159PT gNFIB shCTRL (n = 10), SUM159PT gNFIB sh1 ERO1A (n = 12), or SUM159PT gNFIB sh2 ERO1A (n = 13) cells, two-tailed log-rank test.
The NFIB-ERO1A axis promotes breast cancer metastatic colonization of
disseminated tumour cells (2021)
Figure 5

Figure 5. Ero1l/ERO1A increases Vegfa/VEGFA expression and angiogenesis.

A Bar graph showing the ROS levels in the NFIB models. The fluorescence signal was normalized to the blank. n = 2 biological replicates and n = 2 technical replicates, means ± s.d., two-tailed Student's t-test.

B Left panel: representative images of HIF1α immunofluorescence. HIF1α was stained with Alexa-633 (red) and nucleus with DAPI (blue). Scale bar 100 μm. Right panel: bar graph showing quantification of HIF1α nuclear/cytoplasmatic ratio. n = 5 biological replicates, means ± s.d., two-tailed Mann-Whitney U-test.

C Left panel: Bar graph representing mean VEGFA mRNA expression in SUM159PT gCTRL and SUM159PT gNFIB breast cancer cell lines upon downregulation of ERO1A with two independent shRNAs (sh1 and sh2). Right panel: Bar graph representing mean VEGFA mRNA expression in lung extracts from animals inoculated i.v. with SUM159PT gNFIB shCTRL, SUM159PT sh1 ERO1A, or SUM159PT sh2 ERO1A cells. In both graphs n = 3 biological replicates and n = 2 technical replicates, means ± s.d., two-tailed Student's t-test, FC= fold change.

D Left panel: Representative images of CD31-positive endothelial structures in lungs. Scale bar, 1 mm. Right panel: Bar graphs showing lung metastases quantified as percentage of metastatic area per lung area and quantification of CD31 staining in the metastatic area. Means ± s.d., n = 8 shCTRL, n = 5 per sh1 ERO1A, and n = 6 sh2 ERO1A, two-tailed Student's t-test.

E Left panel: Bar graph showing average of master segments counted after incubation of HUVEC cells with conditioned medium from LM1, HR1, 4T1 (Nfib high-expression and the respective Nfib low-expression or KO controls) and SUM159PT gNFIB shCTRL, SUM159PT gNFIB sh1 ERO1A, SUM159PT gNFIB sh2 ERO1A cells. Human VEGF121(0.4 ng/μl) was added to SUM159PT gNFIB sh1 and sh2 ERO1A cells, n = 2 technical replicates, means ± s.d., two-tailed Student's t-test. Right panel: Representative images showing tube formation four hours after addition of conditioned medium from HR1 gCtrl or g4Nfib.Scale bar 100 μm.
The NFIB-ERO1A axis promotes breast cancer metastatic colonization of
disseminated tumour cells (2021)
Figure 6

Figure 6. NFIB-ERO1A-VEGFA overexpression is associated with aggressive tumours.

A Representative images of NFIB, ERO1L, VEGFA-positive TNBC PDX primary tumours. Scale bar, 1 mm.

B Representative images of NFIB, ERO1L, VEGFA-positive lung metastases from TNBC PDX. Scale bar, 1 mm.

C Higher NFIB expression in iC10 and basal breast cancer subtype compared with the other subtypes using intClust (Dawson et al, 2013) and PAM50 classifications in the METABRIC cohort (n = 1980 across the 10 Intclust, iC). Boxplot represents NFIB expression values comprised between the 1st and the 3rd quartiles with the central band representing median expression value, and whiskers indicating the farthest data points that are still within the distance of 1.5 times the interquartile range (IQR).

D, E Distant metastasis-free survival (D, n = 232) and overall survival (E, n = 241) plots generated using the Kaplan-Meier Plotter based on the mean expression using the signal intensity of the NFIB (213029_at) combined with the ERO1A (218498_s_at) probes. The cut-off was automatically set to split patients into two groups (median), high and low. The plots were generated using the signal intensity of the different probes in Affymetrix microarray gene expression data from TNBC patients of The Cancer Genome Atlas. Number of patients (n) and P values (two tailed log-rank test) are presented in the panels.

Expanded View Figures legends:
The deubiquitylase USP33 discriminates between RALB functions in autophagy and innate immune response (2013)
figf1

(a) Schematic outline of the MAPPIT technique. The indicated RAL mutants (baits) were fused to a leptin receptor fragment that is deficient in STAT3 recruitment sites. The RAL downstream effectors (preys) were tethered to a GP130 cytokine receptor fragment containing functional STAT3-docking sites. Association of bait and prey proteins restores a functional leptin receptor complex. On leptin stimulation, STAT3 activation can be monitored by a STAT3-responsive luciferase reporter. WT, wild type. (b) MAPPIT screen identified RAL-interacting proteins. A MAPPIT array covering close to 8,500 preys was screened with RALA-G23V as bait. Data are shown in a volcano plot of the log ratio of normalized MAPPIT luciferase activity versus
P value Source data can be found in Supplementary Table S1. (c) MAPPIT assay confirmed interaction between the RAL proteins and USP33. RALA-G23V or RALB-G23V baits were introduced into HEK293T cells together with the indicated preys. The results are expressed as a mean of normalized luciferase activity ± s.d (leptin-treated cells versus leptin-untreated cells) for three independent experiments. Statistics source data can be found in Supplementary Table S1. (d,e) USP33 interacts with both RALA and RALB. At 48 h post-transfection with tagged USP33 and RAL expression constructs, the indicated proteins were then immunoprecipitated with anti-Flag (M2) or anti-HA–agarose, followed by immunoblotting using anti-Flag or anti-HA antibodies. WCL, whole-cell lysate; IP, immunoprecipitates. (f) Endogenous USP33 interacts with RALB. Flag–RALB was immunoprecipitated using anti-Flag (M2) agarose and analysed for co-precipitation with endogenous USP33 using anti-USP33 antibody. Cells expressing USP33 shRNA were used as a negative control. (g) GTP binding is not required for interaction between USP33 and the RAL proteins. The GST-tagged RALA and RALB were isolated with glutathione–agarose and mixed with lysates derived from HEK293T cells expressing Flag–USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag-specific antibody. (h) USP33 affects ubiquitylation of RALB, but not RALA. 6xHis-tagged ubiquitin and Flag–RAL-G23V mutants were introduced into HEK293T cells stably expressing GFP shRNA or USP33 shRNA. Ubiquitylated RAL proteins were purified by Co2+ metal affinity chromatography and detected by antibodies specific to RALA or RALB. Uncropped images of blots are shown in Supplementary Fig. S7.
The deubiquitylase USP33 discriminates between RALB functions in autophagy and innate immune response (2013)
figf2

(a) Tandem affinity purification of ubiquitylated RALB. 6xHis–ubiquitin and Flag–RALB-G23V were co-transfected into HEK293T cells, and ubiquitylated RALB was purified using anti-Flag resin followed by Co2+ metal affinity chromatography. Isolated RALB was visualized by immunoblotting using anti-RALB antibody. (b) RALB undergoes ubiquitylation on multiple sites. Schematic diagram of the RALB domains (upper panel). RALB ubiquitylation sites were identified in two independent tandem mass spectrometry analyses with a total coverage of 63% of the RALB amino acid sequence (Supplementary Fig. S4a). The major identified ubiquitylation sites, Lys 47 and Lys 160, are located in the effector-interacting domain (green) and the G5 box (red), respectively. (c) Substitution of arginine for lysine at position 47 significantly inhibits ubiquitylation of RALB. (d) USP33 regulates RALB ubiquitylation at Lys 47. (e) The GTP–GDP status of RALB affects RALB ubiquitylation at Lys 47. For c–e, 6xHis-tagged ubiquitin and the indicated RALB mutants were introduced into HEK293T cells expressing GFP shRNA or USP33 shRNA. Ubiquitylated RALB was purified by Co2+ metal affinity chromatography and detected by antibodies specific to RALB. WT, wild type. (f) Lack of ubiquitylation at Lys 47 inhibits RALB binding to SEC5. The indicated Flag-tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-SEC5 antibody. (g) Ubiquitylation at Lys 47 impairs RALB binding to EXO84. Flag-tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-EXO84 antibody. (h) Ubiquitylation at Lys 47 does not affect RALB activity. The activity of RALB mutants was determined by a RALBP1–RBD binding assay. (i) The Rosetta v3 protocol described in ref. was used for ubiquitin-docking simulations. The plot represents the energies of the produced decoys distributed by the root-mean-square deviation (RMSD) of ubiquitin position relative to RALA from an arbitrarily chosen decoy. (j) Rosetta modelling of ubiquitylated-RALA–SEC5-RBD and ubiquitylated-RALA–EXO84-RBD complexes. Superposition of the top ten refined solutions of ubiquitylated-RALA–SEC5-RBD complex, selected by combined scores of two independent docking simulations of ubiquitylated-RALA–SEC5-RBD and ubiquitylated-RALA (left). Simulated ubiquitin positions linked to Lys 47 of RALA were combined with the RALA–EXO84 complex (right). Uncropped images of blots are shown in Supplementary Fig. S7.
The deubiquitylase USP33 discriminates between RALB functions in autophagy and innate immune response (2013)
figf3

(a) USP33 does not affect RALB activity. The activity of RALB was determined in HEK293T cells expressing GFP shRNA or USP33 shRNA and empty vector (V) or HA-tagged USP33 by RALBP1–RBD binding assay. (b) USP33 modulates interaction between RALB and SEC5. Flag-tagged RALB was overexpressed in HEK293T cells expressing GFP shRNA, USP33 shRNA, empty vector (V) or HA-tagged USP33. The indicated proteins were then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-Flag or anti-SEC5 antibodies. (c) USP33 regulates RALB binding to EXO84. Flag-tagged RALB was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells expressing the indicated constructs. The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti-HA or anti-EXO84 antibody. (d) Activated RALB co-localizes with both EXO84 and SEC5. At 24 h after co-transfection with RALB-G23V–EGFP and HA–EXO84, HeLa cells were immunostained with anti-HA and anti-SEC5 antibodies. Scale bars, 10 μm. The outlined areas are shown at higher magnification in the bottom panels. (e) RALB ubiquitylation controls interaction between EXO84 and SEC5. Flag-tagged SEC5 was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing RALB-G23V or RALB-G23V-K47R. The presence of EXO84 in SEC5 complexes was analysed by immunoblotting using anti-EXO84 antibody. (f) USP33 regulates EXO84–SEC5 complex formation. Flag-tagged SEC5 was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells expressing the indicated constructs followed by immunoblotting using anti-EXO84 antibody. (g) Suppression of SEC5 affects RALB binding to EXO84. HA-tagged RALB-G23V was immunoprecipitated from HEK293T cells expressing the indicated constructs. The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti-EXO84 antibody. For a–c,f,g immunoblotting using antibodies specific for USP33, anti-Flag (M2) or anti-HA determined the levels of USP33 suppression or overexpression. Immunoblot analysis was performed using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S7.
The deubiquitylase USP33 discriminates between RALB functions in autophagy and innate immune response (2013)
figf4

(a) Suppression of USP33 facilitates TBK1–SEC5 complex formation. Flag-tagged SEC5 was introduced into HEK293T cells expressing empty vector (V) or HA-tagged RALB-G23V together with GFP shRNA or USP33 shRNA as indicated, and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using antibody specific for TBK1. (b) RALB undergoes ubiquitylation in response to poly(I:C) treatment. At 48 h after introduction of 6xHis-tagged ubiquitin and Flag–RALB-G23V, 293-hTLR3 cells were treated with 10 μg ml−1 of poly(I:C) for 6 h. Ubiquitylated RALB was purified by Co2+ metal affinity chromatography and detected by immunoblotting using anti-RALB antibody. (c) RALB ubiquitylation at Lys 47 is crucial for formation of the RALB–SEC5 complex in response to poly(I:C) treatment. Flag-tagged SEC5 and the indicated HA-tagged RALB mutants were introduced into 293-hTLR3 cells. At 48 h after transfection cells were treated 100 μg ml−1 of poly(I:C) for 6 h. Flag-tagged SEC5 was immunoprecipitated with anti-Flag (M2), and analysed for co-precipitation by immunoblotting using anti-HA antibody. (d) RALB ubiquitylation at Lys 47 affects activation of TBK1 and IRF3. At 48 h after transfection with empty vector (V), Flag–RALB-G23V and Flag–RALB-G23V–K47R, 293-hTLR3 cells were treated with 10 μg ml−1 of poly(I:C) for 6 h. Immunoblot analysis was performed using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S7.
The deubiquitylase USP33 discriminates between RALB functions in autophagy and innate immune response (2013)
figf5

(a–d) USP33 inhibits assembly of SEC5–beclin-1 complexes. The indicated proteins were immunoprecipitated from cells expressing GFP shRNA or USP33 shRNA with anti-Flag (M2), anti-HA or anti-SEC5 antibody and analysed for co-precipitation by immunoblotting using specific antibodies as indicated. (e) USP33 potentiates the interaction between EXO84 and beclin 1. Endogenous beclin 1 was immunoprecipitated from HEK293T cells, expressing either empty vector (V), HA–USP33 wild type (WT) or the catalytically inactive mutant C194S-H683Q, using anti-beclin 1 antibody. Immunoprecipitates were analysed for co-precipitation by immunoblotting using anti-EXO84 or anti-beclin 1 antibodies. (f,g) RALB but not RALA undergoes deubiquitylation under nutrient deprivation. 6xHis-tagged ubiquitin and the indicated RAL mutants were overexpressed in HEK293T cells. At 48 h after transfection cells were deprived of nutrients and incubated in Hank’s buffered salt solution (HBSS) medium for 90 min. Ubiquitylated RAL proteins were purified by Co2+ metal affinity chromatography and analysed by immunoblotting using anti-RALA or anti-RALB antibodies. (h) Nutrient starvation results in the accumulation of USP33. HEK TE cells were incubated in HBSS medium for the indicated periods of time. Immunoblotting was performed using anti-USP33 antibody. (i) USP33 co-localizes with RALB-positive vesicles in the absence of nutrients. At 48 h after co-transfection with RALB-G23V–EGFP and USP33–mCherry, HeLa cells were incubated in HBSS medium for 90 min. Cells were imaged for EGFP and mCherry fluorescence. Scale bars, 10 μm. The outlined areas are shown at higher magnification in the bottom panels. Arrows indicate USP33 co-localization to RALB-positive vesicles. (j) Beclin 1 co-localizes with USP33. HeLa cells were transfected with beclin-1–HA and USP33–mCherry. At 48 h after transfection, cells were incubated in HBSS medium for 90 minutes and treated as in i. Scale bars, 10 μm. The outlined areas are shown at higher magnification in the bottom panels. Uncropped images of blots are shown in Supplementary Fig. S7.
The deubiquitylase USP33 discriminates between RALB functions in autophagy and innate immune response (2013)
figf6

(a) USP33 depletion inhibits accumulation of LC3–lipid conjugates. HEK TE cells stably expressing GFP shRNA or USP33 shRNA were incubated with DMEM or HBSS medium for 4 h, in the presence or absence of 50 μM of chloroquine (CQ). Cells were then assayed for the relative accumulation of LC3-I and LC3-II by immunoblotting analysis using anti-LC3 antibody. LC3-II/GAPDH ratios have been calculated using ImageJ densitometric analysis. (b–d) USP33 expression levels affect the accumulation of LC3 punctae. Wild-type USP33 or a USP33-shRNA-resistant USP33 mutant (USP33R) was introduced into HEK TE cells stably expressing GFP shRNA or USP33 shRNA. The generated cells were incubated in HBSS medium for 4 h. USP33 was analysed by immunoblotting using anti-USP33 antibody (b). Representative images of LC3 immunostaining are shown (c). Endogenous LC3 punctae were quantified using the In Cell Analyser 2000 automatic imaging system and IN Cell Investigator software. The mean distribution of LC3 punctae per cell is represented as the mean of two independent experiments. Statistics source data for d can be found in Supplementary Table S1 (d). (e) USP33 depletion leads to increased levels of p62. HEK TE cells expressing GFP shRNA or USP33 shRNA were incubated in HBSS medium for the indicated periods of time. Immunoblotting was performed using the indicated antibodies. (f) Overexpression of the RALB-G23V-K47R mutant leads to accumulation of LC3–lipid conjugates. HEK293T cells overexpressing the RALB-G23V or RALB-G23V-K47R mutants were assayed for the relative accumulation of LC3-I and LC3-II by immunoblotting using anti-LC3 antibody. (g) Ubiquitylation of RALB at Lys 47 determines a choice of specific pathways to mediate particular cellular functions. dsRNA, mimicking viral infection, triggers ubiquitylation of RALB. RALB ubiquitylated at Lys 47 preferentially binds SEC5 that drives SEC5–TBK1 complex formation and activation of IRF3 signalling. In contrast, nutrient deprivation induces stabilization and relocalization of USP33 that deubiquitylates RALB at Lys 47. Deubiquitylation of RALB mediates assembly of the RALB–EXO84–beclin-1 complex that initiates autophagy. Uncropped images of blots are shown in Supplementary Fig. S7.
Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites (2020)
Figure 2:

Structural overview of SARS-CoV-2 N-NTDFigure legend: A. Ribbon representation of SARS-CoV-2 N-NTD molecules in one asymmetric unit. The four molecules are highlighted with different color respectively; B. Superimpositions of four molecules in one asymmetric unit. The dash circles indicate the sandwiched structure composed of Loop region 1, β-sheet core, and Loop region 2. The β-strand is labeled with β1 to β7, the 310 helix is labeled with η1; C. Topological style illustration of SARS-CoV-2 N-NTD structure; D. Electrostatic surface of the SARS-CoV-2 N-NTD. Blue denotes positive charge potential, while red indicates negative charge potential. The potential distribution was calculated by Pymol. The values range from −5 kT (red) to 0 (white) and to +5 kT, where k is the Boltzmann constant and T is the temperature.