EMBO/soda-nel huggingface dataset:
The fitness cost and benefit of phase separated protein deposits (2019)
Figure 2

Figure 2. Protein phase separation and selection coefficient

A. Experimental design to measure the selection coefficient (S) upon growing URA3agg in competition with URA3sol.

B. The selection coefficient, S, is proportional to the difference in growth rates between URA3agg and URA3sol (Material and Methods). ω is the growth rate, which corresponds to the inverse of doubling time, τ.

C-D. The S values measured at standard conditions (30°C) indicate that, depending on the media composition (+Ura, -Ura, +5FOA), the formation of protein deposits can be neutral, deleterious or even advantageous for the cell as the function becomes non-essential, essential or toxic, respectively.

E. S values measured in different environments. In all cases the standard error is below 5%. See also Appendix Table S1. Color scale: purple in absence of uracil (-Ura), white in the presence of uracil (+Ura) and orange in the presence of 5FOA (+5FOA). At 37°C, the heat stress together with the inability to fold Ura3p have such a strong fitness effect that the strains barely grew in absence of uracil, impeding the measurement of S. A bar of purple-white diagonal lines indicates a presumed value of S at 37°C without uracil.
The fitness cost and benefit of phase separated protein deposits (2019)
Figure 3

Figure 3. Fitness cost and benefit analysis

A. We measured the intensity of fluorescence emitted by the fluorescent reporter (GFP) to estimate: (i) the amount, (ii) the location and (iii) the deposited/diffused state of the model proteins. Accordingly, for each cell analyzed, we measured the fluorescence intensity of its cytoplasm (FTOTALsol for URA3sol and FCYTOagg for URA3agg) and of its foci (FFOCIagg). We used these measurements to estimate the different effects of protein phase separation on cell fitness. The overall fitness effect of forming a protein deposit depends on three factors: foci formation, loss of function and gain of function (Eq.1). In our system, we can distinguish between the cost of forming Ura3p deposits, cost of losing the essential Ura3p activity and the benefit from protection against Ura3p toxicity. Each cost and benefit effect depend on the ratio of deposited or free protein and the specific environment. Accordingly, we can split these cost and benefit effects into two components: one associated with the amount of deposited or free protein (FFOCIagg/FTOTALagg or FCYTOagg/FTOTALsol) and a magnitude of effect defined by the environmental conditions (α, β or γ) (Eq.2). For the non-essential, essential and toxic roles (+Ura, -Ura and +5FOA) of Ura3p, the number and type of effects applicable is different (Eq.3, Eq.4 and Eq.5).

B. Plots showing the distribution and relationship between the fractions of deposited or free Ura3p, its magnitude of effect on fitness (α/β/γ) and the selection coefficient (S) in different environments.

C. Average of the selection coefficient (left) and average of the cytosolic fluorescence (equivalent to the fractional abundance of the free protein in URA3agg, right).

D. Schematic representation highlighting how phase separation of the same protein can result in different fitness effects depending on the environment.
The fitness cost and benefit of phase separated protein deposits (2019)
Figure 5

Figure 5. Impact on cell-to-cell variability and phenotypic diversity

Panels from A to D show that the variability in foci formation contributes to variability in free protein abundance.

A. Confocal microscopy images of URA3agg and URA3sol incubated for 18 hours at 30°C.

B. Coefficient of variation and p-values (Wilcoxon test) of the total fluorescence measured from populations of URA3agg and URA3sol.

C. Boxplot showing the distribution of foci fluorescence (left) or cytoplasmic fluorescence (right), from the foci containing subset of a population of URA3agg grown at 30ºC. Coefficient of Variation and p-values of each boxplot are shown below.

D. Schematic showing how the formation of protein deposit can affect the abundance of free protein in a cell. Pie charts showing the average fraction of deposited and free protein of URA3agg in different media composition and 30ºC.

E. Confocal microscopy image of URA3agg incubated in the presence of 5FOA after 18 hours at 30°C.

F. Schematic showing that in our system, the phase separation-promoting region can modulate Ura3p activity and thus its phenotypic effect in a cell.

G. Schematic highlighting that higher/lower level of free protein inside the cell results in higher/lower Ura3p activity leading to the manifestation of a stronger/weaker phenotype.

H. Boxplots of the cytoplasmic fluorescence normalized by the cell area (FCYTOagg/cell area) for individual cells of the URA3agg population in 5FOA. Light green box, cells with foci containing at least 70% of the total fluorescence (big foci). Dark green box, cells with foci containing less than 70% of the total fluorescence (small foci).

I-J. Cell cycle arrest phenotype. Density plots showing the fluorescence distribution of individual cells from the URA3agg population stained with propidium iodide to measure their DNA content. The cells were incubated for 18 hours at 30°C without (I) and with 5FOA (J).
Sequentially Acting SOX Proteins Orchestrate Astrocyte and Oligodendrocyte Specific Gene Expression (2018)
Figure 1

Figure 1. scRNA-seq analysis of mouse spinal cord cells.

(A) Experimental setup for scRNA-seq analysis. E11.5 CD133+ spinal cord progenitor cells were isolated using magnetic cell sorting (MACS) whereas E15.5 cells were randomly isolated from dissociated spinal cord tissue. Bioinformatic analyses, including t-SNE, revealed clustering of 350 E11.5 CD133+ progenitor cells and E15.5 cells. Colors in the graph indicate cell origin (pink for E15.5 cells and brown for E11.5 cells). (B) Walktrap community (Igraph) analysis of the cells revealed 7 distinct cell clusters within the t-SNE graph (colored according to the Walktrap clusters), highlighted in blue, yellow, green, red, light-blue, magenta and orange. Differential expression analysis revealed genes specifically expressed in each cell cluster. Examples of two to three specifically expressed genes from each cell cluster are presented. (C) Figures show the presence of differentially expressed genes within the analyzed single cells. Black color indicates cells with RPKM values above 40 for Pax3, 300 for Stmn2, 200 for Slc1a3 and 100 for Mbp. (D) GO-analysis of the differentially expressed genes in each Walktrap cell cluster. (E) Overlap between differentially expressed genes in three Walktrap cells clusters (marked by blue, green and red) and genes expressed in adult murine astrocytes, oligodendrocytes and neurons. Adult neuronal and glial expression profiles were derived from two individual studies [14,17]. P-values (phyper, R) were calculated from the total number of genes for the mm10 assembly (23´389). Non-significant overlaps were excluded.
Sequentially Acting SOX Proteins Orchestrate Astrocyte and Oligodendrocyte Specific Gene Expression (2018)
Figure 2

Figure 2. Glial genes are prebound in NPCs.

(A) Experimental setup for ESC based in vitro derivation of NPCs (4 days of differentiation condition, DDC) and GPCs (11 DDC). Time points for SOX3, SOX9 and H3K27Ac ChIP-seq experiments are as indicated. (B) Expression (RPKM) of Sox3 and Sox9 in NPC and GPC cultures from RNA-seq experiments. Results are represented as mean ± SEM from 3-4 experiments (n=3 for NPC and n=4 for GPC, *** equals p=4x10-5, t.test). (C) Immunohistochemical analysis of SOX3 and SOX9 expression in ESC derived NPC and GPC cultures. Analysis of TUJ1, GFAP and SOX10 protein expression in differentiated cultures revealed that NPC give rise to neurons and GPCs to astrocytes and oligodendrocytes. Scale bar represents 40µm. (D) Bar graph shows enrichment of genes bound by SOX3 in NPCs within the differentially expressed gene sets. Yellow bar represents enrichment within NPC/GPC genes, green bar within neuronal genes, blue bar within astrocytic genes and red bar within oligodendrocytic genes. (E) Bar graph shows enrichment of genes bound by SOX2 in mouse E11 spinal cord within the different gene sets. SOX2 data from [21]. (F) H3K27Ac read density in NPCs or GPCs around DNA-regions bound by SOX3 in NPCs. SOX3 DNA-regions with different H3K27Ac read density patterns in NPCs and GPCs were categorized as group I and II loci. Graphs show the mean read density of H3K27Ac in NPCs and GPCs at group I and II loci. (G) Expression pattern of genes associated with group I and II loci (from Fig. 2E) within differentially expressed gene sets. Significance calculated by prop.test R, (***) P<0.001. (H) Venn diagram shows overlap between SOX3 binding in NPCs and GPCs. Bar graph shows expression pattern of genes continuously bound by SOX3 NPCs and GPCs. (I) Venn diagram shows overlap between SOX3 and SOX9 binding in GPCs. Bar graph shows expression pattern of genes co-bound by SOX3 and SOX9 in GPCs. (J) ChIP-seq peak graphics around the astrocyte gene Fgfbp3. ChIP-seq peaks are derived from three different experiments; SOX3 ChIPs in NPCs, SOX3 ChIPs in GPCs, SOX9 ChIPs in GPCs. Both ChIP-seq reads and called peak regions (underlying black lines) are shown for all data sets. Bar graphs shows the distribution of differentially expressed genes that are bound by all three factors. P-values (phyper, R) were calculated from the total number of protein coding genes in mm10 assembly (23´389).
Sequentially Acting SOX Proteins Orchestrate Astrocyte and Oligodendrocyte Specific Gene Expression (2018)
Figure 4

Figure 4. Enrichment of NFI binding motifs around astrocyte specific genes.

(A) Graphical view showing SOX9 and SOX10 expression among spinal cord cells analyzed with scRNA-seq. (B) Bar graph shows enrichment of genes bound by SOX10 in rat oligodendrocytes within the differentially expressed gene sets. Yellow bar represents enrichment within NPC/GPC genes, green bar within neuronal genes, blue bar within astrocytic genes and red bar within oligodendrocytic genes. SOX10 ChIP-seq data from [37] (C) Venn-diagram shows 2622 SOX9 bound sites in GPCs, which are not co-targeted by SOX3, and their overlap with SOX10 bound sites in mature oligodendrocytes. Bar graph shows that mostly oligodendrocyte-specific genes are sequentially bound by SOX9 in GPCs and by SOX10 in oligodendrocytes. However, SOX9 and SOX10 also bind genes that are selectively expressed in astrocytes. (D) ChIP-seq peak graphics around the oligodendrocyte gene Plekhb1. ChIP-seq peaks are derived from four different experiments; SOX3 ChIP-seq in NPCs, SOX3 ChIP-seq in GPCs, SOX9 ChIP-seq in GPCs and SOX10 ChIP-seq in oligodendrocytes. Both ChIP-seq reads and called peak regions (underlying black lines) are shown for all data sets except for SOX10, for which only called peak regions are shown (black line). Triangle in the SOX10 data set indicate called peak. SOX10 ChIP raw reads could not be transferred from rn5 to mm9 assembly and therefore only SOX10 peak regions are shown in the figure. Scale bar: 5kb. (E) Figure shows most strongly enriched DNA binding motifs within 3 specific peak sets; SOX3 and SOX9 bound regions around astrocyte specific genes, SOX9 and SOX10 bound regions around astrocyte specific genes, and SOX9 and SOX10 bound regions around oligodendrocyte specific genes. (F) Expression of Nfia, Ehf and Zbtb3 among spinal cord cells analyzed with scRNA-seq. Red color indicates cells with RPKM values above 55 for Nfia, 5 for Zbtb3 and 5 for Ehf. n=3 for SOX9 and 2 for SOX3. P-values (phyper, R) were calculated from the total number of genes for mm10 (23´389).
Sequentially Acting SOX Proteins Orchestrate Astrocyte and Oligodendrocyte Specific Gene Expression (2018)
Figure 5

Figure 5. Sequentially acting SOX proteins control neuronal and glial specific CRMs.

(A) Luc-reporter assays in P19 cells. Luc-reporters containing SOX3 and SOX9 bound CRMs around the astrocyte specific genes Fgfbp3 and Ttyh1 were strongly activated by SOX9 in synergy with NFIA; an activation that could be efficiently blocked by SOX3. No activation of these reporters could be detected with SOX10 or SOX11. (B) Luc-reporters containing SOX9 and SOX10 bound DNA-regions around the astrocyte genes Aldh1l1 and Atp1a2, which are not expressed in SOX10+ oligodendrocytes, could not be activated by SOX9 or SOX10. (C) Luc-reporters containing SOX9 and SOX10 bound CRMs around the oligodendrocyte specific genes Plekhb1 and Mid2 were activated in an additive manner by SOX9 and SOX10. No activation could be detected with SOX11. (D) Luc-reporters containing SOX3 and SOX11 bound CRMs of the neuronal genes Tubb3 and Lhx2, which are sequentially bound by SOX3 and SOX11, could be activated by SOX11 and efficiently repressed by SOX3. No activation could be detected with SOX10. (E) Forced expression of SOX3 for 96 hrs in differentiating GPCs blocked the formation of astrocytes but not oligodendrocytes. Scale bar, 25µm. (F) Statistical view of Fig. 5E. Bars represents mean values (of 3 experiments) of cells expressing GFAP or SOX10 in GFP or Sox3-transduced cells. Error bars in figure 5 indicate standard deviation of 3-7 individual samples and three-star significance > 0.001 (t-test). (*) 0,05<P<0,01; (**) 0,01<P<0,001; (***) P<0,001.
Sequentially Acting SOX Proteins Orchestrate Astrocyte and Oligodendrocyte Specific Gene Expression (2018)
Figure 6

Figure 6. SOX3 prevents premature astocyte induction in the chick spinal cord.

(A) SOX3 over-expression experiments in chicken spinal cord at E4. 48 hrs post electroporation, transfected side showed decreased expression of FGFR2, NFIA and Fgfr3. Bar: 50µm. (B) 24 hrs after miss-expression of a dominant negative version of SOXB1 (dnSOXB1), premature expression of FGFR2, NFIA and Fgfr3 were found in the transfected side. Bar: 40µm. (C) 24 hrs after SOX9 over-expression in chicken spinal cord (at E4), increased expression of FGFR2, NFIA and Fgfr3 was shown compared to the un-transfected control side. Bar: 40µm. (D) Sox3 and Sox9 co-electroporation in chicken spinal cord at E4 could rescue the effect of SOX9 overexpression and FGFR2, NFIA and Fgfr3 levels were comparable to the un-transfected control side. Embryos were analyzed 24 hrs post electroporation. Bar: 40µm. (E) Proposed model of sequentially acting SOX proteins in neuronal and glial lineage specification. Silent neuronal, astrocyte and oligodendrocyte genes are all prebound by SOX3 in NPCs. In GPCs transactivating astrocytic enhancers, enriched for NFI motifs, (CRM A) become additionally targeted by SOX9 whereas non-activating astrocytic enhancers (CRMB) or CRMs around oligodendrocyte genes become pre-bound by SOX9 only. While SOX3 represses the activation of prebound genes, neuronal and glial gene expression become activated when SOX3 is downregulated and a cellular context of activating transcription factors has developed, which according to the model can consist of SOX11 in neurons, SOX10 in oligodendrocytes and NFIA and SOX9 in astrocytes. It should be noted that we do not provide any evidence that SOX3/SOX9 or NFIA/SOX9 physically interact or co-bind DNA.
Neutralization of SARS-CoV-2 by highly potent, hyper-thermostable, and mutation-tolerant nanobodies (2021)
Figure 1

Figure 1. Phylogenetic relationship of the anti-SARS-CoV-2 Spike VHH antibodies identified and characterized in this study.

A The sequences of the isolated VHH antibodies (49 anti-RBD and 10 anti-S1ΔRBD nanobodies) were aligned using Clustal Omega (Sievers et al., 2011). The circular phylogram was reconstructed with Dendroscope (Huson and Scornavacca, 2012). Where applicable, sequence classes are indicated. The anti-RBD nanobodies are colored according to the two RBD epitopes identified in Fig 5 (with epitope 1 in green, epitope 2 in red, and asterisks marking nanobodies included in Fig 5). Anti-S1ΔRBD nanobodies are colored in magenta. VHH antibodies that did not neutralize SARS-CoV-2 are shown in parentheses. See main text and Fig 5 for details.

B Vero E6 cells were inoculated with SARS-CoV-2, fixed with paraformaldehyde after two days, stained with 30 nM of the indicated Alexa Fluor 488 (AF488)-labeled anti-RBD VHH antibodies, and imaged by confocal laser scanning microscopy (CLSM).

C Immunofluorescence (IF) staining as in B, but with the indicated AF568-labeled anti-S1ΔRBD VHH antibodies.
Neutralization of SARS-CoV-2 by highly potent, hyper-thermostable, and mutation-tolerant nanobodies (2021)
Figure 3

Figure 3. Fluorescence-based assay to assess the neutralization potency of VHH antibodies.

A Vero E6 cells were infected with SARS-CoV-2, pre-incubated with indicated concentrations of the neutralizing VHH-72 (Wrapp et al., 2020a). Cells were fixed two days after inoculation, stained with sets of anti-RBD (green) and anti-S1ΔRBD (red) nanobodies (see Fig 1B and C), and analyzed by CLSM. We used a cocktail of such fluorophore-labeled VHH antibodies to ensure that negative fluorescence readings truly indicated neutralization and thus absence of viral infection and not just masking of the IF epitope by the tested nanobody. See Appendix Fig S1 for independent biological replicates.

B Vero E6 cells were infected as in A. Viral RNA in the culture supernatants was quantified by reverse transcription (RT)-qPCR. RNA signals are normalized to the respective minus-VHH controls (100%). Note the log10 scale of the y-axis. "Inoculum" (red dotted line) marks the RNA level detected at the time of infection. See also Appendix Fig S1.
Neutralization of SARS-CoV-2 by highly potent, hyper-thermostable, and mutation-tolerant nanobodies (2021)
Figure 5

Figure 5. Epitope-binning with the indicated VHH antibodies.

A HeLa cells were transiently transfected to express the SARS-CoV-2 Spike protein. Following fixation, cells were stained for 1 hour with fluorophore-labeled Re10B10 (5 nM, green) and Re7E02 (15 nM, red) in the presence of the indicated unlabeled VHH competitors. Competitor (150 nM) was added 20 min prior to the labeled nanobodies. The weakly binding competitors Re5A08, Re9F06, Re6B06, and, Re6D06 were added as trimers (see below). Cells were imaged by CLSM. For each competitor, the sequence class and the binding site on the RBD (epitope 1 or 2) are indicated.

B ACE2 competition experiments with selected VHHs from A. 50 nM RBD was mixed with indicated nanobodies (at 500 nM). Binding to ACE2 (immobilized on the sensors) was monitored by BLI.
Neutralization of SARS-CoV-2 by highly potent, hyper-thermostable, and mutation-tolerant nanobodies (2021)
Figure 8

Figure 8. Neutralization of SARS-CoV-2 with symmetry-matched VHH antibodies.

A Left: Schematic representation of VHH antibodies produced in E. coli as monomers or homo-trimeric fusions with the human collagen XVIII trimerization module. The VHH trimers match the three-fold rotational symmetry of the SARS-CoV-2 Spike protein. The molecular weight of the indicated modules is shown. Right: As an example, monomeric and trimeric forms of Re7H02 (a Re6B06 class member, Fig 1A) were analyzed by size exclusion chromatography (SEC, Superdex 200 column) coupled with multi-angle light scattering (MALS). The elution profiles (UV absorbance signal), as well as the calculated molecular weights (per slice), are plotted, and the average molecular weight is indicated.

B Re6B06 monomer and trimer variants (20 nM) were analyzed by BLI for RBD-binding, with 900 s association and 1500 s dissociation steps. Note the avidity effect of Re6B06 trimerization.

C Neutralization of SARS-CoV-2 by the indicated VHH antibodies. The neutralization experiment was performed as described in Fig 3A. Note the gain in neutralization efficacy when a VHH is trimerized. Re6B06, for example, is 30 000-fold more potent when symmetry-matched with the virus Spike.
Neutralization of SARS-CoV-2 by highly potent, hyper-thermostable, and mutation-tolerant nanobodies (2021)
Figure 10

Figure 10. Neutralization of SARS-CoV-2 B.1.351 by VHH antibodies.

A and B Neutralization of SARS-CoV-2 B.1.351/Beta by the indicated VHH antibody constructs. The neutralization experiment was performed as described in Fig 3A, with the difference being that a mutant-optimized anti-RBD/S1 VHH cocktail was used for immunofluorescence staining (see Appendix Fig. S13). Two titrations for the Re9F06 trimer are shown, corresponding to the standard (upper row) or a five-fold higher virus inoculum (lower row) that was used for even more stringent neutralization conditions.
NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2 (2016)
Figure 1

Figure 1 NAT10 interacts with p53 and Mdm2

A U2OS cells were transfected with Flag-NAT10 or control vectors. Forty-eight hours later, cells were harvested and whole cell extracts were immunoprecipitated with Flag-antibody affinity resin. The NAT10-binding proteins were resolved by SDS-PAGE and detected by silver staining.

B-F U2OS, HCT116 p53+/+ or HCT116 p53-/- cell lysates were immunoprecipitated with control IgG, anti-NAT10 (B and F), anti-p53 (C) and anti-Mdm2 (D and E) antibodies. The immunoprecipitates were subsequently immunoblotted with the indicated antibodies.

G Purified NAT10 was incubated with GST, GST-p53 or GST-Mdm2 proteins coupled to Glutathione Sepharose 4B. Proteins retained on the Sepharose were then analyzed by western blot using the antibodies as indicated. The amount of GST fusion proteins are shown in the lower panel.

H Full-length GST-p53 fusion protein, its deletion mutants or GST protein was used to pull-down experiments with purified NAT10 protein. The levels of the GST fusion proteins are shown in the left panel.

I GST pull-down assay was performed using purified GST-NAT10 deletion mutants or GST protein and overexpressed Flag-p53 or Mdm2 protein in HEK293T cells. Schematic diagram represents the constructs of GST-NAT10 deletion mutants (right panel).
NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2 (2016)
Figure 2

Figure 2 NAT10 acetylates p53 at K120

A Recombinant His-p53 was incubated with or without purified Flag-NAT10 as described in Experimental Procedures. The reaction products were resolved by SDS-PAGE and immunoblotted using anti-acetyl-lysine antibodies. Flag-PCAF was used as a positive control.

B In vitro acetylation was performed and the acetylated p53 was detected as in A.

C In vitro acetylation assay was performed as described in A. The reaction products were resolved by SDS-PAGE and acetylated p53 was purified from the SDS-PAGE and subjected to mass spectrometry analysis.

D His-p53 or His-p53-K120R fusion protein was incubated with purified NAT10 or NAT10 GE mutant as described in Experimental Procedures. Reaction mixtures were subjected to western blot using the site specific monoclonal anti-Ac-p53-K120 antibody.

E H1299 cells were transfected with the indicated vectors. Total proteins and the anti-Flag antibody M2-specific immunoprecipitates were analyzed by western blot using the indicated antibodies.