EMBO/soda-nel huggingface dataset:
Consequence of the tumor-associated conversion to cyclin D1b. (2015)
Figure 6

Cyclin D1b expression promotes double-strand breaks and PARP-1 activity

Ccnd1+/+ and Ccnd1KI/KI cells were transfected with a pool of siRNA directed against the N-terminus of the murine cyclin D1 transcript or scramble control for 48 h (as in Figure5). Cells were then fixed and stained for markers of double-strand breaks (p-H2AX and 53BP1, 400× objective). Total number of foci for each cell was quantified and represented as the % of cells with > 10 foci/cell (p-H2AX) or % of cells with > 2 foci/cell (53BP1). Error bars represent ± SEM.

Ccnd1+/+ and Ccnd1KI/KIMAF lines were grown in serum-proficient media for 24 h and probed for expression of auto-modified PARP1 via immunoblot. Gapdh and cyclin D1b serve as controls.

Cells were plated as in (B) and treated with 0.5 μM etoposide for 3 h. Cells were harvested and then probed for markers of DNA damage via immunoblot. Lamin B serves as a control.

Indicated MAF lines were treated as in (A) and harvested 48 h post-transfection. Cells were analyzed for total PAR levels via immunoblot. cyclin D1 levels serve as siRNA validation controls.

cyclin D1b expression was induced in the prostate cancer cell line LNCaP (previously described (Augello et al, 2014)) and stained for p-H2AX and 53BP1 foci as in (A) via immunofluorescence (400× objective). Total number of foci/cell is reported for LNCaP vector control and cyclin D1b-expressing isogenic pairs in biological triplicate.

Isogenic pairs from (E) were grown in serum-proficient media for 24 h, harvested, and probed for total PAR levels via immunoblot.

Data information: Statistical significance was determined using a two-tailed Student's t-test.*P < 0.05 **P < 0.01, ***P < 0.001.
Consequence of the tumor-associated conversion to cyclin D1b. (2015)
Figure 7

Cyclin D1b expression sensitizes cells to senescence induced by therapeutic challenge

Ccnd1+/+ and Ccnd1KI/KIMAF lines were grown in serum-proficient media for 24 h. Cells were then treated with control DMSO or 2.5 μM of the PARP inhibitor ABT-888. One hour post-treatment, the indicated lines were treated with 5 Gy of radiation. Cells were then allowed to recover for 48 h, after which BrdU was added for 1 h, and then harvested for bivariate flow cytometry. Representative traces for each condition are shown (left) and BrdU incorporation of biological triplicates was quantified (right).

Cells were plated and treated as in (A). Forty-eight hours post-treatment, cells were fixed and stained for markers of senescence (β-galactosidase activity). Cells positive for the staining (blue) were quantified for each condition (400× magnification) and reported as a percentage of the total population (right). Plates treated in parallel with both IR and ABT-888 were harvested at 48 h via 1× trypsin and re-plated in serum-proficient media. Cells were allowed to grow for 96 h and were then stained with crystal violet (4× objective, with inset at 200× magnification, boxes highlight area of magnified images).

Data information: Error bars represent ± SEM, and statistical significance was determined using ANOVA (A) or a two-tailed Student's t-test (B). *P < 0.05, ***P < 0.001.
Resolving mechanisms of immune-mediated disease in primary CD4 T cells (2020)
Figure 2

Figure 2. Allele-specific expression-modulating effects in CD4 T cells

A qq plots of the observed -log10(P) values versus the expected -log10(P) values under the null hypothesis for representative resting and stimulated CD4 T cell samples.

B Activity of each allele at 10 positive control SNPs and 1 negative control SNP in stimulated T cells. GATA1, NF-κB, and RUNX1 constructs were designed to include a binding site for the indicated transcription factors (+) or with that site disrupted (-). Fixed effects meta-analysis P value is shown: *<0.05; **<0.01, ***<0.001, ****<0.0001. Box and whisker plots represent median and IQR (box) and min-to-max (whiskers). Exact P values are shown in Appendix Table S4.

C Experimental workflow for validation experiment using a different promoter (EF1α), reporter gene (luciferase) and quantification method (qPCR).

D Expression-modulating effect of each SNP [log2(OR)] as measured in MPRA and validation experiments. OR were calculated using the median activity of allelic constructs and are presented with respect to the risk allele (luciferase: n=5, MPRA: n=12).
Resolving mechanisms of immune-mediated disease in primary CD4 T cells (2020)
Figure 4

Figure 4. MPRA in CD4 T cells identifies an expression-modulating variant that disrupts NF-κB binding and enhancer function

A IBD GWAS results (Liu et al, 2015) at a multi-disease-associated locus on chromosome 6q23.

B Fine-mapping results (Huang et al, 2017) (posterior probabilities) for candidate SNPs at this locus.

C A single variant (rs6927172) has the largest and most significant expression-modulating activity in resting (left panel) and stimulated CD4 T cells (right panel) with the risk allele reducing transcription. Plots represent median and IQR (box) and min-to-max (whiskers). FDR-corrected meta-analysis P value shown.

D Sequence logo for an experimentally-validated NF-κB binding motif (Wong et al, 2011). The genomic sequence around rs6927172 is aligned below.

E Allele-specific NF-κB binding in CD4 T cells from rs6927172 heterozygotes, demonstrating reduced NF-κB binding to the risk allele following stimulation (n=8; one-sample t-test, two-tailed).

F Allele-specific expression of enhancer RNA in heterozygous CD4 T cells. DNA used for technical control (n=6; paired t-test; two-tailed).

G Genome-wide H3K27ac ChIP-seq in stimulated CD4 T cells from major- and minor-allele homozygotes at rs6927172 (n=6). Upper panels show input-normalised H3K27ac signals plotted against enhancer rank. Super-enhancers are defined above the inflection point of the curve. Lower panels show H3K27ac reads from a major (left) and a minor (risk) allele homozygote (right) in a 9kb window around rs6927172.

H Promoter-capture Hi-C plot depicting interactions of the 6q23 super-enhancer in stimulated CD4 T cells.

I Expression of genes on 6q23 in CD4 T cells from 131 patients with active IBD, stratified by rs6927172 genotype (qPCR; one-way ANOVA). Error bars represent SD. Expression of IL20RA and IL22RAR2 not detected.

Data information: Data represent mean+/-SEM, unless indicated. * P<0.05; ** P<0.01, *** P<0.001, **** P<0.0001. Exact P values are shown in Appendix Table S4.
Resolving mechanisms of immune-mediated disease in primary CD4 T cells (2020)
Figure 5

Figure 5. Deletion of the NF-κB binding site, disrupted by rs6927172, dysregulates TNFAIP3 expression and increases CD4 T cell activation.

A Location of gRNAs flanking the NF-κB binding site (highlighted).

B Primary CD4 T cell editing efficiency for indicated combinations of 5' and 3' gRNAs (n=6 for DB, DH, FH, and n=2 for FB). Distribution of indels assessed using ICE.

C Experimental workflow: equimolar amounts of 5' and 3' gRNA-containing RNPs (fluorescently tagged with ATTO-550) were nucleofected into resting CD4 T cells, which were stimulated after 48 hours with anti-CD2/3/28 microbeads and IL-2.

D Expression of genes on 6q23 in EU-containing mRNA (EU added at time of stimulation) showing that deletion of the NF-κB binding site specifically reduces transcription of TNFAIP3, but not other genes at this locus (n=6; one sample t-test). Representative data from the DH gRNA combination.

E Expression of CD69, an activation marker, following CRISPR editing with indicated gRNA combinations or the non-targeting (negative) control (NTC) - data shown for ATTO-550 positive (RNP-containing) and negative cells (n=6; paired t-test, one-tailed). Inset flow cytometry plot depicting representative gating of ATTO-550 positive and negative cells.

F Correlation between editing efficiency (total indel rate) and levels of phosphorylated IκBα in CD4 T cells (normalised to the mean fluorescence intensity in NTC; n=9, linear regression).

G Secretion of effector cytokines following deletion of the NF-κB binding site - Th1 (left panel, IFNγ), Th17 (centre panel, IL-17A) and Th2 subsets (right panel, IL-4) (n=6, paired t-test, one-tailed).

Data information: Data represent mean+/-SEM. * P<0.05; ** P<0.01; **** P<0.0001. Exact P values are shown in Appendix Table S4.

rs6759298 (AS)

rs10865331 (Ps)

rs2327832 (SLE)

rs6920220 (rest)a

rs2836883 (AS, PSC)

rs2836878 (UC, CD)
An IgG-based bispecific antibody for improved dual targeting in PSMA positive cancer (2020)
Figure 1

Figure 1. Binding regions of the PSMA antibodies 10B3 and J591 and comparative reactivity with prostate carcinoma and lung SCC samples.

A. Binding regions of the 10B3 antibody as determined by conjugation mass spectrometry (in dark red) and of J591 (in yellow) Bander et al, 2003a() are labeled in a 3D model of the PSMA-molecule as reported by Davis et al. Davis et al, 2005() in a dimeric (left panel) or monomeric (right panel) representation of the molecule (PDB ID code 1Z8L). Apical, helical and protease domains are colored in green, grey and blue respectively.

B, C. Cryosections of samples from patients with prostate carcinoma and SCC of the lung. Samples were processed as described in the methods section. Ve=vessel, Tu=tumor. Scale 50µm. See also Fig 1D for comparative semi-quantitative analysis of a panel of samples from patients with prostate carcinoma, lung SCC and non-lung SCC.

D. Semi quantitative analysis of binding of the PSMA antibodies 10B3 and J591 to cryosections from different tumor entities. For definition of the H-score reflecting binding intensity, refer to the methods section. Statistical analysis was performed using the paired, non-parametric Wilcoxon test. Sample size (n) and the corresponding P-values are indicated in the graphs.
An IgG-based bispecific antibody for improved dual targeting in PSMA positive cancer (2020)
Figure 2

Figure 2. Characterization of the Fabsc- and IgGsc-molecules.

A. Schematic representation of the Fabsc and IgGsc format.

B. SDS PAGE of IgGsc- and Fabsc-molecules. NR: non-reduced ; R: reduced.

C. Fabsc- and IgGsc-proteins were subjected to analytical chromatography using Superdex S200 Increase 10/300GL column. Representative gel-filtration profiles and the corresponding analysis are presented in the tables below.

D. A fluorescence-based thermal shift assay was performed as described the methods section. The data were analyzed using the first derivative approach and the calculated melting temperatures (Tm) are presented in the table below the profiles.

E, F. Binding of the Fabsc- and IgGsc-molecules to PSMA-expressing LNCaP (E) or 22Rv1low (F) cells was assessed by flow cytometry, as described in the methods section. The EC50 values were calculated using GraphPad Prism software and are presented below the figure.

G-I. Binding of the Fabsc- and IgGsc-molecules to CD3-expressing Jurkat-cells (G) and to CD4 T cells (H) and CD8 T cells (I) in PBMC preparations was assessed by flow cytometry. The results represent the average of three independent experiments performed with different PBMC donors. EC50 values are presented in the table below the figure.

J. Binding of the Fabsc- and IgGsc-molecules to a His-tagged CD3epsilon-delta heterodimer was determined by SPR. CD3 heterodimer protein was immobilized to an NTA sensor chip and the binding kinetics of the IgGsc- or Fabsc-molecules was assessed at 25 °C. The data obtained were analyzed using Biaevaluation software and the corresponding calculated KD values are presented in the table in the lower right.
An IgG-based bispecific antibody for improved dual targeting in PSMA positive cancer (2020)
Figure 7

Figure 7. T-cell activation and PSA levels during treatment of three patients with castrate resistant, metastasized prostate carcinoma.

The IgGsc-molecule was applied in escalating doses, with a starting dose of 20µg (patient 1) or 40µg (patients 2 and 3) to a maximum of approx. 2.6mg, as indicated. PSA values were monitored and T-cell activation was assessed by flow cytometry.

Expanded View Figure legends
SARS-CoV-2 Alpha, Beta and Delta variants display enhanced Spike-mediated Syncytia Formation (2021)
Figure 2

Figure 2. SARS-CoV-2 variant infection increases formation of syncytia in U2OS-ACE2 and Vero GFP-split cells.

(A) U2OS-ACE2 or Vero cells expressing either GFP 1-10 or GFP 11 (1:1 ratio) were infected 24h after plating and imaged 20h (U2OS-ACE2) or 48h (Vero) post-infection.

(B) Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for U2OS-ACE2 20h post infection at MOI 0.001. Right Panel: Representative images of U2OS-ACE2 20h post infection, GFP-Split (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel.

(C) Left Panel: Quantified fusion of Vero cells infected at MOI 0.01. Right Panel: Representative images of Vero cells 48h post infection, GFP-Split (Green) and Hoechst (Blue).

Data information: Scale bars: 200 µm. Data are mean ± SD of 8 independent experiments. Statistical analysis: One‐way ANOVA compared to D614G reference, ns: non‐significant,

**P < 0.01, ****P < 0.0001.
SARS-CoV-2 Alpha, Beta and Delta variants display enhanced Spike-mediated Syncytia Formation (2021)
Figure 3

Figure 3. Alpha and Beta SARS-CoV-2 S proteins induce more robust syncytia formation than D614G.

(A) Vero GFP-split cells were transfected with variant S proteins and imaged 18h post-transfection.

(B) Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Vero GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm.

(C) Left Panel: Quantification of variant S protein mediated fusion in Vero-GFP split cells by video microscopy. Results are mean ± SD from three fields per condition from one representative experiment. Right Panel: Fusion quantification of at least 3 independent video microscopy experiments, 20h post transfection, normalized to D614G.

Data information: Data are mean ± SD of at least 3 independent experiments. Statistical analysis: One‐way ANOVA compared to D614G reference, ns: non‐significant, *P < 0. 05, ***P < 0.001, ****P < 0.0001.
SARS-CoV-2 Alpha, Beta and Delta variants display enhanced Spike-mediated Syncytia Formation (2021)
Figure 4

Figure 4. Mutations associated with Alpha and Beta S proteins differentially affect cell-cell fusion.

(A) Top Panel: Schematic representation of the S protein colour coded for the functional regions: N-terminal domain (NTD), receptor binding domain (RBD), fusion peptide (FP), heptad repeat 1,2 (HR1, HR2), transmembrane anchor (TA), C-terminal domain (CTD). Bottom left Panel: Vero GFP-split cells were transfected with S plasmids containing each of the individual mutations associated with Alpha variant in the D614G background. The amount of fusion was quantified at 20h and normalized to D614G reference plasmid. Bottom right Panel: Quantified fusion for each of the individual S protein mutations associated with the Beta variant. Colour code of each mutation corresponds to S protein functional regions represented in the schematic on the Top Panel. Data set for N501Y and D614G reference mutations are duplicated between bottom left and bottom right panels for presentation as these mutations are common to both variants.

(B) Left Panel: Quantified fusion of the Alpha + E484K variant S protein normalized to D614G S. Right Panel: Representative images of fusion at 20h. Scale bar: 200 µm. Top and bottom are the same images with and without Hoechst channel.

Data information: Data are mean ± SD of at least 4 independent experiments. Top and bottom are the same images with and without Hoechst channel. Statistical analysis: statistics for both left and right panels of A were conducted together. One‐way ANOVA compared to D614G reference, ns: non‐significant, **P < 0.01, ***P < 0.001, ****P < 0.0001.
SARS-CoV-2 Alpha, Beta and Delta variants display enhanced Spike-mediated Syncytia Formation (2021)
Figure 5

Figure 5. ACE2 and monoclonal antibody binding to S proteins with Alpha and Beta associated mutations.

(A) 293T cells were transfected S proteins with each variant-associated mutation for 24h and stained with biotinylated ACE2 and fluorescent streptavidin before analysis by flow cytometry.

(B) Left Panel: EC50 values (concentration of ACE2 needed for 50% binding) for Alpha and associated mutations. Colour code corresponds to location on S protein functional domains and lower EC50 values signifies higher affinity to ACE2 binding. Right Panel: EC50 values for Beta and associated mutations. Data set for N501Y and D614G reference mutations are duplicated between left and right panels as mutations are common to both variants. (C) S protein transfected 293T cells were stained with human monoclonal antibodies targeting the S2 (mAb10), RBD (mAb48 and mAb98), and the NTD (mAb71). Cells were analyzed by flow cytometry. The percentage of positive cells is indicated.

Data information: Data are mean of at least 3 independent experiments. Statistical analysis: One‐way ANOVA compared to D614G reference, ns: non‐significant, *P < 0. 05, **P < 0.01, ****P < 0.0001.
SARS-CoV-2 Alpha, Beta and Delta variants display enhanced Spike-mediated Syncytia Formation (2021)
Figure 6

Figure 6. Delta SARS-CoV-2 S protein induces more syncytia formation and binds more to ACE2 than D614G.

(A) Vero GFP-split cells were transfected with variant S proteins and imaged 18h post-transfection. Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Vero GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm.

(B) Left Panel: Quantification of Delta S protein mediated fusion in Vero-GFP split cells by video microscopy. Results are mean ± SD from three fields per condition from one representative experiment. Right Panel: Fusion quantification of 3 independent video microscopy experiments, 20h post transfection, normalized to D614G.

(C) 293T cells were transfected S proteins with each variant-associated mutation for 24h and stained with biotinylated ACE2 and fluorescent streptavidin before analysis by flow cytometry. Left Panel: Representative ACE2 binding dilution curves for the Delta variant in relation to Alpha and D614G. Right Panel: EC50 values (concentration of ACE2 needed for 50% binding) for Alpha for the Delta variant.

(D) Caco2 GFP-split cells were transfected with variant S proteins and imaged 18h post-transfection. Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Caco2 GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm

Data information: Data are mean ± SD of at least 3 independent experiments. Statistical analysis: One‐way ANOVA compared to D614G reference, *P < 0. 05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
N-glycosylation of PD-1 promotes binding of camrelizumab (2020)
Figure 1

Fig. 1 N-glycan profiles of PD-1 and glycosylation binding dependency of camrelizumab

(A) N-glycan composition of PD-1 protein expressed from 293T cells by ESI-MS analysis. N-glycans of representative peaks from ESI-MS were identified by MS/MS analysis. H, mannose; N, N-acetylglucosamine; F, fucose. Structural formulas: blue square, N-acetylglucosamine; red triangle, fucose; green circle, mannose; yellow circle, galactose. (B,C) Accelerated thermal stability of PD-1 proteins obtained from 293T cells (green line), insect cells (pink line) or E. coli (blue line) is characterized in all-in-one UNcle platform. Protein unfolding is observed as an increase in barycentric mean (B) while small particle formation of the samples is observed as an increase in SLS intensity at 266 nm (C).

(D) SPR analysis of the binding characteristics of camrelizumab with PD-1 expressed in 293T cells, insect cells or E. coli cell, which enabled different levels of glycosylations. The binding affinity is presented as KD. The data presented here is a representative of two independent experiments with similar results.
N-glycosylation of PD-1 promotes binding of camrelizumab (2020)
Figure 2

Fig. 2 Overall structure of PD-1 camrelizumab-scFv complex

(A) Gel filtration profiles of PD-1 (orange), camrelizumab-scFv (blue), and the PD-1/camrelizumab-scFv complex (red) are analyzed by size-exclusion chromatography as indicated. The SDS-PAGE analyses are shown with peak 1 of camrelizumab-scFv, peak 2 of camrelizumab-scFv/PD-1 complex, and peak 3 of PD-1.

(B) The structure of camrelizumab-scFv/PD-1 complex. PD-1 is shown as surface representation (white). Camrelizumab-scFv is shown as cartoon representation. VL is colored in yellow and VH in deep salmon. The CDR1, CDR2, and CDR3 loops of the VL (LCDR1, LCDR2, and LCDR3) are colored in slate, warm pink and violet purple, respectively. The CDR1, CDR2, and CDR3 loops of VH (HCDR1, HCDR2, and HCDR3) are colored in marine, orange and cyan, respectively. Hydrogen bond interactions between PD-1 and camrelizumab-scFv are shown as sticks and labeled. Hydrogen bonds are shown as dashed red lines.
N-glycosylation of PD-1 promotes binding of camrelizumab (2020)
Figure 4

Fig. 4 N-glycan of N58 on PD-1 is crucial for binding to camrelizumab

(A) The overall structure of N-glycan on N58 of PD-1 is presented with N-glycans on N58 of PD-1 highlighted in sticks in green. The 2 Fo-Fc electron density map of the N58 N-glycans contoured at 1.0 sigma is represented in orange on the right. The β strands were labeled according to canonical IgV-strand designations.

(B) Location of N58 glycosylation in the interaction with camrelizumab-scFv. VL of camrelizumab-scFv is colored in yellow and VH in deep salmon.

(C) Detailed interactions of N-glycans on N58 of PD-1 with camrelizumab-scFv. The observed glycans on N58 of PD-1 are highlighted in sticks and colored in green. Residues with hydrogen bond and van der Waals force interaction are shown as sticks and labeled while Hydrogen bond and van der Waals force interaction are shown as dashed red lines.

(D) Binding paratope on VH and VL of camrelizumab by PD-1 is presented with residues in contact with glycan of N58 highlighted in green.

(E) SPR analysis of the binding of camrelizumab with N58A mutated PD-1 expressed in 293T cells.

(F)

The SPR analysis of the binding of camrelizumab with WT (upper) or PNGase F treated PD-1 proteins (lower) expressed in 293T cells.

(G, H) The SPR analysis of the binding of nivolumab (G) with WT (upper) or N58A mutated (lower) PD-1 expressed in 293T cells, and the binding of pembrolizumab (H) with WT (upper) or N58A mutated (lower) PD-1 proteins.
N-glycosylation of PD-1 promotes binding of camrelizumab (2020)
Figure 5

Fig. 5 Reduced blocking efficiency of camrelizumab to N58 glycosylation deficient PD-1.

(A,B) The blocking of the binding of WT PD-1-mFc (A) or N58A mutated PD-1-mFc (B) to PD-L1 transiently expressed on the surface of 293T cells are analyzed with varied concentrations of camrelizumab. The PD-L1 expressing 293T cells stained with PBS alone is used as negative controls, whereas the cells stained with WT or N58A mutated PD-1-mFc proteins are used as a positive control. The frequency of PD-1-mFc staining positive cells in PD-L1-GFP positive cells is labeled in the upright corner. The reduced frequency of the PD-L1-mFc-staining-positive subpopulation compared with positive control indicates the blockage of the PD-1/PD-L1 interaction. The density of events at a given position in the plot is color-coded, with red representing the highest number of events at that point in the graph, while yellow, green, and blue represent progressively lower event densities. The results presented here are representative of three independent experiments with similar results.