EMBO/soda-nel huggingface dataset:
Bacterial protein domains with a novel Ig-like fold bind human CEACAM receptors (2020)
Figure 2

Figure 2: The IgSF domain in the β protein of GBS binds to the N-terminal domain of CEACAMs. A) Schematic representation of the β protein (Hedén et al, 1991). The first residue of each domain is indicated. XPZ is a proline-rich repeat region (Areschoug et al, 2002a). Known human ligand binding partners are indicated above each respective domain. IgA binds to a region denoted IgABR. Factor H binds the β75K region, but does not bind via XPZ.

B) Staining of recombinant β protein domains (B6N, IgABR, B6C, β-IgSF and β75KN) after separation by SDS-PAGE. Recombinant proteins were expressed in E. coli. β protein purified from GBS is used as a control. Dotted boxes signal proteins of expected size.

C) Binding of rCC1-His or rSiglec5-Fc to dynabeads (DB) coated with β protein domains. Mean and SD values are reported for n = 2 replicates.

D) Binding of varying concentrations of β-IgSF or human serum albumin (HSA) coupled to streptavidin-PE (SPE), to DB coated with CC1 (DB.CC1) or control protein (DB). Mean and SD values are reported for n = 6 replicates.

E) Structure of the relevant (Siglec-like sialic acid-binding) domain of the highest-scoring template 5KIQ (SrpA binding region), and predicted three-dimensional model of the IgSF domain from the β protein, constructed from the Hhpred alignment using MODELLER.(Webb & Sali, 2016)

F) Inhibition of β-IgSF binding to DB.CC1 by anti-CC1-N mAb (clone CC1/3/5-Sab), but not by anti-CC1-A1B1 mAb (clone B3-17). β-IgSF-biotin was coupled to SPE. Mean and SD values are reported for n = 6 replicates. G) Concentration-dependent binding of β-IgSF to DB coated with 100 μg/ml CC1 (DB.CC1), CC1-N (DB.CC1-N) or CC1-A1B1A2 (DB.CC1-A1B1A2). Assays utilized proteins coupled to SPE. Mean and SD values are reported for n = 6 replicates.

Data information: Fluorescence of DB in (C, D and F) was measured by flow cytometry.
Bacterial protein domains with a novel Ig-like fold bind human CEACAM receptors (2020)
Figure 3

Figure 3. β-IgSF binds with high-affinity to human CEACAM1 and CEACAM5.

A) Representative isothermal titration calorimetry (ITC) binding curves of CEACAM-N domains and β-IgSF duplicates. Experiments were performed using β-IgSF and N domains of (r)CEACAM1 (CC1), CEACAM3 (CC3), CEACAM5 (CC5), CEACAM6 (CC6) and CEACAM8 (CC8).

B) Representative flow cytometry plots analyzing the interaction of rCC1, CC3, CC5, CC6 or CC8 (10 μg/ml) with GBS A909. Fluorescence of bacteria was measured by flow cytometry. Representative data from n = 3 independent experiments.
Bacterial protein domains with a novel Ig-like fold bind human CEACAM receptors (2020)
Figure 4

Figure 4: GBS exploit human CEACAM for enhanced adhesion to epithelial cells.

A) Adherence of GBS A909 to human CEACAM1 (CC1)-, CEACAM3 (CC3)-, CEACAM5 (CC5)-, CEACAM6 (CC6)- or CEACAM8 (CC8)-expressing, or an empty vector control, HeLa transfectants at MOI of 10 for 30 mins. Mean and SD for n = 4 independent replicates are shown.

B) Confocal imaging of FITC-labelled GBS A909 strain adhesion to human CC1-expressing or empty vector control HeLa transfectants. Cell membranes were stained with AF-647 conjugated wheat germ agglutinin (WGA) (red) and nuclei with DAPI (blue). Scale bar: 26 μM.

C) Adherence of A909 to human CC1-, CC3-, CC5-, CC6- or CC8-expressing, or an empty vector control, CHO transfectants at MOI of 10 for 30 mins. Mean and SD from n = 5 independent replicates is displayed.

D) Adhesion of WT, ∆bac and ∆bac + pLZ.bac complemented A909 strains to human CC1- or CC5-expressing CHO transfectants, or empty vector control cells. Mean and SD from n = 5 independent replicates is displayed.

E, F) Adhesion of FITC-labelled A909 to human CC1- -expressing, or control, CHO transfectants at an MOI of 10. Cell lines were detached and then incubated with FITC-labelled A909 for 30 mins at 4oC. The percentage of fluorescent cells was calculated for each population. Representative flow cytometry plots are shown (E), and the integrated results displaying the mean and SD from 3 independent replicates are reported in (F).

G) Adhesion of FITC-labelled A909 strains to human CC1- expressing or control CHO transfectants at MOI of 10. Cell lines were detached and then incubated with FITC-labelled A909 strains for 30 mins at 4oC. Mean and SD for each population for n = 6 independent replicates is displayed

Data information: Fluorescence of cells in (E, F and G) was measured by flow cytometry analysis, where the percentage of fluorescent cells was calculated. The number of adhered bacteria to cell monolayers in (A, C and D) were quantified by growth on Todd-Hewitt agar and enumeration of colony forming units (CFU). Data in (A, C, D, F and G) was analyzed by repeated One-way ANOVA with Sidak's multiple comparisons. *P<0.05, **P<0.01.
Bacterial protein domains with a novel Ig-like fold bind human CEACAM receptors (2020)
Figure 6

Figure 6. The IgI3 domain of β protein binds CEACAM1 through the unique α-helix and CD loop.

A) Molecular structure of CEACAM1 (CC1)-N, in which each strand of the IgV structure is coloured differenty.

B) Molecular structure of the β-IgI3 & CC1 N-terminal domain co-complex. Residues that form the β-IgI3-CC1 interface are highlighted.

C) Close-up of the β-IgI3 (light blue) interface with CC1-N (pink), including highlighting of residues important for forming β-IgI3 (blue) & CC1-N (red) interactions. The GFCC′C′′ strands of CC1-N are shown and labelled, whilst the C to D strands of β-IgI3 are shown and labelled.

D) Binding of rCC1-N to dynabeads (DB) coated with β-IgI3 wild type (WT) or mutants. Mutation of β-IgI3 residues 42, 46 and 53 lead to significant changes in CC1-N binding capacity.

E) Binding of β-IgI3 coupled to Streptavidin-PE to DB coated with rCC1-N wildtype or variants. Mutation of CC1-N residues 29, 44, 89, 91 and 95 lead to significant changes in β-IgI3 binding capacity.

Data information: Fluorescence of DB in (D and E) was measured by flow cytometry. Mean and SD values in (D and E) are displayed for n = 3 independent replicates. Statistical significance was calculated using t test for (D and E), where *, p<0.05; **, p<0.01.
Bacterial protein domains with a novel Ig-like fold bind human CEACAM receptors (2020)
Figure 7

Figure 7: The unique IgI3 structure is present in Gram-positive bacterial species and evidence for binding CEACAM1.

A) Amino acid based Maximum Likelihood tree of β-IgI3 homologs. Clades of related IgI3 domains are shown, with clade I representative of β-IgI3 domain sequences. Branches are coloured by bacterial species from which the sequence originates, except those with no species designated. Maximum likelihood values bootstrap values > 60 are shown as a circle, where larger circles indicate a higher bootstrap support value.

B) Superposition of the IgI3 domain from β protein (blue) onto predicted IgI3 domain structure of clade II sequence of S. pyogenes R28 protein (red).

C) Close-up of the truncated C strand, α-helix and D strand of β-IgI3 (blue) and R28-IgI3 (red), indicating the corresponding R28-IgI3 residues present at sites in β-IgI3 that bind CEACAM1 (CC1).

D) Binding of recombinant (r)CC1-HIS to dynabeads (DB) coated with β-IgI3, R28-IgI3 or control. Mean and SD is shown for n = 6 independent replicates.

E) Binding of β-IgI3, R28-IgI3 or human serum albumin (HSA) coupled to PE-conjugated streptavidin (SPE), to DB coated with CC1 (DB.CC1). Mean and SD is shown for n = 6 independent replicates.

F) Binding of recombinant rCC1-HIS (10 μg/mL) to R28-expressing GBS strain H1147 and R28-expressing S. pyogenes strain AL368. Data representative of n = 3 replicates.

G) The per residue free energy contribution spectrums of β-IgI3/CC1-N interface and R28-IgI3/CC1-N interface. The residues with binding free energy contributions lower than -2.0 kcal/mol are identified as key (favourable) residues and coloured blue (for β-IgI3) or red (for R28-IgI3). Free energy contributions at -2.0 kcal/mol and 2.0 kcal/mol are indicated by dashed lines. The position of the truncated C strand, α-helix and D strand is shaded for each protein. The mean and standard deviation is representative of estimates from 50 docked models..

Data information: Statistical significance was calculated using a One-way ANOVA with Sidak's multiple comparisons for (D and E), where **, p<0.01. Fluorescence of DB (D and E) and bacteria (F) was measured by flow cytometry.

Extended View Figure Legends
Human-specific ARHGAP11B ensures human-like basal progenitor levels in hominid cerebral organoids (2022)
Figure 2

Figure 2 - Expression of ARHGAP11B in chimpanzee cerebral organoids increases the abundance of cycling BPs.

A Double immunofluorescence for GFP (green) and the BP marker TBR2 (yellow), combined with DAPI staining (white), of a 57 days-old chimpanzee cerebral organoid 2 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and TBR2+ double-positive cells. Scale bar, 50 µm.

B Quantification of the proportion of GFP+ cells that are TBR2+ in 57 days-old chimpanzee cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 9 control and 9 ARHGAP11B-transfected cerebral organoids of two independent batches each; error bars indicate SD; ***, P <0.001 (two-sided Student's t-test).

C Triple immunofluorescence for GFP (green), the cycling cell marker Ki67 (magenta) and TBR2 (yellow), combined with DAPI staining (white), of a 59 days-old chimpanzee cerebral organoid 4 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+, Ki67+ and TBR2+ triple-positive cells. Scale bar, 50 µm.

D Quantification of the proportion of GFP+ cells in the SVZ/NL that are Ki67+ and TBR2+ double-positive in 59 days-old chimpanzee cerebral organoids 4 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 8 control and 8 ARHGAP11B-transfected cerebral organoids of four independent batches each; error bars indicate SD; *, P <0.05 (one-sided Wilcoxon rank sum test).
Human-specific ARHGAP11B ensures human-like basal progenitor levels in hominid cerebral organoids (2022)
Figure 3

Figure 3 - Expression of ARHGAP11B in chimpanzee cerebral organoids increases the abundance of HOPX-positive cells in the SVZ.

A Double immunofluorescence for GFP (green) and the radial glia marker HOPX (yellow), combined with DAPI staining (white), of a 59 days-old chimpanzee cerebral organoid 4 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and HOPX+ double-positive cells. Scale bar, 50 µm.

B Quantification of the proportion of GFP+ cells in the SVZ/NL that are HOPX+ in 59 days-old chimpanzee cerebral organoids 4 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 10 control and 10 ARHGAP11B-transfected cerebral organoids of three independent batches each; error bars indicate SD; **, P <0.01 (one-sided Wilcoxon rank sum test).

C Double immunofluorescence for GFP (green) and HOPX (yellow), combined with DAPI staining (white), of a 61 days-old chimpanzee cerebral organoid 10 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and HOPX+ double-positive cells. Scale bar, 50 µm.

D Quantification of the proportion of GFP+ cells in the SVZ/NL that are HOPX+ in 61 days-old chimpanzee cerebral organoids 10 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 5 control and 7 ARHGAP11B-transfected cerebral organoids of three independent batches each; error bars indicate SD; *, P<0.05 (two-sided Student's t-test).
Human-specific ARHGAP11B ensures human-like basal progenitor levels in hominid cerebral organoids (2022)
Figure 4

Figure 4 - Expression of ARHGAP11B in chimpanzee cerebral organoids differentially affects the generation of deep-layer vs. upper-layer cortical neurons.

A Double immunofluorescence for GFP (green) and the neuron marker Hu (magenta), combined with DAPI staining (white), of a 61 days-old chimpanzee cerebral organoid 10 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and Hu+ double-positive cells. Note that the same electroporated regions are depicted in Figure EV2 with a different marker (PCNA). Scale bar, 50 µm.

B Quantification of the proportion of GFP+ cells that are Hu+ in 61 days-old chimpanzee cerebral organoids 10 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 7 control and 11 ARHGAP11B-transfected cerebral organoids of two independent batches each; error bars indicate SD; **, P <0.01 (two-sided Student's t-test).

C Double immunofluorescence for GFP (green) and the deep-layer neuron marker CTIP2 (magenta), combined with DAPI staining (white), of a 59 days-old chimpanzee cerebral organoid 4 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and CTIP2+ double-positive cells. Scale bar, 50 µm.

D Quantification of the proportion of GFP+ cells in the SVZ/NL that are CTIP2+ in 59-days old chimpanzee cerebral organoids 4 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 10 control and 10 ARHGAP11B-transfected cerebral organoids of two independent batches each; error bars indicate SD; ***, P <0.001 (two-sided Student's t-test).

E Double immunofluorescence for GFP (green) and the upper-layer neuron marker SATB2 (magenta), combined with DAPI staining (white), of a 66 days-old chimpanzee cerebral organoid 15 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and SATB2+ double-positive cells. Scale bar, 50 µm.

F Quantification of the proportion of GFP+ cells in the SVZ/NL that are SATB2+ in 66 days-old chimpanzee cerebral organoids 15 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 6 control and 4 ARHGAP11B-transfected cerebral organoids of two independent batches each; error bars indicate SD; **, P <0.01 (one-sided Wilcoxon rank sum test).
Human-specific ARHGAP11B ensures human-like basal progenitor levels in hominid cerebral organoids (2022)
Figure 5

Figure 5 - Dominant-negative ARHGAP11A220 reduces the level of cycling BPs in ARHGAP11B-expressing human cerebral organoids to the chimpanzee level but has no effect on BP levels in chimpanzee cerebral organoids.

A Double immunofluorescence for GFP (green) and the thymidine analog BrdU (magenta, see Fig 1A), combined with DAPI staining (white), of 57 days-old human (top panels) and chimpanzee (bottom panels) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (upper panels) or ARHGAP11A220 expression plasmid (lower panels). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and BrdU+ double-positive cells. Scale bar, 50 µm.

B Quantification of the proportion of GFP+ cells in the SVZ/NL that are BrdU+ in 57 days-old human (blue) and chimpanzee (red) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (dark blue and dark red) or ARHGAP11A220 expression plasmid (light blue and light red). Data are the mean of 12 human control, 12 human ARHGAP11A220-transfected, 9 chimpanzee control and 8 chimpanzee ARHGAP11A220-transfected cerebral organoids (grown from one human and one chimpanzee iPSC line, respectively) of six (human) and four (chimpanzee) independent batches each; error bars indicate SD; n.s., not significant; **, P <0.01 (Kruskal Wallis test).

C Double immunofluorescence for GFP (green) and TBR2 (yellow), combined with DAPI staining (white), of 57 days-old human (top panels) and chimpanzee (bottom panels) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (upper panels) or ARHGAP11A220 expression plasmid (lower panels). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and TBR2+ double-positive cells. Scale bar, 50 µm.

D Quantification of the proportion of GFP+ cells in the SVZ/NL that are TBR2+ in 57 days-old human (blue) and chimpanzee (red) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (dark blue and dark red) or ARHGAP11A220 expression plasmid (light blue and light red). Data are the mean of 9 human control, 9 human ARHGAP11A220-transfected, 10 chimpanzee control and 10 chimpanzee ARHGAP11A220-transfected cerebral organoids (grown from one human and one chimpanzee iPSC line, respectively) of four (human) and three (chimpanzee) independent batches each; error bars indicate SD; n.s., not significant; *, P <0.05 (Kruskal Wallis test).
Human-specific ARHGAP11B ensures human-like basal progenitor levels in hominid cerebral organoids (2022)
Figure 6

Figure 6 - ARHGAP11B knockout leads to a substantial reduction of bRG abundance in human forebrain organoids.

A Double immunofluorescence for GFP (green) and the bRG marker PTPRZ1 (magenta), combined with DAPI staining (white), of 60 days-old human forebrain organoids, generated from ARHGAP11A plus ARHGAP11B double-knockout clone 16. Images were obtained 10 days after electroporation with GFP expression plasmid plus either ARHGAP11A plus ARHGAP11B expression plasmids (ARHGAP11A+B-rescue, referred to as Control), ARHGAP11B expression plasmid only (ARHGAP11B-rescue, referred to as ARHGAP11A-KO), or ARHGAP11A expression plasmid only (ARHGAP11A-rescue, referred to as ARHGAP11B-KO). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and PTPRZ1+ double-positive cells. Scale bar, 50 µm.

B Quantification of the proportion of GFP+ cells in the SVZ/NL that are PTPRZ1+ in 60 days-old ARHGAP11A plus ARHGAP11B double-knockout (11A+11B KO) human forebrain organoids. Organoids were generated from clone 16 (left) and clone j (right), and data were obtained 10 days after the indicated electroporation with GFP expression plasmid plus either ARHGAP11A (11A) plus ARHGAP11B (11B) expression plasmids (referred to as Control, as this constitutes an ARHGAP11A+B double-rescue in the 11A+11B KO organoids; dark blue), ARHGAP11B (11B) expression plasmid only (referred to as 11A-KO, as this constitutes a selective ARHGAP11B rescue in the 11A+11B KO organoids; light blue), or ARHGAP11A (11A) expression plasmid only (referred to as 11B-KO, as this constitues a selective ARHGAP11A rescue in the 11A+11B KO organoids; dark red). For clone 16, data are the mean of 8 control, 8 11A-KO and 8 11B-KO human forebrain organoids of two independent batches each; for clone j, data are the mean of 9 control, 10 11A-KO and 8 11B-KO human forebrain organoids of three independent batches each; error bars indicate SD; n.s., not significant; *, P <0.05; **, P <0.01; ***, P <0.001 (Kruskal Wallis test).

Expanded View Figure legends
Tousled-like kinase 2 regulates recovery from a DNA damage-induced G2 arrest (2016)
Figure 1

Figure 1. An siRNA-based screening approach identifies potential regulators of recovery. Plot of one of the screens (the duplicates are shown in Fig. EV1C). U2OS cells were transfected, synchronized at the G1/S-border by thymidine for 24 hours and then either released into nocodazole for 16 hours (unperturbed mitotic entry) or released for 7 hours, treated with 0.5 μM Adriamycin for one hour and after 16 hour G2 arrest were induced to recover by addition of caffeine (checkpoint recovery). Mitotic index was scored based on the percentage of Histone H3-pS10 positive DAPI-nuclei and normalized to the untransfected controls. Black diamonds indicate individual siRNA-targeted genes from the library, light grey diamonds indicate positive (Wip1-depleted) and negative (untransfected and GAPDH-depleted) controls and dark grey diamonds indicate the hits based on the two screens. Dotted lines indicate selection criteria for recovery-specific genes.
Tousled-like kinase 2 regulates recovery from a DNA damage-induced G2 arrest (2016)
Figure 2

Figure 2. Tlk2 kinase activity is required for recovery from a DNA damage-induced arrest. A) U2OS cells were transfected with four independent siRNA's from the pools used in the screen, treated as in figure 1 and analyzed for mitotic index. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, ** for P≤0.01) B) U2OS cells were synchronized with a single thymidine block, released into G2 and damaged with 0.5 μM Adriamycin for one hour. After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for Tlk2 protein levels. C) Live cell imaging of thymidine synchronized unperturbed or damaged G2 cells. Cumulative percentage of cells entering mitosis were scored and plotted. D) U2OS cells were transfected with either a control siRNA or Tlk2 siRNA #3, synchronized and damaged in G2. Cells were either harvested or treated with caffeine for 8 hours before harvest and cell cycle distribution was analyzed by FACS. Percentages of cells in each quadrant are indicated. E) Tlk2Δ cells were generated using CRISPR/Cas9 genome editing. Cells were synchronized in G2 by thymidine release, damaged with 0.5 μM Adriamycin for one hour. After a 16-hour G2 arrest cells were induced to recover by addition of caffeine for 8 hours and analyzed by FACS. Error bars represent SD, n=4. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, *** for P≤0.001). F) U2TR cells stably expressing Tlk2 siRNA #3-insensitive tetracycline-inducible FLAG-Tlk2-wt or FLAG-Tlk2-D613A were thymidine synchronized and damaged in G2. Tetracycline was present form the start of the experiment where indicated. G) Caffeine induced recovery assay of cell lines shown in 2F. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, ** for P≤0.01, *** for P≤0.001).