EMBO/soda-nel huggingface dataset:
Distinct Notch1 and BCL11B requirements mediate human γδ/αβ T cell development (2020)
Figure 1

Figure 1. Human TCRγδ T cell development is more Notch1/DLL4/JAG2 dependent compared to TCRαβ T cell development.

(A) Flow cytometry analysis of CD34+CD1a- thymocytes cultured in FTOCs, in the presence of a control or anti-Notch1 blocking antibody. Numbers in dot plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells.

(B, C) (B) Mean frequency and (C) absolute cell numbers of human CD4+CD8β+ DP (black shades), CD3+TCRαβ+ (blue shades) and CD3+TCRγδ+ (red shades) thymocytes developed in corresponding cultures from A (n=6 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a non-parametric paired Wilcoxon test).

(D) Flow cytometry analysis of CD34+CD1a- thymocytes submitted to FTOC with fetal thymic lobes deficient for Dll4 and/or Jag2 (Cre+) as indicated above the dot plots. Numbers in dot plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells.

(E, F) (E) Mean frequency and (F) absolute cell numbers of CD3+TCRαβ+ (blue) and CD3+TCRγδ+ (red) thymocytes developed in corresponding cultures from D (n=5-7 independent experiments, depending on the mouse genotype, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a non-parametric Mann-Whitney U test).
Distinct Notch1 and BCL11B requirements mediate human γδ/αβ T cell development (2020)
Figure 4

Figure 4. Notch signaling impacts the miRNA landscape in both ex vivo isolated as in vitro cultured thymocytes.

(A) Line plots displaying averaged NOTCH1, MYC and HES1 expression across T cell development (n=2 biological replicates and error bars indicate SEM).

(B) Heatmap displaying transcript wise scaled expression of NOTCH1, HES1, MYC and miRNAs from ex vivo isolated subsets correlating (spearman rho >= 0.5) with NOTCH1 gene expression as described in Materials and Methods. Red arrows indicate all 6 members of the miR-17~92 cluster (n=2).

(C) Heatmap visualizing Notch1 regulated gene expression of in vitro cultured thymocytes across multiple culturing conditions (OP9-control, OP9-JAG1, OP9-JAG2, OP9-DLL1 and OP9-DLL4) and time points (day 0, 5 and 10).

(D) NOTCH1 correlating miRNAs (spearman rho >= 0.3) of in vitro cultured thymocytes across multiple culturing conditions and timepoints. The miRNA expression levels were scaled transcript wise and red arrows indicate members of miR-17~92 cluster (n=2).

(E) CHIPseq data for Notch1 and RBPj binding at the miR-17~92 locus in CUTTL1 cells. Red line underneath the ChIPseq peaks indicate significant enrichment compared to the input sample.
Distinct Notch1 and BCL11B requirements mediate human γδ/αβ T cell development (2020)
Figure 5

Figure 5. Regulation of BCL11B expression in thymocytes through miR-17.

(A) Schematic representation of the potential miR-17 (blue) and miR-92a (green) binding sites in the 3'UTR of the BCL11B gene. 3 nucleotides in the 3 putative miR-17 binding sites which are targeted for mutagenesis, used in a 3'UTR luciferase reporter assay described in B, are depicted in red.

(B) Transfection of miR-17 Pre-miR miRNA Precursor, but not of miR-92a, specifically targets the wild-type (black bars) 3'UTR of BCL11B in a 3'UTR luciferase reporter assay following cotransfection of HEKT cells in comparison to the controls (transfection of no miRNA or a control miRNA). Mutation of the predicted miR-17 binding sites in the 3'UTR BCL11B reporter construct prevents miR-17 dependent targeting (grey bars). Graphs show mean firefly luciferase values (from 3'UTR reporter constructs) following normalization to the cotransfected control Renilla luciferase and relative to control sample without miRNA (n=5 independent experiments, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a paired t-test).

(C) Quantitative RT-PCR gene expression analysis of BCL11B in control (black bar) or miR-17 (grey bar) transduced CD34+ thymocytes after 24h of culture on coated Fc-DLL1 plates, relative to the geometric mean of ACTIN and GAPDH and relative to the control-transduced sample (n=3 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).

(D) Quantitative real-time RT-PCR gene expression analysis of BCL11B in control (black bar) or miR-17 sponge (white bar) transduced CD34+ thymocytes, relative to the geometric mean of ACTIN and GAPDH (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).
Distinct Notch1 and BCL11B requirements mediate human γδ/αβ T cell development (2020)
Figure 6

Figure 6. Notch dependent regulation of miR-17 and BCL11B.

(A) Gene expression pattern of BCL11B (red dots) and HES1 (blue squares) during human T cell development, derived from microarray data as shown in Figure 2. (n=2 biological replicates and error bars indicate SEM).

(B-C) Quantitative RT-PCR expression analysis of BCL11B in human (B) CB CD34+Lin- and (C) CD34+ thymocyte progenitors after 5 and 2 days, respectively, of coculture on OP9 stromal cells expressing different Notch ligands. Graphs show mean expression, relative to the geometric mean of ACTIN and GAPDH and relative to the OP9-DLL1 cultured cells (n=4 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a paired t-test).

(D) Quantitative RT-PCR expression analysis of BCL11B in human CD34+ thymocyte progenitors after 2 days of coculture on OP9-DLL1 stromal cells in the presence of 5 µM DAPT (GSI). Graphs show mean expression, relative to the geometric mean of ACTIN and GAPDH and relative to sample cultured in the presence of control DMSO (n=4 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).

(E-F) Quantitative RT-PCR expression analysis of miR-17 in human (E) CB CD34+Lin- and (F) CD34+ thymocyte progenitors after 5 and 2 days, respectively, of coculture on (E) OP9 stromal cells expressing different Notch ligands or (F) OP9-DLL1 stromal cells in the presence of 5 µM DAPT (GSI). Graphs show mean expression, relative to the geometric mean of SNORD61, SNORD95 and RNU6-2 and relative to the OP9-DLL1 cultured cells (E) or to the sample cultured in the presence of control DMSO (F) (n=2 independent experiments with different donors; error bars indicate SEM - the two data points are shown).

(G) Quantitative RT-PCR gene expression analysis of BCL11B in control (black bar), GATA3 shRNA or TCF7 shRNA (grey bars) transduced CD34+ thymocytes after 48h of coculture on OP9-DLL1 stromal cells, relative to the geometric mean of ACTIN and GAPDH and relative to the control-transduced sample (n=6 independent experiments with different donors for GATA3 shRNA, n=4 independent experiments with different donors for TCF7 shRNA, error bars indicate SEM and * indicates p<0.05 in a paired t-test).
Distinct Notch1 and BCL11B requirements mediate human γδ/αβ T cell development (2020)
Figure 7

Figure 7. Continuous miR-17 expression expands human thymocytes and enhances human TCRγδ T cell development.

(A) Flow cytometry analysis of control and miR-17 transduced CD34+ thymocytes, cultured in ATOs for 3 weeks. Numbers in contour plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells.

(B) Graphs show the mean frequency of CD4+CD8β+ DP, CD3+TCRαβ+ and CD3+TCRγδ+ thymocytes of control (black bar) or miR-17 (grey bar) transduced CD34+ thymocytes in these cultures (n=6 and n=8 independent experiments for control and miR-17, respectively. Error bars indicate SEM and ns (not significant) indicates p>0.05 in a non-parametric Mann-Whitney U test).

(C) Graphs show the mean absolute cell numbers of total, CD4+CD8β+ DP, CD3+TCRαβ+ and CD3+TCRγδ+ thymocytes that developed in these cultures (n=6 and n=8 independent experiments for control and miR-17, respectively. Error bars indicate SEM and * indicates p<0.05 in a non-parametric Mann-Whitney U test).
Distinct Notch1 and BCL11B requirements mediate human γδ/αβ T cell development (2020)
Figure 8

Figure 8. Human TCRαβ T cell development is more BCL11B dependent compared to TCRγδ T cell development.

(A) Flow cytometry analysis showing mean of Median Fluorescence Intensity (MFI) of BCL11B protein levels in Jurkat cells, 72h post transduction with control shRNA (black bar), BCL11B shRNA1 or BCL11B shRNA2 (grey bars), each normalized to the FMO sample (BCL11B stained - BCL11B unstained sample). (n=2 independent experiments; error bars indicate SEM - the two data points are shown).

(B) Flow cytometry analysis showing mean of MFI of BCL11B protein levels in CD4+CD8β+ DP (black bar) and CD4-CD8β- DN CD3+TCRγδ+ (grey bar) ex vivo thymocytes, normalized to FMO sample (BCL11B stained - BCL11B unstained sample) (n=4 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).

(C) Mean total cell numbers of control shRNA (black bar), BCL11B shRNA1 or BCL11B shRNA2 (grey bars) transduced CD34+ thymocytes, cultured in ATOs for 3 weeks (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a non-parametric paired Wilcoxon test).

(D) Flow cytometry analysis of CD4+CD8β+ DP, CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) thymocytes that developed in corresponding cultures from C. Numbers in contour plots indicate the percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells.

(E) Graphs show the mean frequency of CD3+TCRαβ+ and CD3+TCRγδ+ thymocytes that developed in these cultures (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).

(F-G) Mean absolute cell numbers of (F) CD3+TCRαβ+ and (G) CD3+TCRγδ+ thymocytes that developed in these cultures (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a non-parametric paired Wilcoxon test).

(H) CD3+TCRγδ+/CD3+TCRαβ+ ratio from cultures depicted in D (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).
Stochastic transcription in the p53-mediated response to DNA damage is modulated by burst frequency (2019)
Figure 1

Figure 1 - Single cell quantification of RNA expression by smFISH highlights strong heterogeneity of p53 target gene expression.

A p53 has been show to response with a series of undamped pulse to ionizing irradiation leading to cell cycle arrest while intrinsic DNA damage during cell cycle does not induce regular pulsatile p53 and subsequent gene expression programs. Schematic representations of p53 dynamics in both cellular conditions are shown.

B We selected p53 target genes that are involved in different cell fate programs ranging from apoptosis (BAX), DNA repair (DDB2) cell cycle arrest (CDKN1A), proliferation control (SESN1) and the regulation of the p53 network itself (PPM1D and MDM2).

C Induction of selected p53 target genes after DNA damage induction in A549 wild-type and p53 knockdown cells. RNA levels were measured by qRT-PCR before and 3h after treatment with 10 Gy R. Fold changes relative to basal levels are shown for each cell line.

D Fluorescence microscopy images of smFISH probes labelled with CAL-Fluor 610 (grey) overlayed with Hoechst 33342 stainings (blue) for the indicated target genes in untreated A549 cells. Scale bar corresponds to 10 µm distance, images were contrast and brightness enhanced for better visualization.

E Histograms of quantitative analysis of RNAs per cell for each target gene in the absence of DNA damage (basal). smFISH staining and quantitative analysis of p53 targets shows broad variability of RNA counts per cell for all genes in basal conditions. Dashed line: median; solid line: probabilty density estimate (see Data Visualization section), m: median, CV: coefficient of variation, Fano: Fano factor, n: number of cells analyzed.
Stochastic transcription in the p53-mediated response to DNA damage is modulated by burst frequency (2019)
Figure 4

Figure 4 - Promoter archetypes change upon modulation of p53 dynamics through small molecule inhibitors.

A Chk2 inhibition with the small molecule BML-277 induces transient p53 dynamics with only one pulse after 10 Gy IR . A schematic illustration of the experimental setup and quantification of p53 levels in A549 wild type cells after irradiation with 10 Gy IR and addition of 10 µM BML-277 by immunofluorescence staining (see Methods section for details) are shown as box plots (see Data Visualization section).

B/C We quantified promoter activity of BAX (B, sustained archetype) and PPM1D (C, pulsatile archetype) before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after irradiation with 10 Gy IR and inhibition of the second p53 pulse by Chk2 inhibition. Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The fraction of active promoters was reduced at 6 h and 9 h after irradiation; the transcription rate was not notably affected.

D Sequential treatment with Nutlin-3 converts pulsatile p53 dynamics into sustained nuclear levels. A schematic illustration of the experimental setup and quantification of p53 levels in A549 wild type cells after irradiation with 10 Gy IR and sequential treatment with 0.75 µM Nutlin-3 at 2.5 h, with 2.25 µM at 3.5 h and 4 µM at 5.5 h post IR based on immunofluorescence staining (see Methods section for details) are shown as box plots (see Data Visualization section).

E/F We quantified promoter activity of MDM2 (E, transient archetype) and CDKN1A (F, transient archetype) before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after irradiation with 10 Gy IR and sequential Nutlin-3 treatment. Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The relative fraction of active promoters strongly increased, changing transient to sustained archetypes. The transcription rate increased as well both compared to basal levels and to previous experiments with pulsatile p53 dynamics (inset, fold change relative to IR alone for each time point).
Liver gene therapy with intein-mediated F8 trans-splicing corrects mouse hemophilia A (2022)
Figure 2

Figure 2. Comparison of human F8 variants in vitro.

A Schematic representation of the four different F8 variants that were cloned into an AAV plasmid: wild-type F8; N6 containing 11 amino acids of the modified SQ amino acid linker (SQm) followed by the human N6 B domain; SQ containing the SQ amino acid linker; V3 containing the V3 peptide in the middle of the SQ linker. ITR, inverted terminal repeats; CMV, cytomegalovirus promoter; star symbol, 3xFlag tag; PolyA, short synthetic polyadenylation signal; Ntds, nucleotides; SP, signal peptide. Details on the exact amino acid differences in the B domain can be found in Fig EV1.

B Western blot analysis of lysates of HEK293 cells 72 hours post-transfection with the various F8 variants. Neg, non-transfected cells.

C Chromogenic assay of F8 activity in the medium of transfected cells are reported as International Units/deciliter (IU/dl). Data are presented as mean±SEM. Each dot within the same group corresponds to a biological replicate: Neg n=5; Wild-type n=8; N6 n=8; SQ n=7; V3 n=6. Significant differences between groups were assessed using The Kruskal-Wallis test followed by the posthoc analysis: Nemenyi's All-Pairs Rank Comparison Test. The Kruskal-Wallis test p value = 2.88e-05. **indicates the significant difference between the N6 and the Wild-type groups: p value ≤ 0.01.*indicates the significant difference between the V3 and the Wild-type groups: p value ≤ 0.05. All p values are reported in Appendix Table S1.
Liver gene therapy with intein-mediated F8 trans-splicing corrects mouse hemophilia A (2022)
Figure 3

Figure 3. In vitro F8-N6 (N6) intein expression and activity.

A Western blot (WB) of protein lysates of HEK293 cells 72 hours post-transfection (n=3; biological replicates) with either Npu inteins or Heterologous (N-intein DnaB + C-intein DnaE) split-inteins. I + II, N6 split-intein proteins; I+II (Het.), heterologous split-intein proteins; I, 5'N6 coding sequence (CDS)-N-DnaE protein; I (N-int B), 5'N6 CDS-N-DnaB. II, C-DnaE-3'N6 CDS protein. Excised inteins (∼12kDa) are present only in the down part of the blot when I + II (Npu inteins) are provided. Arrows indicate the full-length N6 protein, single halves and excised inteins.

B WB of medium of the transfected cells showing the secreted proteins (n=3; biological replicates). Arrows indicate the full-length N6 protein as well as single halves and excised inteins.

C Chromogenic assay performed on the medium of transfected cells to detect F8 activity levels reported as International Units/deciliter (IU/dl). Data are presented as mean ±SEM. Each dot within each group represent a different biological replicate (n): I + II n=3; I+II (Het.) n=3; I (N-int E) n=3; I (N-int B) n=3; II (C-int E) n=3; Neg n=4. Significant differences between groups were assessed using Kruskal-Wallis rank sum test Kruskal−Wallis, p value = 0.013. *indicates the significant difference between the I+II and the I (N-int E) groups: p value ≤ 0.05.

***indicates the significant difference between the I+II and the Neg groups: p value ≤0.001. All p values are reported in Appendix Table S1.
Liver gene therapy with intein-mediated F8 trans-splicing corrects mouse hemophilia A (2022)
Figure 4

Figure 4. Codon optimisation of the N6 split-intein improves F8 activity levels.

A Western blot (WB) of protein lysates of HEK293 cells 72 hpt with the AAV-N6 split- intein plasmids and with the codon-optimised set. I + II, N6 split-intein proteins; I, 5' N6 CDS-N-DnaE protein; II, C-DnaE-3'N6 CDS protein. (n=3; biological replicates). Arrows indicate the full-length N6 protein, excised inteins and both single halves. Codop: codon-optimised.

B WB of medium from the transfected cells showing increased secretion of the Codop-N6 full-length protein compared to the non-codon-optimised. (n=3; biological replicates).

Arrows indicate the full-length N6 protein, excised inteins and both single halves.

C Chromogenic assay performed on the medium from transfected cells to measure F8 activity levels reported as International Units/deciliter (IU/dl) (n=3; biological replicates).Data are presented as mean ±SEM. Significant differences between groups were assessed using Kruskal-Wallis test p value = 0.027. *indicates the significant difference between the I+II (N6 split-intein) and the II (C-DnaE-3'N6 CDS) groups: p value ≤ 0.05. **indicates the significant difference between the Codop I+II and the I (5' N6 CDS-N-DnaE) codop groups: p value ≤ 0.01. **indicates the significant difference between the Codop I+II and the II (C-DnaE-3'N6 CDS) codop groups: p value ≤ 0.01. All p values are reported in Appendix Table S1.

D Peptides sequences obtained by LC-MS analysis which include the N6 splitting point which is correctly reconstituted; S: Ser 962 (n=5; biological replicates).
Liver gene therapy with intein-mediated F8 trans-splicing corrects mouse hemophilia A (2022)
Figure 5

Figure 5. AAV-N6 intein administration results in therapeutic F8 activity levels.

A Alkaline gel Southern blot analysis of AAV DNA. AAV DNA was hybridized to a probe specific for the HLP promoter. Neg, AAV DNA treated with Dnase I; CodopV3, AAV-CodopV3; I, AAV-5'N6-N-intein; II, AAV-C-intein-3' N6.

B Chromogenic assay performed on plasma samples to detect F8 activity in AAV-treated mice compared to controls groups. Data are presented as mean ±SEM. The same animals were analysed at the various time-points. Each dot within the same group and within different groups of treatment corresponds to a single animal. The baseline includes all animals (n=20) before the treatment: CodopV3: n=8; N6 intein: n=5; CodopN6 intein: n=5. The statistical difference between groups has been assessed at 4weeks post-injection (wpi) with the One-way Anova p value = 0.056; at 8 wpi with the Kruskal-Wallis test p value = 0.68; at 12wpi with the Kruskal-Wallis test p value = 0.45; at 16wpi with the Kruskal-Wallis test p value = 0.58. All p values are reported in Appendix Table S1.

C Activated partial thromboplastin time (aPTT) assay performed on plasma samples both at the baseline and at the last time-point of the analysis (16 wpi). Data are presented as mean ±SEM. Each dot within each group of treatment corresponds to different animals: The baseline includes all animals before the treatment n=21; CodopV3: n=8; N6 intein: n=5; CodopN6 intein: n=5. Significant differences between groups were assessed using the Kruskal-Wallis test. **indicates the significant difference between the baseline and the N6 intein groups: p value ≤ 0.01.*indicates the significant difference between the baseline and the CodopN6 intein groups: p value ≤ 0.05. All p values are reported in Appendix Table S1.

D Tail-clip assay reported as bleeding time in seconds. Significant differences between groups were assessed using One-way Anova test p value is = 0.11.Data are presented as mean±SEM. Each dot corresponds to a different animal. Knockout (K.O.) group: n=4; CodopV3: n=4; N6 intein: n=4; CodopN6 intein: n=6.

E Haemoglobin content measured as Optical Density (OD) at 416 nm after the tail-clip assay. Significant differences between groups were assessed using One-way Anova test. Data are reported as mean±SEM. Each dot corresponds to a single animal. Knockout (K.O.) group: n=4; CodopV3: n=4; N6 intein: n=4; CodopN6 intein: n=6.

F Western blot analysis of liver lysates (100 µg) from either CodopN6 intein-treated mice (n=6) or untreated haemophilic mice (n=2). A lysate from HEK293 cells transfected with the N6 full-length plasmid was used as positive control (50 µg). Arrows point at N6 full-length protein (N6), the 5' half of N6 (5'N6) and 3' half of N6 (3'N6).