EMBO/soda-nel huggingface dataset:
Metabolic memory underlying minimal residual disease in breast cancer (2021)
Figure 1

Figure 1. A multi-omics approach reveals characteristic features of residual organoids in vitro and their metabolic resemblance to tumor organoids

A, Representative immunofluorescence staining of normal, tumor, and residual 3D organoids grown from primary mammary cells of a preclinical mouse model of breast cancer (Methods). Similar morphology of normal and residual organoids shown on the left with polarity markers ITGA6 (red), ZO-1 (green), GM-130 (magenta); DAPI (blue). Right: human MYC oncoprotein (green) is expressed only in tumor cells, CDH1 (red). Scale bar: 25 μm.

B, C, Principal component (PC) analyses of normal (blue), tumor (orange) and residual (green) cells based on (B) RNA-sequencing data (normal, n=8; tumor and residual, n=4 each) and (C) lipidomics data (n=4).

D, Heat map of untargeted metabolomics results showing the clustering of the normal - wild type (dark blue), normal - uninduced (light blue), tumor (yellow) and residual (green) populations and the most altered metabolic pathways (n=4). Hierarchical clustering was based on the complete linkage method using the Euclidean distance metric. Significance is defined as a false discovery rate < 0.05 versus the normal population, calculated using unpaired two-sided t-tests and adjusted for multiple hypothesis testing according to Storey's and Tibshirani's method (Storey & Tibshirani, 2003) (Materials and Methods). PPP, pentose phosphate pathway; TCA, tricarboxylic acid cycle; OXPHOS, oxidative phosphorylation; AA, amino acids.

E, Principal component analysis of normal and WT (blue), tumor (orange), and residual (green) populations based on intracellular metabolomics targeted at central carbon metabolism (n=8 each; WT n=2).

F, Selection of the most profoundly altered metabolites in extracellular spent growth medium from normal (normal and WT, n=4 and n=2, respectively), tumor (n=4), and residual (n=4) populations, based on metabolomics analysis targeted at central carbon metabolites. Values represent metabolite abundance levels as quantified by the area under the curve (AUC) of the marker fragment ions/transitions for each metabolite. Statistics were calculated using the limma package in R (Ritchie et al., 2015). Significant results (marked with *) correspond to a Benjamini-Hochberg adjusted p-value ≤ 0.05 (residual or tumor compared to normal). ns, not significant. Box plots: midline, median; box, 25-75th percentile; whisker, minimum to maximum.

Data information: A-F, Number of replicates corresponds to individual mice used to derive organoids. B, C, E, Centroids represent the mean and concentration ellipses represent one standard deviation (level=0.68) of an estimated t-distribution based on the first two principal components.
SAMHD1-mediated dNTP degradation is required for efficient DNA repair during antibody class switch recombination (2020)
Figure 1

Figure 1. SAMHD1 was identified as a novel regulator of antibody class switch recombination through locus specific proteomics.

(a) Schematic representation of the IgH locus of WT and of 8X-LexA binding element (LexA-BE) knocked-in CH12F3-2A cells. The a, b and c bars indicate the position of primers used for ChIP-qPCR in Fig. 1b. A protein in which the DNA-binding domain (DB) of LexA was tagged with 3X-FLAG and a nuclear localization signal (NLS) (FNLDD) was expressed in WT (FNLDD alone) and Sα-engineered (FNLDD- Sα-LexA) CH12F3-2A cells.

(b) Chromatin immunoprecipitation using α-FLAG antibody was performed to confirm the pull-down of Sα chromatin from the Sα-engineered (FNLDD-Sα-LexA) CH12F3-2A cells. The a, b, and c bars indicate the primers used for ChIP-qPCR as shown in Fig. 1a (n=3; mean + sd; two tailed unpaired Student's t-test).

(c) Schematic representation of the iChIP coupled with SILAC based-mass spectrometry.

(d) Functional classification of the Sα region binding proteins identified by iChIP.

(e) Bar graph showing the proteins with ≥2-fold enrichment as calculated by the heavy to light SILAC ratio.

(f) Effect of the siRNA-mediated KD of histone chaperones and chromatin remodelers on IgM to IgA switching in CH12F3-2A cells. Data are presented as the % IgA switching relative to the IgA switching in cells transfected with control siRNA (siCONT) (n=2; mean + sd).
SAMHD1-mediated dNTP degradation is required for efficient DNA repair during antibody class switch recombination (2020)
Figure 2

Figure 2. SAMHD1 is required for efficient immunoglobulin class switch recombination.

(a) Top: Scheme of the IgA-switching assay in CH12F3-2A cells. After electroporation of siRNAs, cells were cultured for 24 h, and then stimulated by CIT, cultured for another 24 h, and subjected to FACS analysis. Bottom: Confirmation of the siRNA-mediated KD of SAMHD1 in CH12F3-2A cells.

(b) FACS profile of the percentage of CH12F3-2A cells undergoing IgA switching after transfection of the indicated siRNAs into CH12F3-2A cells cultured with (+) or without (-) CIT stimulation for 24 h. The number in the FACS plot represents the percentage of cells expressing IgA on their surface.

(c) Summary of the FACS data obtained from three independent experiments. Data are shown as the % IgA switching relative to the IgA switching in cells transfected with control siRNA (siCONT) (n=3; mean + sd; two tailed unpaired Student's t-test).

(d) Quantitative RT-PCR analysis of the μGLT, αGLT, Aid, and Samhd1 expression in the indicated samples. The data were normalized to Gapdh, and represents as the mean of the two independent experiments with standard deviations. Statistical significance as determined by two-tailed unpaired Student's t-test is shown. ns = not significant.

(e) Confirmation of the CRISPR/Cas9 mediated KO of Samhd1 in CH12F3-2A cells. Beta-actin was used as a loading control.

(f) CSR time course analysis of WT and Samhd1 KO clones. The data are shown as percentage of IgA expressing cells (n=2; mean ± sd; two tailed unpaired Student's t-test; **, p<0.01).

(g) Proliferation of WT and Samhd1 KO clones after stimulation with CIT (n = 2; mean ± sd; two tailed unpaired Student's t-test; ns = not significant).

(h) Analysis of the cell proliferation of WT or Samhd1 KO clones of CH12F3-2A. Histograms show the CFSE dye dilution derived from the indicated samples. Aphidicolin (2 μg/mL) was also used as a positive control.

(i) Top: Schematic representation of the IgG1 or IgG3 switching assay in spleen B cells. Bottom: Western blot analysis of the SAMHD1 expression in spleen B cells derived from WT or Samhd1 KO mice. Tubulin was used as a loading control.

(j) Bar graphs show the compilation of IgG1 and IgG3 CSR assays from five independent experiments. The data are shown as percentage of IgG1 (left) or IgG3 (right) expressing cells (n=5; mean ± sd; two-tailed unpaired Student's t-test).
SAMHD1-mediated dNTP degradation is required for efficient DNA repair during antibody class switch recombination (2020)
Figure 3

Figure 3. SAMHD1 depletion in CH12F3-2A cells decreases the frequency of IgH/c-Myc chromosomal translocations.

(a) Top: Schematic illustration of the experimental design for the translocation assay in CH12F3-2A cells. Bottom: PCR amplification scheme to detect IgH/c-Myc chromosomal translocations. Triangles represent the positions of nested PCR primers used to amplify the rearranged regions. The position of the Myc probe used in the Southern blot hybridization is shown as a horizontal black bar.

(b) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. CH12F3-2A cells were transfected with the indicated siRNAs.

(c) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test).

(d-g) Analysis of the IgH/c-Myc translocation frequency in CH12F3-2A cells expressing Tet-inducible miRNA against Top1 mRNA. Schematic illustration of the experimental design for the translocation assay (d). Western blot analysis of the KD of SAMHD1 or TOP1 by the transfection of CH12F3-2A cells by SAMHD1 siRNA or by tetracycline (50 nM) treatment, respectively. Expression of Tubulin is shown as a loading control (e: left). Relative expression of TOP1 and SAMHD1 as determined by densitometric quantification of western blot images in Fig. 3e (e: right). Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments (f). Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test) (g).

(h-j) Analysis of the IgH/c-Myc translocation frequency in WT and Samhd1 KO CH12F3-2A cells. (h) Schematic illustration of the experimental design for the translocation assay. (i) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. (j) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test).

(k-m) Analysis of the IgH/c-Myc translocation frequency in spleen B cell derived from WT (Samhd1+/+) or Samhd1 KO (Samhd1-/-) mice. (k) Schematic illustration of the experimental design for the translocation assay. (l) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. (m) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test).
SAMHD1-mediated dNTP degradation is required for efficient DNA repair during antibody class switch recombination (2020)
Figure 4

Figure 4. SAMHD1 depletion does not affect the formation of AID-induced DNA double strand breaks or synapsis.

(a) Top: schematic representation of the Sµ region of CH12F3-2A cells. Bottom: schematic representation of the Sµ region of CH12F3-2A cells. The 5′ Sµ region analyzed for SHM is underlined. The genomic DNA isolated from WT or Samhd1 KO CH12F3-2A cells from three independent experiments were pooled prior to SHM analysis.

(b) Mutation frequency in the 5′ Sµ region of WT or Samhd1 KO CH12F3-2A cells. Statistical significance as evaluated by Fisher's exact test is shown. ns = not significant.

(c) Details of the mutation analysis of the 5′ Sµ region.

(d) LM-PCR analysis of the AID-induced DNA double-strand breaks in the Sµ region of WT or Samhd1 KO CH12F3-2A cells. Wedges indicate a three-fold increase in the DNA amount. Amplification of Gapdh was used as an internal loading control.

(e) Gamma-H2AX-ChIP signal in the Sµ region is shown as the fraction of immunoprecipitated DNA (% IP) normalized to the total amount of DNA used for the immunoprecipitation (n=3; mean + sd; two-tailed unpaired Student's t-test; ns = not significant).

(f) Left: Scheme of long-range interactions between Sμ-Sα elements in the IgH locus before and after AID activation. Right: Representative gel picture of the 3C assay detecting the Sμ-Sα interaction in WT or Samhd1 KO CH12F3-2A cells with or without CIT stimulation for 24 h. Gapdh was amplified as a loading control.
SAMHD1-mediated dNTP degradation is required for efficient DNA repair during antibody class switch recombination (2020)
Figure 5

Figure 5. SAMHD1 suppresses insertions during DNA double strand break repair end-joining.

(a-d) Analysis of Sμ-Sα recombination junctions from WT or CH12F3-2A cells after transfection with control (siCONT) or Ligase 4 (siLIG4) siRNA. The genomic DNA isolated from WT or Samhd1 KO cells from three independent experiments were pooled prior to junction analysis. A total of 62 (siCONT) and 73 (siLIG4) junctions from WT, and 60 (siCONT) and 60 (siLIG4) junctions from Samhd1 KO cells were sequenced and analyzed. Statistical significance as evaluated by Fisher's exact test (b) or two-tailed unpaired Student's t-test (d) or is shown. Schematic representation of the Sμ-Sα junction analysis (a). Summary of the characteristics of Sμ-Sα recombination junctions (b). Sequences at Sμ-Sα recombination junctions with insertions (c). Summary of the average length of insertions at Sμ-Sα recombination junctions (d).

(e-i) Analysis of the CRISPR/Cas9 induced chromosomal translocations between the Rosa26 (Chr6) and H3f3b (Chr 11) genes. The genomic DNA isolated from WT or Samhd1 KO cells from three independent experiments were pooled prior to chromosomal translocation or junction analysis. Schematic representation of the chromosomal translocations (e). Representative images of agarose gels after PCR-amplification of the Rosa26/H3f3b translocation from WT or Samhd1 KO CH12F3-2A cells (f). Relative frequency of Rosa26/H3b3f chromosomal translocations (g). Frequency of junctions containing insertions (h) and average length of insertions (i). Statistical significance as evaluated by two-tailed unpaired Student's t-test (g, i) or Fisher's exact test (h) is shown.
SAMHD1-mediated dNTP degradation is required for efficient DNA repair during antibody class switch recombination (2020)
Figure 6

Figure 6. Accumulation of purine deoxyribonucleotides is responsible for the defects in CSR and IgH/c-Myc translocations.

(a) Top: Schematic representation of GFP-fused WT- or mutated-SAMHD1 proteins. Bottom: Schematic representation of the CSR complementation assays.

(b) Confirmation of the expression of GFP-tagged human SAMHD1 (WT, and DN, DA, TA, and RE mutants). Histone H3 expression is shown as a loading control.

(c) CSR complementation assay in Samhd1 KO cells. Data are shown as the % IgA switching relative to the IgA switching in WT cells transfected with empty vector, and are derived from three independent experiments (mean ± sd; two-tailed unpaired Student's t-test).

(d) Effects of SAMHD1 depletion or BRD2 depletion on the level of metabolites involved in nucleotide metabolism as analyzed by CE-TOF-MS. BRD2 knockdown used as an unrelated control. The deoxyribonucleoside triphosphates or the metabolites derived from them are boxed (n=2; mean ± sd).

(e) Effect of deoxyribonucleoside supplementation individually or in combination on the rate of IgM to IgA switching in CH12F3-2A cells. Data are presented as % IgA switching relative to the IgA switching in cells treated with an equivalent volume of the solvent in which the deoxyribonucleosides were dissolved (n=3; mean ± sd; two-tailed unpaired Student's t-test; ** = p ≤ 0.01; *** = p ≤ 0.001).

(f) Effect of deoxyribonucleoside supplementation individually or in combination on the frequency of IgH/c-Myc translocations in CH12F3-2A cells (n = 2; mean ± sd; Fisher's exact test; ns = not significant; *** = p ≤ 0.001). The concentration dNTPs used for supplementation either individually or in combination were as follows: 2′-deoxyguanosine (dG), 0.15 mM; 2′-deoxyadenosine (dA), 1.0 mM; 2′-deoxycytidine (dC), 12 mM; and 2′-deoxythymidine (dT), 1.6 mM.
SAMHD1-mediated dNTP degradation is required for efficient DNA repair during antibody class switch recombination (2020)
Figure 7

Figure 7. SAMHD1 promotes the CSR from staggered DSBs.

(a) Top: Schematic representation of the IgH locus depicting Sμ and Sα region with the position of guide RNAs (gRNA) that were used to induce blunt end DSBs using WT SpCas9. Bottom: IgH locus depicting Sμ and Sα region with the position of guide RNAs (gRNA) that were used to induce staggered DSBs with 5′ overhangs using D10N mutants of SpCas9 (Cas9n).

(b) FACS profile of the percentage of WT and Samhd1 KO CH12F3-2A cells undergoing IgA switching following transfection with the indicated plasmids. The number in the FACS plot represents the percentage of cells expressing IgA on their surface.

(c) Summary of the FACS data obtained from two independent experiments. The data are shown as percentage of IgA expressing cells upon CSR induced by blunt (left) or staggered (right) DSBs (n = 2; mean + sd; two-tailed unpaired Student's t-test); ns = not significant.

(d) Length of resected DNA at Sμ-Sα junctions obtained from the joining of DSBs with blunt ends. Statistical significance as evaluated by two-tailed unpaired Student's t-test is shown.

(e) Summary of the characteristics of Sμ-Sα recombination junctions resulting from the joining of DSBs with blunt ends.

(f) Length of resected DNA at Sμ-Sα junctions obtained from the joining of staggered DSBs. Left: Length of Sμ and Sα overhangs were 35 nt and 98 nt, respectively. Right: Length of Sμ and Sα overhangs were 35 nt and 187 nt, respectively.

(g) Summary of the characteristics of Sμ-Sα recombination junctions resulting from the joining of staggered DSBs. Statistical significance as evaluated by Fisher's exact test is shown.

(h) Summary of the average length of insertions at Sμ-Sα recombination junctions. Statistical significance as evaluated by two-tailed unpaired Student's t-test is shown.

(i) Sequences of Sμ-Sα recombination junctions with insertions. The middle column indicates the insertions (in) found in different clones at the junctions.

(j-m) Analysis of the effect of TdT expression on the frequency of CSR as well as characteristics of Sμ-Sα recombination junctions resulting from the joining of DSBs with blunt ends. (j) Top: Schematic representation of the experimental design to analyze the effect of TdT expression on CSR in WT and Samhd1 KO cells. Bottom: Western blot analysis of the expression of TdT in WT and Samhd1 KO cells. Tubulin was used as a loading control. (k) FACS profile of the percentage IgA switching in WT or Samhd1 KO CH12F3-2A cells with or without TdT expression. (l) Summary of the FACS data obtained from two independent experiments (n = 2; mean + sd; two-tailed unpaired Student's t-test; * = p ≤ 0.05). (m) Summary of the characteristics of Sμ-Sα recombination junctions. Statistical significance as evaluated by Fisher's exact test is shown. ns = not significant.
E2F1 proteolysis via SCF-Cyclin F underlies synthetic lethality between Cyclin F loss and Chk1 inhibition (2019)
Figure 1

Figure 1. KCGS screen identifies synthetic lethality between Chk1 inhibitors and cyclin F Knock-Out (K/O)

HeLa and cyclin F Knock-Out (CCNF K/O) cells were treated with Kinase ChemoGenomic Set (KSGS) in 384 well format. After 72 Hours (h) viability was measured using rezasurin. Flowchart representation (left). Robust Z-score difference is plotted for data acquired at 1 μM concentration (right).

Cell survival measured using rezasurin and compared to controls treated with DMSO (expressed as relative proliferation %). Cells were treated with the indicated inhibitors at 0.1 and 1 μM concentration.

Cell survival measured using rezasurin and compared to controls (0) treated with DMSO (expressed as relative proliferation %). Cells were treated with UCN-01 at the indicated concentrations.

Differences in viability of HeLa and CCNF K/O cells after treatment with specific Chk1i (LY2603618) at the indicated concentrations plotted on a log10 scale to measure differences in IC50.

Number of cells was measured in HeLa and CCNF K/O cells left untreated or treated with Chk1i (LY2603618) for 24h, 48h, or 72h as indicated. (n.s. = non-significant).

Cell viability measurements using Propidium Iodide staining for HeLa and CCNF K/O cells after treatment with Chk1i (LY2603618).

U-2-OS cells were seeded and transfected with non-targeting siRNA siNC (Negative Control) or siCyc F. 24 hours after transfection, cells were treated with UCN-01, or Chk1i (LY2603618) at indicated concentrations for 3 days before viability was measured.

Cell viability measurements using Propidium Iodide staining in U-2-OS cells transfected with non-targeting siNC and siCyc F and treated with Chk1i (LY2603618) as indicated.

Data information: (C, D, E-H) Data are presented as mean ± standard deviation (SD), with at least 3 independent experiments. p-values (* p<0.05, ** p<0.005, *** p<0.0005, ****p<0.00005) were calculated by paired and two-tailed t test. NT= treatment with DMSO.
E2F1 proteolysis via SCF-Cyclin F underlies synthetic lethality between Cyclin F loss and Chk1 inhibition (2019)
Figure 2

Figure 2. Loss of cyclin F causes DNA replication catastrophe after Chk1 inhibition

Cells treated with 1μM Chk1i (LY2603618) for the indicated hours (h) were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by Western blot (WB). TFIIH was used as a loading control.

Percentage (%) of γ-H2AX positive cells in G1 cells after Chk1i (LY2603618) treatment for the indicated hours (h) measured using Fluorescence Activated Cell Sorting (FACS). G1 cells were considered having DAPI 2n staining and EdU negative.

Percentage (%) of γ-H2AX positive cells in replicating (EdU+) cells after Chk1i (LY2603618) treatment at the indicated time points.

Percentage (%) of γ-H2AX positive cells in G2/M cells after Chk1i (LY2603618) treatment. G2/M cells were considered having DAPI 4n staining and EdU negativity.

Intensity of γ-H2AX measured as Relative Fluorescence Units (RFU) in early S phase cells treated with Chk1i. Mean intensity of γ-H2AX fluorescence was evaluated from EdU+ cells with 2N DAPI content.

Intensity of γ-H2AX measured as Relative Fluorescence Units (RFU) in replicating cells treated with Chk1i. Mean intensity of γ-H2AX fluorescence was evaluated from EdU+.

Overall, cell cycle profile of HeLa and CCNF K/O cells after Chk1i treatment. Cells with 2n DAPI staining but EdU- were gated as G1 cells; cells with >2n but <4n DAPI staining but EdU- were gated as EdU- S cells; cells with >2n but <4n DAPI staining but EdU+ were gated as EdU+ S phase; cells with 4n DAPI and pH3 S10 negative staining were gated as G2 phase and cells with 4n DAPI and pH3 S10 positive as M cells. Data from at least 3 independent replicates and plotted as Mean ± SD.

Representative plots of γ-H2AX vs RPA2 signal of HeLa and CCNF K/O cells after treatment with 1μM Chk1i (LY2603618) at the indicated hours (h). Blue labeled dots represent γ-H2AX positive cells. Relates to materials section for details on representation.

Data information: Data from at least 3 independent biological replicates and data were plotted with Mean ±SD (B-D) or Mean RFU ± SD (E, F).
E2F1 proteolysis via SCF-Cyclin F underlies synthetic lethality between Cyclin F loss and Chk1 inhibition (2019)
Figure 3

Figure 3. Loss of cyclin F causes SSBs and DSBs after Chk1 inhibition

Representative plots of γ-H2AX intensity vs DAPI signal of CCNF K/O cells transfected with empty GFP and GFP-CCNF untreated (top panels) and after treatment with 1μM Chk1i (LY2603618) for 20h (bottom panels).

Normalised γ-H2AX intensity in CCNF K/O cells transfected with either GFP empty vector or GFP-Cyc F and treated with 1μM Chk1i for 20 hours. GFP positive cells were gated. Data from 3 independent experiments were plotted with Mean % ± SD.

Representative images of neutral and alkaline comet assay from HeLa and CCNF K/O cells treated with Chk1i. Cells were treated with 1μM Chk1i for 24h before being harvested for neutral and alkaline comet assays as indicated.

Quantification of comets in alkaline gels. At least 100 cells, across two slides, were analysed in each condition in two biological replicates. Data are shown as medians, with 25/75% percentile range (box) and 10-90% percentile range (whiskers). Fold changes of median values are shown in bold. p-values were calculated using the Mann-Whitney test (two-tailed). Olive tail moment = (Tail.mean - Head.mean)*Tail%DNA/100.

Quantification of comets in neutral gels. At least 100 cells, across two slides, were analysed in each condition in two biological replicates. Data are shown as medians, with 25/75% percentile range (box) and 10-90% percentile range (whiskers). Fold changes of median values are shown in bold. p-values were calculated using the Mann-Whitney test (two-tailed). Olive tail moment = (Tail.mean - Head.mean)*Tail%DNA/100.