EMBO/soda-nel huggingface dataset:
PrimPol-mediated repriming facilitates replication traverse of DNA interstrand crosslinks (2021)
Figure 4

Figure 4. PrimPol ablation impairs cell proliferation and DNA synthesis in the presence of ICLs.

A. CellTiter-Glo viability assays in WT and PRIMPOL KO cells 6 days after treatment with 1 µg/ml MMC (2 h) or TMP-UVA (2 µM TMP, 2 h followed by 5 s irradiation). Histograms represent the total number of viable cells (average and SD of three assays) at the end of the experiment, expressed as percentage of the number of untreated cells. Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA and Bonferroni post-test. p-values of individual comparisons are indicated.

B. Flow cytometry profiles of BrdU incorporation (y-axis) vs DNA content (PI; x-axis) in WT (blue) and PRIMPOL KO (orange) cells, either collected without treatment (UNT) or at the indicated times after exposure to TMP-UVA (2 µM TMP for 2 h followed by 5 s irradiation). DNA content profiles are shown on top of the boxes (cell count). Dashed horizontal lines inside panels are included for comparative purposes between WT and KO BrdU incorporation levels. Histograms (right panel) represent the percentage of cells in S-phase in each condition (average and SD of three assays). Circle dots in each column represent the values of individual replicates. Statistical analysis in all cases was conducted with one-way ANOVA and Bonferroni post-test. p-values of individual comparisons are indicated.

C. Representative confocal microscopy images of EdU staining in WT and PRIMPOL KO cells, 8 h after UVA (control) or TMP-UVA treatment (2 µM TMP, 2 h followed by 5 s irradiation). Nuclear DNA is counterstained with DAPI. When indicated, V5-PrimPol was reintroduced into KO cells. Scale bar, 25 µm. Dot plots indicate the distribution of nuclear EdU intensity, and the mean values are indicated by horizontal red lines. Data from the combination of three replicates (n ≥200 cells per condition). Statistical analysis was conducted with Kruskal-Wallis test and Dunns post-test. p-values of individual comparisons are indicated.
PrimPol-mediated repriming facilitates replication traverse of DNA interstrand crosslinks (2021)
Figure 5

Figure 5. PrimPol deficiency leads to ICL repair defects.

A. Confocal microscopy images of FANCD2 immunofluorescence staining in control (UVA) or TMP-UVA-treated (2 µM TMP, 2 h followed by 5 s irradiation) WT and PRIMPOL KO cells. Nuclear DNA is counterstained with DAPI. Scale bar, 25 µm.

B. Dot plot (left panel) shows the distribution and mean (horizontal red line) of FANCD2 foci number per cell in each condition. Data pooled from three replicates are represented (n ≥100 cells per condition). Statistical analysis was conducted with Kruskal-Wallis test and Dunn´s post-test. p-values of individual comparisons are indicated. Histograms (right panel) show the average foci number and SD of three assays in each case. Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.

C. Metaphase spreads stained with Leishman, obtained from control (-) or TMP-UVA-treated cells (2 µM TMP, 2 h followed by 5 s irradiation). Scale bar, 10 µm. Representative images of chromosomal breaks and fusions are shown at higher magnification.

D. Histograms show the number of chromosomal aberrations per metaphase (average and SEM) in each condition. Data taken from three replicates are combined (50 metaphases per replicate). Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.
PrimPol-mediated repriming facilitates replication traverse of DNA interstrand crosslinks (2021)
Figure 6

Figure 6. PRIMPOL KO mice are hypersensitive to MMC.

A. Kaplan-Meier survival curves of WT and PRIMPOL KO mice after the administration of a single dose of MMC (7.5, 10 or 15 mg/kg). Dashed vertical lines represent median survival values for WT (blue) and KO (orange) mice. Statistical analysis was conducted with Log-rank (Mantel-Cox) test; p-value for the highest dose of MMC (15 mg/kg) is indicated.

B. Haematoxylin-eosin (H&E) and Ki-67 immunohistochemistry (IHC) stainings of bone marrow sections derived from WT and PRIMPOL KO mice following the administration of 10 mg/kg MMC. Control mice (PBS) were sacrificed when MMC-injected mice reached the humane endpoint. Scale bar, 2.5 mm. In each image, a relevant area is shown at higher magnification (10x).

C. Histograms show Ki-67-positive cellular area in the conditions shown in (B). Results are shown with three pairs of age- and gender-matched WT and KO littermates. When combined, the differences observed between MMC-treated WT and KO mice were statistically significant (two-way ANOVA and Bonferroni post-test, p-value 0.0044).
PrimPol-mediated repriming facilitates replication traverse of DNA interstrand crosslinks (2021)
Figure 7

Figure 7. Loss of backup origins exacerbates the effect of PrimPol loss in ICL repair.

A. Immunoblots showing the levels of MCM3 and PrimPol proteins in PRIMPOL WT and KO cells. MEK2 and Ponceau-S are shown as loading controls.

B. Schematic of experimental design and examples of replicative structures (fork/ first-label origin/ terminations) in stretched DNA fibers. Scale bar, 10 µm.

C. Percentage of first-label origins in each condition (average and SD of three assays). Circle dots in each column represent the values of individual replicates. Statistical analysis: one-way ANOVA followed by Bonferroni post-test. p-values are indicated.

D. Histograms show the percentage of the different replication patterns observed in each experimental condition (average and SD of three assays). Circle dots in each column represent the values of individual replicates. Statistical analysis: two-way ANOVA followed by Bonferroni post-test. p-values of the indicated comparisons are shown.

E. IF detection of FANCD2 foci in WT and PRIMPOL KO cells 48 h after TMP-UVA (2 µM TMP, 2 h followed by 5 s irradiation) with or without MCM3 downregulation. Nuclear DNA is counterstained with DAPI. Scale bar, 25 µm.

F. Top, dot plot shows the distribution and mean value (horizontal red line) of the number of FANCD2 foci per cell in each condition. The pool of three replicates is represented (n ≥100 cells per condition). Statistical analysis was conducted with Kruskal-Wallis test and Dunn's post-test. p-values are indicated. Bottom, histograms show the average foci number and SD of three assays. Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of the indicated comparisons are shown.

G. Cell survival assays of WT and PRIMPOL KO cells with or without MCM3 downregulation in the presence of increasing amounts of MMC or TMP (followed by 90 sec UVA irradiation) for 72 h. The same control curves without MCM3 downregulation are also shown as part of Figure EV6D. Each point in the curve represents the percentage of surviving cells (average and SD of three assays for MMC and two assays for TMP). Statistical analysis was conducted with two-way ANOVA followed by Bonferroni post-test. p-values of comparisons between KO and KO-siMCM3 are indicated.

H. Model for the participation of PrimPol in the ICL traverse reaction (left branch), and the alternative fork convergence mechanism (right branch), both of which generate a suitable template for FA-mediated ICL repair. In the absence of PrimPol, ICL traverse is much less efficient and cells rely on dormant origin activation to promote fork convergence at the ICL. When dormant origins are not available, a fraction of the ICLs remain unrepaired, generating chromosomal instability. See text for details.

Expanded View Figure Legends
Microglia promote glioblastoma via mTOR-mediated immunosuppression of the tumour microenvironment. (2020)
Figure 1

Figure 1 Upregulation of mTOR signalling in TAM-MG but not TAM-BMDM in GBM mouse models.

(A) Representative images of a PDGFB tumour: H&E on the right to identify the tumour, an adjacent section stained for Iba1 (red), p-S6 (green) and DAPI (blue) at the centre and the region selection for quantification on the left (non-tumour in light blue, tumour edge in orange and tumour core in dark blue).

(B) Representative images of core, edge and adjacent non-tumour brain tissue for Iba1, p-S6 and DAPI staining in Pten-/-p53-/- (right) and GL261 (left) tumours. Percentage of Iba1+ cells co-expressing p-S6 in the three defined regions. Five high grade glioma models were analysed - GL261 (n=3), PDGFB (n=3), Ntv-a;PDGFB+shp53 (n=2), Pten-/-p53-/- (n=3), and Pten-/-p53-/-Idh1mut (n=3) (mean ±SEM; Two-Way ANOVA Tukey test).

(C) Venn diagram identifying significantly deregulated genes in healthy versus GL261 TAM-MG and/or TAM-BMDM (GSE68376 dataset).

(D) Top-most deregulated canonical pathways in GL261 TAM-MG, as identified by the IPA software. Threshold indicates p≤0.05. Z-score indicates the orientation of the deregulation. Ratio indicates the number of deregulated genes in the pathway.

(E) mTOR-related deregulated canonical pathways in GL261 TAM-MG and TAM-BMDM, as identified by the IPA software.

(F,G) Expression levels of (F) Rps6 and (G) Eif4ebp1 in healthy versus GL261 microglia and macrophage (n=3; mean ±SEM, likelihood ratio test in edgeR).

Data information: *p≤0.05, **p≤0.01, ***p≤0.001.
Microglia promote glioblastoma via mTOR-mediated immunosuppression of the tumour microenvironment. (2020)
Figure 2

Figure 2 Microglia and BMDM are differently conditioned by mGIC.

(A) Schematic of the in vitro model whereby microglia and BMDM were pre-treated with Torin, LY294002 as indicated and stimulated with mGL261, mGICPten-/-;p53-/--CM or mNSC-CM.

(B-I) Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 4hr (B, D and F) and 0.5hr (H) and normalised against non-phosphorylated protein and vinculin analysed on the same blot. Flow cytometry analysis was carried out in microglia for (C) p-S6 S240/244; (E) p-4EBP1 T37/46; (G) p-AKT S473; and (I) p-AKT T308. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin, mGIC-CM+LY) was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.
Microglia promote glioblastoma via mTOR-mediated immunosuppression of the tumour microenvironment. (2020)
Figure 3

Figure 3 Genetic inhibition of mTORC1 signalling in Cx3cr1+ TAM impacts tumour growth and survival.

(A) Schematic of the generation and analysis of the Cx3cr1-Cre;Rheb1-loxp GL261 model.

(B) Survival analysis for Cx3cr1-Rheb1Δ/Δ (n=7) and Rheb1fl/fl (n=7) mice. Chi square test.

(C) Representative images and quantification of the tumour volume with MRI of Rheb1fl/fl (n=8) compared to Cx3cr1-Rheb1Δ/Δ (n=8) mice. (mean ±SEM; Unpaired parametric t test).

(D) H&E staining showing representative histological features (overview and high magnification of microvascular proliferation) of Rheb1fl/fl and Cx3cr1-Rheb1Δ/Δ GL261 tumours. Scale bar is 125 μm (top, H&E) and 80 μm (all other images)

(E) Percentage of GFP+ CD45- GL261 tumour cells in Cx3cr1-Rheb1Δ/Δ (n=5) and Rheb1fl/fl (n=7) tumours, with representative FACS plot (mean ±SEM; Unpaired parametric t test).

Data information: **p≤0.01, and ***p≤0.001
Microglia promote glioblastoma via mTOR-mediated immunosuppression of the tumour microenvironment. (2020)
Figure 4

Figure 4 Genetic inhibition of mTORC1 signalling in TAM affects the immune profile of GL261 tumours

(A) Significantly deregulated canonical pathways in Cx3cr1-Rheb1Δ/Δ versus Rheb1fl/fl GL261 tumours, as identified by the IPA software from differentially regulated genes and divided in three categories: Adaptive immunity, Cytokine signalling, and Innate immunity. Size of bubbles is indicative of ratio of differentially regulated genes. The x-axis represents -log10(p-value) with a threshold of p≤0.05 applied.

(B) Levels of TAM-MG (P2RY12+ CD49d-) and TAM-BMDM (P2RY12- CD49d+) gated from CD45+ population in GL261 tumours from Rheb1fl/fl (n=4) and Cx3cr1-Rheb1Δ/Δ (n=5) mice by flow cytometry (representative flow cytometry plots on top and quantification at the bottom; mean ±SEM; Two-Way ANOVA Tukey test).

(C) Percentage of CD8 CTL (top) and CD4 Th (CD4) cells in GL261 tumours from Rheb1fl/fl (n=4) and Cx3cr1-Rheb1Δ/Δ (n=5) mice assessed by flow cytometry (representative flow cytometry plots, left and quantification, right; mean ±SEM; Unpaired t test).

(D) Tumour tissues from Rheb1fl/fl (n=4) and Cx3cr1-Rheb1Δ/Δ (n=4) were stained for CD8, CD4 and FoxP3. DAB staining was quantified as % of positive cells using Definiens software. Representative images of the stained tissues are shown. (mean ±SEM; unpaired parametric t test). Scale bar is 100µm.

Data information: *p≤0.05, and **p≤0.01
Microglia promote glioblastoma via mTOR-mediated immunosuppression of the tumour microenvironment. (2020)
Figure 5

Figure 5 Genetic inhibition of mTORC1 signalling in TAM affects the immune reactivity of T cells.

(A) Schematic of the analysis of bulk GL261 RNAseq data from Cx3cr1-Rheb1Δ/Δ (n=3) and Rheb1fl/fl (n=3) mice. ssGSEA enrichment scores for signalling pathway signatures and CIBERSORT cell fractions were calculated for each sample. Pearson correlation score was then calculated comparing signalling pathway enrichment scores and CIBERSORT cell fractions. Finally, a differential correlation analysis was run between the Cx3cr1-Rheb1Δ/Δ (n=3) and Rheb1fl/fl (n=3) samples.

(B) Heatmap of the differential correlation analysis between CIBERSORT cell fractions and signatures enrichment scores (ssGSEA) for lymphocytes in the Cx3cr1-Rheb1Δ/Δ (n=3) vs Rheb1fl/fl (n=3) GL261 tumours.

(C,D) CD4 Th cells (C) and CD8 CTL cells (D) expression of Ki67, perforin, granzyme b, and IFNγ, in Cx3cr1-Rheb1fl/fl (n=5) and Rheb1Δ/Δ (n=5) GL261 tumours. Representative FACS plots show control samples (tumour-derived cells stimulated in culture in the absence of protein inhibitor cocktail - in orange) compared to stimulated cells derived from Rheb1fl/fl (fl/fl, red) and Cx3cr1-Rheb1Δ/Δ (Δ/Δ, blue) GL261 tumours (cells stimulated in culture with protein inhibitor cocktail) (mean ±SEM; Two-Way ANOVA Tukey test).

(E,F) Expression of CD44 (E) and CD62L (F) by CD4 Th and CD8 CTL in Cx3cr1-Rheb1fl/fl (n=5; fl/fl, red) and Rheb1Δ/Δ (n=5; Δ/Δ, blue) GL261 tumours with representative FACS plots show control samples (mean ±SEM; Two-Way ANOVA Tukey test).

Data information: *p≤0.05, **p≤0.01, ***p≤0.001
Microglia promote glioblastoma via mTOR-mediated immunosuppression of the tumour microenvironment. (2020)
Figure 6

Figure 6 mTOR inhibition in TAM-MG induces a pro-inflammatory tumour microenvironment via the regulation of STAT3 and NF-ĸB transcription factors

(A) Significantly deregulated canonical pathways in GL261 Rheb1fl/fl versus Rheb1Δ/Δ TAM-MG, as identified by the IPA software from differentially regulated genes. Size of bubbles is indicative of ratio of differentially regulated genes. The x-axis represents -log10(p-value) with a threshold of p≤0.05 applied. On the right, number of up and down regulated genes in each identified pathway is indicated in blue and red respectively.

(B) Frequency of occurrence of differentially regulated genes across significantly deregulated canonical pathways compared to the Log fold change (Log(FC)).

(C) Upstream regulators as identified by the IPA software from differentially regulated genes.

(D) Heatmap of Pearson correlation analysis between ssGSEA signatures enrichment scores in the Rheb1fl/fl versus Cx3cr1-Rheb1Δ/Δ GL261 tumours.

(E) Microglia were pre-treated with Torin, or medium as indicated and then stimulated with mNSC-CM, mGICPten-/-;p53-/--CM, or GL261-CM. p-NF-ĸB (p-P65) and p-STAT3 were analysed by immunoblotting of whole cell lysates collected at 4hr.

(F) Immunofluorescence images of NF-ĸB (P65) in conditioned microglia. Quantification of nuclear translocation of NF-κB from staining. Units represent the % of voxels in the NF-κB channel colocalised with DAPI (n=3; mean ±SEM; One-Way ANOVA Tukey test - right). Scale bar is 50µm.

(G) Production of Il-12 (Il-12p40), Tnf, Il6, and Il10 by conditioned microglia was determined by qPCR. Each treatment was normalised to housekeeping gene and the unconditioned control. (n=3; mean ±SEM; Two-Way ANOVA Tukey test).

(H,I) MFI levels of pSTAT3 in TAM-MG (CD45+ P2RY12+ CD49d-) (H) and p-NF-κB (p-P65) in TAM-BMDM (CD45+ P2RY12- CD49d+) (I) from Rheb1fl/fl (fl/fl, blue, n=4) and Cx3cr1-Rheb1Δ/Δ (Δ/Δ, red, n=5) GL261 tumours. Representative FACS plots are displayed for each cell type (mean ±SEM, Two-Way ANOVA Tukey test).

Data information: *p≤0.05, **p≤0.01, ***p≤0.001.
Microglia promote glioblastoma via mTOR-mediated immunosuppression of the tumour microenvironment. (2020)
Figure 7

Figure 7 mTOR signalling in TAM-MG promotes immune evasion mechanisms in human glioblastoma.

(A) Correlation between ssGSEA enrichment scores for the mTOR signature versus TAM-MG or TAM-BMDM signatures in TCGA GBM transcriptomic data. Comparison carried out on all IDH-wild-type samples, and in a subgroup specific manner according to Wang's classifier. Size of circle is indicative of Rsquare value and bold outline represents a p-value ≤0.05.

(B) Separation of IDH-wildtype GBM samples between those displaying high mTOR and microglia enrichment (+ve correlation, group 1 in orange) and those without this signature (group 2 in green).

(C) CIBERSORT cell fractions calculated from the TPM of TCGA IDH-wild type GBM samples from group 1 compared to those from group 2.

(D) Heatmap of Pearson correlation analysis comparing ssGSEA enrichment scores of TAM-MG and TAM-BMDM signatures versus signalling pathway signatures, calculated from the TPM of IDH-wild type GBM samples with the signature from group 1.

(E) Schematic of the experimental setup to derive patient-matched EPSC and GIC lines.

(F) Staining by flow cytometry of EPSC, YS-EBs and matched-iMGL with indicated markers (n=3; mean ±SEM).

(G,H) Mature iMGL were immunostained for (G) PU1 and CD11b and (H) Iba1 and TREM2. Scale bar 20µm.

(I-K) (I) P-S6, (J) p-STAT3, and (K) p-NF-ĸB (p-P65) were analysed by flow cytometry in iMGL, pre-treated with Torin, as indicated and then stimulated with matched patient EPSC- or hGIC-CM. (n=3; mean ±SEM, Two-Way ANOVA Tukey test).

Data information: **p≤0.01, ***p≤0.001.

Expanded View Figure Legends
Kit ligand has a critical role in mouse yolk sac and aorta-gonad-mesonephros hematopoiesis (2018)
Figure 1

Figure 1. YS Sl/Sl EMPs fail to reach normal numbers after their initial generation

A. Number of EMPs per embryo equivalent (e.e.) of wild type and Sl/Sl E9.5 and E11.5 YS, determined by flow cytometry (panels in B,D). E9.5 Kit+ CD41+ CD16/32+ EMP numbers are the mean±SD from 4 wild type and 3 Sl/Sl biological replicates, with each replicate consisting of single or 2 pooled YS of the same genotype. Total number of embryos analyzed: 7 +/+ (14-23sp), 5 Sl/Sl (17-25sp). E11.5 Kit+ CD41+ EMP numbers are the mean±SD of 5 +/+ and 6 Sl/Sl YS analyzed individually over 2 independent experiments. Total live cells per YS: 1.7±0.3x105 (wt), 1.7±0.2x105 (Sl/Sl) at E9.5; 4.2±1.4x105 (wt), 4.0±2.2x105 (Sl/Sl) at E11.5.

B. Example of flow cytometry analysis of E9.5 YS EMPs quantified in panel (A). Analyses were performed on Ter119- live cells.

C. Number of CFU-C in wild type and Sl/Sl E9.5 and E11.5 YS. E9.5 data (mean±SD) are from 3 wild type and 2 Sl/Sl biological replicates plated in duplicate, with each replicate consisting of single or 2 pooled YS of the same genotype. Total number of analyzed embryos: 5 +/+ (17-23sp), 3 Sl/Sl (17-25sp) over 2 independent experiments. For E11.5, data are from 4 wild type and 6 Sl/Sl biological replicates plated in duplicate, with each replicate consisting of single or 2 pooled YSs of the same genotype. Total number of embryos analyzed: 6 +/+, 7 Sl/Sl over 4 independent experiments. GEMM: granulocyte, erythroid, monocyte/macrophage, megakaryocyte; G/M/GM: granulocyte, monocyte/macrophage; Ery: erythroid.

D. Example of flow cytometry analysis of E11.5 YS EMPs quantified in panel (A). Analyses were performed on Ter119- live cells.

E. Confocal WM-IF analysis of CD31+ pHH3+ Kit+ proliferating EMPs (arrowheads) in E9.5 wild type and Sl/Sl YS. A single 2.5μm-thick Z section is shown. For each YS, CD31+ Kit+ pHH3+ cells were counted in 3 to 5 fields using a 25x objective and the percentage of pHH3+ proliferating EMPs was calculated. Bar graphs represent the mean±SD from 3 wild type (21-24sp) and 3 Sl/Sl (22-23sp) YS. Scale bars: 50μm.

F. Cell cycle analysis of wild type and Sl/Sl E11.5 YS EMPs. The percentage of EMPs (Ter119- Kit+ CD41+) in G0/G1 or S/G2/M was determined by flow cytometry on the basis of BrdU and 7-AAD incorporation. a: apoptotic (percentage not shown). Embryos were analyzed individually. N=6 (+/+), N=5 (Sl/Sl) over 3 independent experiments. E11.5 embryos were staged according to tail somite counts as described in [76] and were: 12-17 (+/+); 12-17 (Sl/Sl). Bar graphs show mean±SD, and FACS plots representative results.

Data information: *P<0.05 (Unpaired two-tailed Student t-test).
Kit ligand has a critical role in mouse yolk sac and aorta-gonad-mesonephros hematopoiesis (2018)
Figure 2

Figure 2. Kitl is required for the early FL expansion of EMPs and their progeny

A-C. Flow cytometry analysis of EMPs (Ter119- Kit+ CD41+ CD16/32+) and early erythroid progenitors (Early E: Ter119- Kit+ CD41- CD71+ CD44+) in wild type and Sl/Sl E11.5 FL. EMP and early E data are the mean (±SD) of 6 wild type and 4 Sl/Sl biological replicates, with a total of 10 wild type and 6 Sl/Sl embryos analyzed in pools of 1-3 FL per genotype. Bar graphs on the right show corresponding absolute cell numbers (nr) per FL. Total FL cell counts are the mean (±SD) of 12 wild type and 13 Sl/Sl embryos. Tail somite range: 10-16 (wild type), 12-15 (Sl/Sl). Note that although EMP frequency in Sl/Sl FL is increased, their absolute number is severely decreased.

D. CFU-C numbers in wild type and Sl/Sl E11.5 FL. Data are the mean (±SD) of 10 wild type and 10 Sl/Sl biological replicates plated in duplicate, with each replicate consisting of cell from 1-2 FLs. A total of 12 wild type and 11 Sl/Sl FL were plated over 6 independent experiments. GEMM: granulocyte, erythroid, monocyte/ macrophage, megakaryocyte; G/M/GM: granulocyte, monocyte/macrophage; Ery: erythroid.

E. Left: Total cell numbers (nr) of E12.5 wild type (n=7) and Sl/Sl (n=8) FL. Right: CFU-E numbers per wild type and Sl/Sl E12.5 FL. Data are the mean (±SD) of 3 biological replicates analyzed over 2 independent experiments. Each biological replicate consisted of 1-2 FL samples pooled according to genotype and was plated in duplicate into a methylcellulose based medium containing EPO; colonies were scored after 2 days. Total number of FLs analyzed: 4 wild type and 6 Sl/Sl.

F. Quantification of erythroid lineage cells in E12.5 wild type and Sl/Sl FL. Absolute cell counts (top) and percentages (bottom) of proerythroblasts (ProE), basophilic erythroblasts (BasoE), polychromatic erythroblasts (PolyE) and orthochromatic erythroblasts (OrthoE) were determined by imaging flow cytometry analysis. The number (nr) of cells per FL was based on total cellularity and their relative proportions. Data are the mean (±SD) of 3 biological replicates for each genotype, with replicates consisting of single or 2 pooled FL samples. A total of 4 wild type, 5 Sl/+ and 6 Sl/Sl FLs were analyzed.

G. Representative examples of maturing erythroblast series in wild type and Sl/Sl E12.5 FL, as identified by imaging flow cytometry. Scale bars: 10μm.

Data information: *P<0.05 (Unpaired two-tailed Student t-test).