EMBO/soda-nel huggingface dataset:
NPC1 regulates the distribution of phosphatidylinositol 4-kinases at Golgi and lysosomal membranes (2021)
Figure 1

Fig 1. PtdIns4P accumulates in Golgi and lysosomal compartments following loss of NPC1 function.

A. Confocal images of live control (top) and U18-treated (bottom) tsA201 cells expressing P4M-YFP. Black and orange rectangles show expanded views of PtdIns4P in perinuclear and endo/lysosomal compartments, respectively.

B. Quantification of perinuclear P4M-YFP mean fluorescence intensity normalized to the cytoplasm. Control: n=19; U18: n=16.

C. Confocal images of control and U18-treated hippocampal neurons transfected with P4M-YFP. The inset panels show expanded views of PtdIns4P in perinuclear regions.

D. Quantification of perinuclear YFP-P4M mean fluorescence intensity normalized to the cytoplasm. Control: n=11; U18: n=10.

E. Control (top) and NPC1I1061T (bottom) patient fibroblasts transfected with P4M-YFP (left), TGN38-RFP, and merge (right). Black rectangles show expanded views of PtdIns4P in TGN38 regions. Dashed white lines were used for intensity profile analysis (F).

F. Line scan analysis taken from white dashed lines in (E) showing intensity changes of P4M and TGN38 signal from control (top) and NPC1I1061T (bottom) cells.

G. Quantification of P4M signal in TGN38 region (P4MTGN38). Control: n=12; NPC1I1061T: n=19.

H Same as (E) only LAMP1-CFP expressed instead of TGN38.

I. Same as (F) only LAMP1 instead of TGN38.

J. Quantification of P4M signal in LAMP1 regions (P4MLAMP1). Control: n=14; NPC1I1061T: n=16.

K. Heat map of PtdIns, PtdInsP and PtdInsP2 levels from 4 different murine brain regions. The colors from green to red indicate the relative contents of lipid isoforms in different brain regions from NPC1I1061T mice relative to wild-type brains; n=3 for both WT and NPC1I1061T animals.

Data information: Data are all expressed as mean ± SEM from individual cells. *P < 0.05; **P < 0.01; ****P < 0.0001 with unpaired t test.
NPC1 regulates the distribution of phosphatidylinositol 4-kinases at Golgi and lysosomal membranes (2021)
Figure 2

Fig 2. NPC1 regulates the contents of ER-TGN membrane contact sites.

A. Diagram of the molecular elements within ER-TGN membrane contact sites.

B Representative western blot for OSBP in control and U18-treated tsA201 cells. Protein levels were determined by densitometry with β-actin normalization. Each point (n=3) represents protein levels from U18-treated cells normalized to control bands.

C. Confocal images of control and U18-treated tsA201 cells fixed and stained for OSBP (left), TGN46 (middle), and merge (right). Black rectangles show expanded views of OSBP and TGN46 regions.

D. Quantification of OSBP signal in TGN46 regions (OSBPTGN46). Control: n=40; U18: n=39.

E. Confocal images of control and U18-treated hippocampal neurons fixed and stained for OSBP. Dashed black lines represent the outline of each neuron.

F. Quantification of OSBP in perinuclear region of control and U18-treated neurons. Control: n=10; U18: n=14.

G. Confocal images of control and U18-treated tsA201 cells fixed and stained for VAPA (left), TGN46 (middle), and merge (right). Black rectangles show expanded views of VAPA and TGN46 regions.

H. Quantification of VAPA signal in TGN46 regions (VAPATGN46). Control: n=44; U18: n=44.

I. Representative western blot of SAC1 in control and U18-treated tsA201 cells. Protein levels were normalized to β-actin, n=4.

J. Confocal images of control and U18-treated GFP11-SAC1 HEK293 cells (top) and control and NPC1I1061T patient fibroblasts immunolabeled with anti-SAC1 antibody (bottom). Insets show an enlarged view of SAC1 centered on a perinuclear region.

K. Quantification analyses of GFP11-SAC1 (top; Control: n=10; U18: n=10) and anti-SAC1 (bottom; Control: n=7; NPC1I1061T: n=8) mean fluorescence intensities in Golgi normalized to the cytoplasm.

Data information: Data are all expressed as mean ± SEM from individual cells and all statistical analyses were conducted using unpaired t test with *P < 0.05; **P < 0.01; ***P < 0.001; and **** P < 0.0001.
NPC1 regulates the distribution of phosphatidylinositol 4-kinases at Golgi and lysosomal membranes (2021)
Figure 3

Figure 3. PI4KIIα and PI4KIIIβ are recruited to Golgi and lysosome membranes in NPC1 disease.

A. Diagram showing PtdIns4P metabolizing enzymes.

B. Representative western blot for PI4KIIα and PI4KIIIβ in control and U18-treated tsA201 cells. Protein levels were normalized to β-actin. Individual points (PI4KIIα: n=3; PI4KIIIβ: n=4) represent protein levels of each +U18 band normalized to control.

C. Left: Confocal images of control (left) and U18-treated (right) tsA201 cells expressing PI4KIIα -GFP. Bottom panels show an enlarged view of the probe centered on a perinuclear region. Right: Quantification of perinuclear PI4KIIα intensity relative to the cytoplasm. Control: n=12; U18: n=14.

D. Same as (C) only PI4KIIIβ. Control: n=12; U18: n=15.

E. Confocal images of control and U18-treated tsA201 cells fixed and stained for PI4KIIα (left), TGN46 (middle), and merge (right). Black rectangles show expanded views of PI4KIIα and TGN46 regions.

F. Quantification of PI4KIIα signal in TGN46 regions (PI4KIIα TGN46). Control: n=36; U18: n=36.

G, H same as (E, F) only stained with PI4KIIIβ. Control: n=35; U18: n=43.

I, J same as (C, D) except using hippocampal neurons. (I) Control: n=7; U18: n=8. (J) Control: n=6; U18: n=6.

Data information: Data are all expressed as mean ± SEM from individual cells and all statistical analyses were conducted using unpaired t test with **P < 0.01; ***P < 0.001; ****P < 0.0001.
NPC1 regulates the distribution of phosphatidylinositol 4-kinases at Golgi and lysosomal membranes (2021)
Figure 4

Figure 4. NPC1 loss of function increases DHHC3-mediated PI4KIIα recruitment and PtdIns4P on Golgi and lysosome membranes.

A, Diagram illustrating the Golgi recruitment mechanisms of PI4KIIα.

B, Confocal images of wild-type and PI4KIIα-depleted (sgPI4K2A) HEK293-CAS9 cells expressing P4M-YFP, treated with or without U18. Bottom panels show enlarged views of PtdIns4P in a Golgi region.

C, Quantitative analyses of P4M-YFP mean fluorescence intensity distributed in Golgi normalized to the cytoplasm under different conditions. Control: n=19; U18: n=13, sgPI4K2A: n=19; sgPI4K2A+U18: n=17; sgDHHC3: n=11; sgDHHC3+U18: n=11. * represents a significant difference (P<0.05) between control and other non-U18 treated groups; # represents a significant difference (P<0.05) between U18-treated and other U18-treated sgRNA groups.

D. Confocal images of control (top) and NPC1I1061T (bottom) patient fibroblasts immunolabeled for DHHC3 and Giantin. Insets show enlarged views of DHHC3 and Giatin regions.

E. Analysis of anti-DHHC3 mean fluorescence intensity in a Golgi region relative to cytoplasm. Control: n=19; PC1I1061T: n=17.

F. Confocal images of control (1st column) and NPC1I1061T fibroblasts (2nd column) expressing mCherry-P4M or control fibroblasts expressing P4M-mCherry and GFP-DHHC3 (3rd column).

G. Quantification of Golgi mCherry-P4M. Control: n=11; NPC1I1061T: n=14; control+DHHC3: n=12.

H. Control or GFP-DHHC3 expressing tsA201 cells fixed and stained for PI4KIIα or PI4KIIIβ. Bottom panels show enlarged views of DHHC3, PI4KIIα, or PI4KIIIβ at Golgi regions.

I. Quantification of PI4KIIα (left; control: n=42, DHHC3: n=24), or PI4KIIIβ (right; control: n=39, DHHC3: n=26) intensity relative to cytoplasm.

J. Top: HEK293-CAS9 cells expressing P4M and either vehicle control, sgDHHC3, U18, and U18 with sgDHHC3. Bottom: expanded views of P4M in the black dashed rectangles..

K. Control (left) and NPC1I1061T (right) patient fibroblasts expressing P4M-YFP (top row) or P4M-YFP and DHHC3C157S (DN-DHHC3; bottom row). Inset panels show enlarged views of P4M-YFP distribution at Golgi.

L. Quantification of P4M-YFP signal in the Golgi region relative to cytoplasm. NPC1I1061T: n=20; NPC1I1061T+sgDHHC3: n=14.

M. Confocal images of control (top row), NPC1I1061T (middle row), and NPC1I1061T treated with sgDHHC3 (bottom row) patient cells expressing P4M, LAMP1 and CAS9. Insets are zoomed regions from LAMP1 positive areas.

N. Quantification of P4M changes at endomembranes. Control: n=149, U18: n=251, sgPI4KIIa +U18: n=131, sgDHHC3+U18: n= 271. Each n represents a single enodolysosome.

Data information: All the data are expressed as mean ± SEM from individual cells with the exception of (N) which represents values from individual subcellular structures. Statistical analyses include two-way ANVOA (C, N), one-way ANVOA (G), unpaired t-test (E, I), or Mann Whitney test (L). *P < 0.05; **P < 0.01; ****P < 0.0001.
NPC1 regulates the distribution of phosphatidylinositol 4-kinases at Golgi and lysosomal membranes (2021)
Figure 5

Figure 5. ACBD3-mediated PI4KIIIβ recruitment contributes to increased PtdIns4P levels at Golgi membranes following loss of NPC1 function.

A, Diagram illustrating the Golgi recruitment mechanisms of PI4KIIIβ.

B. Representative western blot for ACBD3 in control and NPC1I1061T patient fibroblasts. Protein levels were normalized to β-actin. Control: n=26; NPC1I1061T: n=27. A paired t test was used for the statistical analysis with *P < 0.05.

C. Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for ACBD3. Black and orange squares show expanded views of anti-ACBD3 in Golgi and endomembrane compartments, respectively.

D. Analyses of ACBD3 intensity distributed in Golgi (left graph; Control: n=9; NPC1I1061T: n=9) or in endomembranes (right graph; Control: n=1360; NPC1I1061T: n=1645 from 15 cells), normalized to the cytoplasm.

E. Confocal images of control (1st column) and NPC1I1061T fibroblasts (2nd column) expressing mCherry-P4M or control fibroblasts expressing P4M-mCherry and GFP-ACBD3 (3rd and 4th columns).

F. Quantification of Golgi mCherry-P4M intensity relative to cytoplasm. Control: n=11; NPC1I1061T: n=14; Control + ACBD3: n=12.

G. Confocal micrographs from control or GFP-ACBD3 expressing tsA201 cells fixed and stained for PI4KIIα or PI4KIIIβ. Bottom panels show enlarged views of ACBD3, PI4KIIα, or PI4KIIIβ at Golgi regions.

H. Quantification of PI4KIIα (left; control: n=42; ACBD3: n=28), or PI4KIIIβ (right; control: n=39; ACBD3: n=20) intensity relative to cytoplasm.

I. Confocal images of control and sgACBD3 HEK293-CAS9 cells expressing P4M-YFP, treated with or without U18. Bottom panels show enlarged views of PtdIns4P in Golgi and in endomembrane compartments.

J. Quantitative analyses of P4M-YFP mean fluorescence intensity at Golgi (left graph) or endomembranes (right graph) normalized to the cytoplasm. * represents a significant difference (P<0.05) between control and other non-U18 treated groups; # represents a significant difference (P<0.05) between U18-treated and other U18-treated sgRNA groups. For left graph; Control: n=19; U18: n=13; sgPI4K3B: n=12; sgPI4K3B+U18: n=11; sgPI4K2A + sgPI4K3B: n=12; sgPI4K2A + sgPI4K3B +U18: n= 12; sgACBD3: n=15; sgACBD3+U18: n=16. For right graph; control: n=149; U18: n=251; sgACBD3: n=138; sgACBD3+U18: n=190. Please note that 'control' and 'U18' datasets in Fig 5J are identical to those in Fig. 4C. Data for all CRISPR experiments were collected on the same days.

Data information: All the data are expressed as mean ± SEM from individual cells with the exception of (D, right) and (J, right) which represents values from individual subcellular structures, n=10 cells and n=13 cells, respectively. Statistical analyses include unpaired t-test (D left graph, H left graph), Mann Whitney test (D right graph, H right graph), or one-way ANOVA (F), *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. As determined by a Grubbs' test, with an alpha value of 0.05 (see methods), significant outliers were excluded from (J, right).
NPC1 regulates the distribution of phosphatidylinositol 4-kinases at Golgi and lysosomal membranes (2021)
Figure 6

Figure 6. PtdIns4P produced by PI4KIIIβ regulates mTORC1 recruitment to endomembrane compartments.

A. Confocal images of control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts expressing P4M-YFP. Black and orange squares show expanded views of PtdIns4P in Golgi and endo/lysosomal compartments respectively.

B. Top graph: analysis of Golgi P4M-YFP intensity normalized to the cytoplasm in control and NPC1I1061T patient fibroblasts and NPC1I1061T fibroblasts lacking either ACBD3 ('sgACBD3'), PI4KIIα ('sgPI4KIIα') or PI4KIIIβ ('sgPI4KIIIβ'). Control: n=9; NPC1I1061T: n=12; NPC1I1061T + sgACBD3: n=9; NPC1I1061T + sgPI4KIIα: n=5; NPC1I1061T + sgPI4KIIIβ: n=8. Bottom graph: analysis of P4M-YFP intensity in endomembrane compartments normalized to the cytoplasm in NPC1I1061T patient fibroblasts. NPC1I1061T: n=406; NPC1I1061T + sgACBD3: n=172.

C. Confocal images of control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts immunolabeled with mTORC1 antibody. Bottom images show expanded views of mTORC1 immunolabeling in the regions indicated by the dashed squares in the main images. White dashed lines represent the outline of each cell.

D. Quantification of mTORC1 mean integrated density normalized by cell area in control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts. Control: n=15; NPC1I1061T: n=14; NPC1I1061T+sgACBD3: n=11.

Data information: All the data are expressed as mean ± SEM from individual cells with the exception of (B, bottom) which represents values from individual subcellular structures. Statistical analyses include one-way ANVOA (B, top), Mann Whitney test (B, bottom), and one-way anova (D). *P < 0.05; **P < 0.01; ****P < 0.0001.
NPC1 regulates the distribution of phosphatidylinositol 4-kinases at Golgi and lysosomal membranes (2021)
Figure 7

Figure 7. SREBP inhibition alleviates NPC1-dependent increase of PI4KIIα, PI4KIIIB and PtdIns4P levels at Golgi membranes.

A. qPCR analysis from control (black bars), NPC1I1061T (pink), and NPC1I1061T patient cells treated overnight with the SREBP inhibitor, PF-429242 (1 μM; blue bars). Error bars represent SEM from 6 technical and 2 biological replicates.

B. Confocal images of tsA201 cells expressing PI4KIIα-GFP. Cells were treated with either vehicle (control), U18, or U18 and PF for 24h prior imaging. Bottom panels show an enlarged view of the enzyme in a perinuclear area.

C. Quantification of PI4KIIα-GFP mean fluorescence intensity in Golgi normalized to the cytoplasm. Control: n=12; U18: n=14; U18+PF: n=12.

D, E. Same as (B, C) but in tsA-201 cells transfected with PI4KIIIβ-GFP. Control: n=18, U18: n=22, U18+PF: n=23.

F, G. Same as (B, C) but in tsA-201 cells transfected with P4M-YFP. Control: n=39, U18: n=42, U18+PF: n=32.

H, I. Same as (F, G) but in wild-type and SCAP-/- CHO cells. Control: n=12, U18: n=11, SCAP-/- +U18: n=10.

Data information: All the data are expressed as mean ± SEM from individual cells and all statistical analyses were conducted using a two-way ANOVA (A) or one-way ANVOA (C, E, G, I). *P < 0.05; **P < 0.01 and ****P < 0.0001.
NPC1 regulates the distribution of phosphatidylinositol 4-kinases at Golgi and lysosomal membranes (2021)
Figure 8

Figure 8. SREBP inhibition alleviates vesicular trafficking defects in NPC1 disease.

A. Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for Giantin and CI-M6PR. Insets show expanded views of each immunolabeling in the Golgi region (upper insets) or centered on endomembranes (bottom inset, merged signals). Dashed grey and white lines represent the outline of each cell.

B. Analysis of the mean area of M6P and Giantin colocalizing puncta (top graph; Control: n=23; NPC1I1061T: n=18) and of their mean fluorescence intensity (bottom graph; Control: n=18; NPC1I1061T: n=18) in control and NPC1I1061T patient fibroblasts.

C. Confocal images of control and NPC1I1061T patient fibroblasts expressing ARF1-GFP. Insets show enlarged views of ARF1 in the Golgi area. Tubular structures were observed in NPC1I1061T patient fibroblasts as indicated by the black arrows.

D. Quantification of ΑRF-1 fluorescence intensity in Golgi normalized to the cytoplasm in control and NPC1I1061T patient fibroblasts. Control: n=10; NPC1I1061T: n=11.

E. Live confocal images of control and U18-treated cells transfected with Str-KDEL_TNF-SBP-mCherry. Images are taken at the indicated time points after biotin addition (at time 0 s) and the corresponding distribution of the TNF-SBP-mCherry within the cell is illustrated in the upper row.

F. Average time-course of mean TNF-SBP-mCherry fluorescence intensity in Golgi normalized to the initial fluorescence value. Shaded area represents the SEM. Control: n=11; U18: n=15.

G. Analysis of the time constants for TNF-SBP-mCherry to reach and to leave the Golgi (Tauarrive and Tauleave Golgi, respectively) and the full duration at half maximum (50:50 Duration), calculated from the upper traces. Error bars represent SEM. Control: n=10; U18: n=13-15.

H. Same as (E) except images are TIRF footprints of tsA201 cells treated either with vehicle (control) or with U18.

I. Averaged time-course of the mean plasma membrane fluorescence intensity normalized to the initial fluorescence value in tsA201 cells treated either with vehicle (control), with U18 alone, or together with PF. Shaded area represents the SEM, n=9.

J. Quantitative analysis of the time constants for TNF-SBP-mCherry to reach the plasma membrane (Tauarrive PM) and the maximal change in fluorescence intensity relative to the initial fluorescence value (Intensity Change TIRF F/F0). Error bars represent SEM. Control: n=6-9; U18: n=8-9; U18+PF: n=5-9.

Data information: All the data are expressed as mean ± SEM from individual cells and all statistical analyses were conducted using unpaired t test with *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
MYBL2 and ATM suppress replication stress in pluripotent stem cells (2021)
Figure 1

Figure 1. Mybl2Δ/Δ ESCs display fork instability and a replication stress phenotype leading to unreplicated DNA, Increase replication-associated DSB and genome instability

A) Distribution curve of replication fork rates for Mybl2+/+ and Mybl2Δ/Δ ESCs. Statistical analysis was performed using the Mann Whitney U test. n=6 experimental replicates. A minimum of 490 replication forks were counted.( **** p< 0.0001).

B) Left panel: representative elongating replication forks (stable and unstable). Quantification of the average IdU/CldU ratio in the Mybl2+/+ and Mybl2Δ/Δ ESCs. Percentage of highly unstable forks (ratio above 2) are indicated. Y-axis was cut to change scale and display large values. Statistical analysis was performed using the Mann Whitney U test. (**** p< 0.0001). At least 450 ratios were recorded from 6 experimental replicates.

C) The length of both CldU labels surrounding IdU tracks (newly fired origins) was measured before calculating a ratio representing symmetry around new origins. Quantification of the average positive ratio in the Mybl2+/+ and Mybl2Δ/Δ ESCs. Y-axis was cut to change scale and display large values. Statistical analysis was performed using the Mann Whitney U test. 55 and 75 ratios were recorded in the Mybl2+/+ and Mybl2Δ/Δ respectively from 3 replicates ( **** p< 0.0001).

D) Representative image showing the presence of UFB by positive Immunostaining for PICH. (Scale bar 10μm). Frequency of anaphases positive for UFBs relative to all anaphases in the Mybl2+/+ and Mybl2Δ/Δ ESCs. At least 75 anaphases were counted per experimental group from 3 independent experiments. Statistical analysis was performed using an unpaired two-tailed t-test (**** p< 0.0001).

E) Representative images for comets in Mybl2+/+ and Mybl2Δ/Δ untreated and Mybl2+/+ and Mybl2Δ/Δ plus CPT (20x magnification). (Scale bar 50μm).

F) Quantification of the mean and distributions of olive tail moments in Mybl2+/+ and Mybl2Δ/Δ untreated and CPT-treated. Y-axis was cut to change scale and display large values. Statistical analysis using Mann Whitney U tests. At least 300 comets were measured for each group from 5 experimental replicates (**** p< 0.0001).

G) Frequency of EdU positive cells with over 6 53BP1 foci in Mybl2+/+, Mybl2Δ/Δ untreated and Mybl2+/+ and Mybl2Δ/Δ plus CPT. Error bars represent SEM. Statistical analysis using unpaired two-tailed t-test. At least 200 EdU positive nuclei were analysed from 4 experimental repeats.
MYBL2 and ATM suppress replication stress in pluripotent stem cells (2021)
Figure 2

Figure 2. MYBL2-ablated ESCs fail to activate the DNA-damaged activated, G2/M checkpoint

A) Cells were treated for 1.5 hours with 5µM CPT, before sequential addition of IdU and CldU for 20 minutes each. Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with CPT (n=3 independent experiments; Error bars indicate SEM). Statistical analysis using two- tailed unpaired t-test.

(B) Distribution of replication rate in Mybl2+/+ and Mybl2Δ/Δ ESC treated or not with CPT as above. Statistical analysis was carried out using unpaired Mann Whitney U tests. A minimum of 200 forks were quantified for each of the genotypes and conditions shown from 3 independent repeats (*p < 0.05; **p< 0.01; ***p< 0.001; **** p< 0.0001).

C) ESCs were treated with or without CPT for 4 hours before 0.1ug/ml colcemid was added during the final 3.5 hours of treatment. Representative image of H3-pS10 positive cells (turquoise). (Scale bar 10μm). Frequency of H3-pS10 positive cells in Mybl2+/+ and Mybl2Δ/Δ ESCs. Data from three independent experiments. Error bars represent SEM. Statistical analysis was carried out using a two-tailed unpaired t-test (*p < 0.05; **p< 0.01; ns= no significant).

D) P-CDK1 (Tyr15), CDK1 and Beta-actin expression levels of Mybl2+/+ and Mybl2Δ/Δ ESCs with or without CPT treatment analyzed by immunoblotting. Bar graph represents average band density of P-CDK1 in Mybl2Δ/Δ ESCs relative to loading control and relative to the MYBL2+/+ from 6 repeats. Error bars represent SEM. Statistical analysis was carried out using a two-tailed unpaired t-test (**p< 0.01).
MYBL2 and ATM suppress replication stress in pluripotent stem cells (2021)
Figure 3

Figure 3. Proficient ATR dependent CHK1 activation in MYBL2- ablated ESC.

A) Immunoblot showing the levels of phosphorylated CHK1 (Ser345) and GAPDH in the Mybl2+/+ and Mybl2Δ/Δ ESCs with or without CPT treatment (2.5µM CPT for 4 hours).

B) Immunoblot showing the levels of P-CHK1 (Ser345) and CHK1 in irradiated Mybl2+/+, and Mybl2Δ/Δ ESCs with or without 3 hours inhibitor treatment: ATR (AZ20, 5µM) and ATM (Ku60019, 10µM). Cells were also exposed to 5Gy irradiation before the final hour of inhibitor treatment. Bar graph (lower panel) represents average band density of P-CHK1 in Mybl2Δ/Δ ESCs relative to CHK1 and relative to untreated cells. Mybl2+/+ from two independent repeats and Mybl2Δ/Δ ESCs from three independent repeats. Error bars represent SD. Statistical analysis was carried out using a two-tailed unpaired t-test (*p< 0.05).

C) Cells were treated for 1.5 hours with 5µM AZ20, before sequential addition of IdU and CldU for 20 minutes each. Dot plot representing the effect of ATR inhibition upon replication rate in Mybl2+/+ and Mybl2Δ/Δ ESC. Statistical analysis of distributions was carried out using unpaired Mann Whitney U tests. A minimum of 240 forks were quantified for each of the genotypes and conditions shown from 2 independent repeats (**** p< 0.0001)

D) Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with ATR inhibitor AZ20. At least 500 replication structures were counted per treatment from two independent repeats. Statistical analysis using two- tailed unpaired t-test.(*p< 0.05; ns= no significant).

E) Distribution and average of IdU/CldU fork ratio in Mybl2+/+ and Mybl2Δ/Δ ESCs with or without ATR inhibitor treatment. Percentage of highly unstable forks (ratio above 2) are indicated. Dotted line at 1 indicates positive ratio. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using the Mann Whitney U test. At least 150 ratios were calculated per treatment group from two independent repeats (*p < 0.05; **p< 0.01; ***p< 0.001; **** p< 0.0001).