Figure 3c ) Kaplan - Meier curves representing disease free ( DSF , top panel ) and overall survival ( OS , bottom panel ) probability for groups of patients P1 ( N = 7 ) , P2 ( N = 5 ) , P3 ( N = 4 ) , P4 ( N = 9 ) and P5 ( N = 15 ) obtained in a ) . Time is represented in years . Statistical significance among groups was determined by log - rank test .

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Figure 3c) Kaplan-Meier curves representing disease free (DSF, top panel) and overall survival (OS, bottom panel) probability for groups of patients P1 (N = 7), P2 (N = 5), P3 (N = 4), P4 (N = 9) and P5 (N = 15) obtained in a). Time is represented in years. Statistical significance among groups was determined by log-rank test.


Figure 5a ) Effect of Vincristine ( VCR ) treatment on cell viability of RPMI8402 cells transduced with sh β - catenin or sh - control . Dose - response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments . * * * p ≤ 0 . 001 of IC50 comparison with respect to sh control Data information : For all applicable figure panels , data represent mean ± SD of three independent experiments . Statistical significance was determined by two - sided Student ' s t test for a ( at day 0 ) Effect of VCR ( b ) treatment in the presence or absence of increasing doses of the β - catenin inhibitor ICG - 001 . Dose - response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , * * p ≤ 0 . 01 , * p ≤ 0 . 05 of IC50 comparison with respect VCR - treated cells . Data information : For all applicable figure panels , data represent mean ± SD of three independent experiments . Statistical significance was determined by two - sided Student ' s t test for , b , ( at day 0 ) Methotrexate ( MTX ) ( c left ) , L - Asparaginase ( L - ASP ) ( c central ) or Cytarabine ( Ara - C ) ( c right ) treatment in the presence or absence of increasing doses of the β - catenin inhibitor ICG - 001 . Dose - response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , * * p ≤ 0 . 01 , * p ≤ 0 . 05 of IC50 Data information : For all applicable figure panels , data represent mean ± SD of three independent experiments . Statistical significance was determined by two - sided Student ' s t test for c , ( at day 0 )

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Figure 5a) Effect of Vincristine (VCR) treatment on cell viability of RPMI8402 cells transduced with sh β-catenin or sh-control. Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. *** p ≤ 0.001 of IC50 comparison with respect to sh control Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for a (at day 0)Effect of VCR (b) treatment in the presence or absence of increasing doses of the β-catenin inhibitor ICG-001. Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 of IC50 comparison with respect VCR-treated cells. Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for , b, (at day 0)Methotrexate (MTX) (c left), L-Asparaginase (L-ASP) (c central) or Cytarabine (Ara-C) (c right) treatment in the presence or absence of increasing doses of the β-catenin inhibitor ICG-001. Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 of IC50 Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for c, (at day 0)


Figure 6c ) Percentage of RPMI8402 cells PB , BM , spleen and liver at sacrifice . RPMI8402 cells identified as human CD45 + HLA - ABC + CD5 + by flow cytometry and gated on DAPI negative cells . Control : animals left untreated ( n = 2 ) ; Chemo + DMSO : animals receiving chemotherapy and vehicle ( n = 4 ) ; Chemo + ICG - 001 : animals receiving chemotherapy and the β - catenin inhibitor ICG - 001 ( n = 4 ) Data information : For all applicable figure panels , data from Chemo + DMSO and Chemo + ICG - 001 groups are mean ± SD and dots represent individual values per animal . Control group data is only represented by individual values per animal . Statistical significance between Chemo + DMSO and Chemo + ICG - 001 groups was determined by two - sided Student ' s t test . g ) H & E staining of representative images per each mouse treated as indicated . h ) Percentage of liver area covered by cellular colonies per 10X micrograph . 4 different images were quantified per animal and mean values per animal are represented by dots . Control : animals left untreated ( n = 2 ) ; Chemo + DMSO : animals receiving chemotherapy and vehicle ( n = 4 ) ; Chemo + ICG - 001 : animals receiving chemotherapy and the β - catenin inhibitor ICG - 001 ( n = 4 ) Data information : For all applicable figure panels , data from Chemo + DMSO and Chemo + ICG - 001 groups are mean ± SD and dots represent individual values per animal . Control group data is only represented by individual values per animal . Statistical significance between Chemo + DMSO and Chemo + ICG - 001 groups was determined by two - sided Student ' s t test .

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Figure 6c) Percentage of RPMI8402 cells PB, BM, spleen and liver at sacrifice. RPMI8402 cells identified as human CD45+HLA-ABC+CD5+ by flow cytometry and gated on DAPI negative cells. Control: animals left untreated (n = 2); Chemo + DMSO: animals receiving chemotherapy and vehicle (n = 4); Chemo + ICG-001: animals receiving chemotherapy and the β-catenin inhibitor ICG-001 (n = 4) Data information: For all applicable figure panels, data from Chemo + DMSO and Chemo + ICG-001 groups are mean ± SD and dots represent individual values per animal. Control group data is only represented by individual values per animal. Statistical significance between Chemo + DMSO and Chemo + ICG-001 groups was determined by two-sided Student's t test.g) H&E staining of representative images per each mouse treated as indicated. h) Percentage of liver area covered by cellular colonies per 10X micrograph. 4 different images were quantified per animal and mean values per animal are represented by dots. Control: animals left untreated (n = 2); Chemo + DMSO: animals receiving chemotherapy and vehicle (n = 4); Chemo + ICG-001: animals receiving chemotherapy and the β-catenin inhibitor ICG-001 (n = 4) Data information: For all applicable figure panels, data from Chemo + DMSO and Chemo + ICG-001 groups are mean ± SD and dots represent individual values per animal. Control group data is only represented by individual values per animal. Statistical significance between Chemo + DMSO and Chemo + ICG-001 groups was determined by two-sided Student's t test.


Figure 1Representative micrographs of a time - lapse microscopy of K and KM non - regressed tumour cells ( H2B - GFP green ) . Top : Mitotic cell with a chromatin bridge ( yellow arrow ) . Bottom : Mitotic cell with misalignment and cytokinesis failure resulting in binucleation ( yellow arrow ) . Percentage of mitotic errors in K and KM primary tumours and in K and KM non - regressed tumours . Percentage of cells in K and KM primary tumours and in K and KM non - regressed tumours with the indicated mitotic errors . Scale bar 20 µm .

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Figure 1Representative micrographs of a time-lapse microscopy of K and KM non-regressed tumour cells (H2B-GFP green). Top: Mitotic cell with a chromatin bridge (yellow arrow). Bottom: Mitotic cell with misalignment and cytokinesis failure resulting in binucleation (yellow arrow).Percentage of mitotic errors in K and KM primary tumours and in K and KM non-regressed tumours. Percentage of cells in K and KM primary tumours and in K and KM non-regressed tumours with the indicated mitotic errors. Scale bar 20 µm.


Figure 2Somatic copy number alterations in each individual Kras and Kras / Mad2 non - regressed tumours . 7 K non - regressed and 16 KM non - regressed tumours were sequenced . Focal deletion ( DEL , green ) , Focal amplification ( AMP , light green ) , whole chromosome gain ( WCG , dark grey ) and loss ( WCL , light grey ) , partial chromosome gain ( PCG , dark blue ) and loss ( PCL , light blue ) and gross chromosomal rearrangement ( GCR , red ) . Overlaying of the amplifications in chromosome 6 across multiple regions showing that all amplicons contained the cMet oncogene . Colour of the block corresponds to the segment mean ( the degree to which a genome segment is lost ( blue ) or gained ( red ) . Immunostaining of phospho - cMet in non - regressed tumours carrying or not the amplification in chromosome 6 . Yellow numbers indicate the total number of cMet positive cells / total number of cells counted . Scale bar 100 µm .

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Figure 2Somatic copy number alterations in each individual Kras and Kras/Mad2 non-regressed tumours. 7 K non-regressed and 16 KM non-regressed tumours were sequenced. Focal deletion (DEL, green), Focal amplification (AMP, light green), whole chromosome gain (WCG, dark grey) and loss (WCL, light grey), partial chromosome gain (PCG, dark blue) and loss (PCL, light blue) and gross chromosomal rearrangement (GCR, red).Overlaying of the amplifications in chromosome 6 across multiple regions showing that all amplicons contained the cMet oncogene. Colour of the block corresponds to the segment mean (the degree to which a genome segment is lost (blue) or gained (red).Immunostaining of phospho-cMet in non-regressed tumours carrying or not the amplification in chromosome 6. Yellow numbers indicate the total number of cMet positive cells/total number of cells counted. Scale bar 100 µm.


Figure 3Tumour diameter before and after doxycycline withdrawal in 4 K and 5 KM breast tumours with cMet amplification . 0 indicates when doxycycline was removed . Each colour represents one tumour . Blue and green squares indicate the timeframe between doxycycline withdrawal and the moment in which tumours resumed growth . Genome - wide log2 - ratio plots of chromosome 6 of two primary tumour biopsies and their corresponding non - regressed tumour showing no amplification in the primary tumour ( upper panels ) and a small amplification in the non - regressed tumours ( bottom panels , yellow arrow ) . Immunostaining of phospho - cMet in 3 biopsied primary tumours ( PT ) and their corresponding non - regressed tumours ( KM5 , KM7 and KM8 , which are also shown in Fig 2C ) . Scale bar 100 µm . Representative two - dimensional scatter plots constructed with overlaid dPCR data of the reference ( VIC ) and cMet ( FAM ) from one tumour without cMet amplification and one with cMet amplified . Dots represent results of independent PCR reactions in the wells of a digital PCR chip . Reactions in the bottom left corner ( yellow ) are negative for both targets , while the ones in the top right corner ( green ) are double positives . Reactions in the top left ( blue ) and bottom right ( red ) corners are positive for cMet and the reference targets , respectively . Representative photographs of FISH staining with a probe for Met ( red signal ) and a probe for a reference gene EML4 ( green signals ) The upper panel is a negative example for cMet amplification containing 2 red and 2 green dots ( white arrows ) . The lower panel shows an example of a tumour with cMet amplification ( several red dots ) and 2 green dots ( yellow arrow ) . Scale bar 10 µm .

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Figure 3Tumour diameter before and after doxycycline withdrawal in 4 K and 5 KM breast tumours with cMet amplification. 0 indicates when doxycycline was removed. Each colour represents one tumour. Blue and green squares indicate the timeframe between doxycycline withdrawal and the moment in which tumours resumed growth.Genome-wide log2-ratio plots of chromosome 6 of two primary tumour biopsies and their corresponding non-regressed tumour showing no amplification in the primary tumour (upper panels) and a small amplification in the non-regressed tumours (bottom panels, yellow arrow).Immunostaining of phospho-cMet in 3 biopsied primary tumours (PT) and their corresponding non-regressed tumours (KM5, KM7 and KM8, which are also shown in Fig 2C). Scale bar 100 µm.Representative two-dimensional scatter plots constructed with overlaid dPCR data of the reference (VIC) and cMet (FAM) from one tumour without cMet amplification and one with cMet amplified. Dots represent results of independent PCR reactions in the wells of a digital PCR chip. Reactions in the bottom left corner (yellow) are negative for both targets, while the ones in the top right corner (green) are double positives. Reactions in the top left (blue) and bottom right (red) corners are positive for cMet and the reference targets, respectively.Representative photographs of FISH staining with a probe for Met (red signal) and a probe for a reference gene EML4 (green signals) The upper panel is a negative example for cMet amplification containing 2 red and 2 green dots (white arrows). The lower panel shows an example of a tumour with cMet amplification (several red dots) and 2 green dots (yellow arrow). Scale bar 10 µm.


Figure 4Relative volume of tumours grown in 12 Rag2 - / - animals after treatment with Tepotinib or vehicle control ( 3K and 3KM tumours for each condition ) . No statistical significance was found by One - way ANOVA . Mean + / - SEM .

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Figure 4Relative volume of tumours grown in 12 Rag2-/- animals after treatment with Tepotinib or vehicle control (3K and 3KM tumours for each condition). No statistical significance was found by One-way ANOVA. Mean +/- SEM.


Figure 5Relative volume of tumours grown in Rag2 - / - animals after treatment with Tepotinib or vehicle control . One - way ANOVA , Tukey ' s multiple comparison . Ns : not significant ; * * p < 0 . 024 . Mean + / - SEM . A total of 19 Rag2 - / - mice injected with cMet expressing tumours were treated with Tepotinib and 14 used as controls . For tumours with no cMet , 13 Rag2 - / - mice were treated with Tepotinib and 9 were used as control . Immunostaining of phospho - cMet in the same tumours grown in Rag2 - / - mice after treatment with Tepotinib or vehicle control . Scale bar 200 µm . Quantification of pH3 in tumours grown in Rag2 - / - animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control . Quantification of caspase 3 in tumours grown in Rag2 - / - animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control .

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Figure 5Relative volume of tumours grown in Rag2-/- animals after treatment with Tepotinib or vehicle control. One-way ANOVA, Tukey's multiple comparison. Ns: not significant; ** p<0.024. Mean +/- SEM. A total of 19 Rag2-/- mice injected with cMet expressing tumours were treated with Tepotinib and 14 used as controls. For tumours with no cMet, 13 Rag2-/- mice were treated with Tepotinib and 9 were used as control.Immunostaining of phospho-cMet in the same tumours grown in Rag2-/- mice after treatment with Tepotinib or vehicle control. Scale bar 200 µm.Quantification of pH3 in tumours grown in Rag2-/- animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control.Quantification of caspase 3 in tumours grown in Rag2-/- animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control.


Figure 1A , B Representative size - exclusion chromatography profiles of the MICU1 - MICU2 complex and chimaera in 2 mM EGTA ( A ) and 2 mM CaCl2 ( B ) conditions .

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Figure 1A, B Representative size-exclusion chromatography profiles of the MICU1-MICU2 complex and chimaera in 2 mM EGTA (A) and 2 mM CaCl2 (B) conditions.


Figure 4A The binding of fluorescently ( GFP ) labelled MICU1 and mutants to EMRE peptide shown by MST in the presence of EGTA . B The binding of fluorescently ( GFP ) labelled MICU1 and mutants to EMRE peptide were analysed with MST in the presence of Ca2 + . C Comparison of binding of MICU1 and EMRE peptide in both conditions . D MICU1 - MICU2 heterodimer interacts with the EMRE peptide at a 1 : 1 ratio . E Carbachol ( CCh ) - induced representative traces of mitochondrial calcium uptake in HEK cells treated with mt - EMRE and scramble peptides . Solid lines , mean ; shaded regions , ± SEM , n = 3 . F Resting mitochondrial Ca2 + levels in mt - EMRE and scramble peptides treated cells , evaluated via ratiometric imaging of the mitochondrial targeted mt - riG6m . Mean ± s . d . , student t - test , * * p < 0 . 01 , n = 3 . Data information : Error bars for some points are too small to be visible . G Mean mitochondrial Ca2 + peaks response to 100 μM CCh . Mean ± s . d . , student t - test , * * * p ≤ 0 . 001 , n = 3 . Data information : Error bars for some points are too small to be visible . H Representative [ Ca2 + ] c clearance traces of U2OS cells treated with PBS and mt - EMRE peptide . I Quantification of mitochondria Ca2 + uptake rate in response to 100 μM histamine . Mean ± s . d . , student t - test , n . s . , not significant , n = 4 . Data information : Error bars for some points are too small to be visible .

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Figure 4A The binding of fluorescently (GFP) labelled MICU1 and mutants to EMRE peptide shown by MST in the presence of EGTA. B The binding of fluorescently (GFP) labelled MICU1 and mutants to EMRE peptide were analysed with MST in the presence of Ca2+. C Comparison of binding of MICU1 and EMRE peptide in both conditions.D MICU1-MICU2 heterodimer interacts with the EMRE peptide at a 1:1 ratio.E Carbachol (CCh)-induced representative traces of mitochondrial calcium uptake in HEK cells treated with mt-EMRE and scramble peptides. Solid lines, mean; shaded regions, ± SEM, n = 3.F Resting mitochondrial Ca2+ levels in mt-EMRE and scramble peptides treated cells, evaluated via ratiometric imaging of the mitochondrial targeted mt-riG6m. Mean ± s.d., student t-test, **p < 0.01, n = 3. Data information: Error bars for some points are too small to be visible.G Mean mitochondrial Ca2+ peaks response to 100 μM CCh. Mean ± s.d., student t-test, ***p ≤ 0.001, n = 3. Data information: Error bars for some points are too small to be visible.H Representative [Ca2+]c clearance traces of U2OS cells treated with PBS and mt-EMRE peptide.I Quantification of mitochondria Ca2+ uptake rate in response to 100 μM histamine. Mean ± s.d., student t-test, n.s., not significant, n = 4. Data information: Error bars for some points are too small to be visible.


Figure 15C or mES medium was used during reprogramming . On day 15 , Oct4GFP + colonies were counted ( A ) . 5C or mES medium was used during reprogramming . The percentages of Oct4GFP + cells were also determined with FACS ( B ) . ( C ) The expression of several core pluripotency markers was determined with qPCR at different time points during reprogramming and normalized against those in MEFs . ( D - E ) Energy metabolism was analyzed on day 6 during reprogramming with the Seahorse instrument . Increase in extracellular acidification rate ( ECAR ) after adding glucose was considered as glycolysis ability of the cells ( D ) , while the decrease in oxygen consumption rate ( OCR ) was considered as the ATP production ability of the cells ( E ) . ( F - G ) Expression of Hif1α was modulated with overexpression or sh - RNA - mediated knockdown via a retrovirus system during reprogramming ( F ) . The numbers of Oct4GFP + colonies were determined on day 15 ( G ) . ( H - I ) Expression of Pdk1 / 2 was modulated with a retrovirus system , and oligomycin ( 1 µM ) and 2 - DG ( 5 mM ) were used during reprogramming ( H ) . The numbers of Oct4GFP + colonies were determined on day 15 ( I ) .

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Figure 15C or mES medium was used during reprogramming. On day 15, Oct4GFP+ colonies were counted (A).5C or mES medium was used during reprogramming. The percentages of Oct4GFP+ cells were also determined with FACS (B).(C) The expression of several core pluripotency markers was determined with qPCR at different time points during reprogramming and normalized against those in MEFs.(D-E) Energy metabolism was analyzed on day 6 during reprogramming with the Seahorse instrument. Increase in extracellular acidification rate (ECAR) after adding glucose was considered as glycolysis ability of the cells (D), while the decrease in oxygen consumption rate (OCR) was considered as the ATP production ability of the cells (E).(F-G) Expression of Hif1α was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming (F). The numbers of Oct4GFP+ colonies were determined on day 15 (G).(H-I) Expression of Pdk1/2 was modulated with a retrovirus system, and oligomycin (1 µM) and 2-DG (5 mM) were used during reprogramming (H). The numbers of Oct4GFP+ colonies were determined on day 15 (I).


Figure 2 ( A - B ) 5C and mES medium were used during reprogramming . Representative alkaline phosphatase ( AP ) staining on day 15 was provided in ( A ) . AP + Oct4GFP - and AP + Oct4GFP + colonies were counted on day 15 ( B ) . ( C ) Thirty - two epigenetic factors were selected because they had higher expression in 5C - Oct4GFP - than in mES - Oct4GFP - . Their expression in ESCs , iPSCs , and pre - iPSCs in two previous reported assay ( GSE14012 and GSE10871 ) was also listed . ( D ) The expression of Bmi1 , Ctcf , Ezh2 , Kdm2b , or Wdr5 was modulated with overexpression or sh - RNA - mediated knockdown via a retrovirus system during reprogramming . AP + Oct4GFP - and AP + Oct4GFP + colonies were counted on day 15 . During reprogramming with mES medium , Bmi1 , Ctcf , Ezh2 , Kdm2b , and Wdr5 were overexpressed simultaneously with the four Yamanaka factors ( OKMS + 5F ) or Oct4 ( O + 5F ) . All factors were delivered simultaneously via a retrovirus system on day 0 and 1 . Reprogramming with only Yamanaka factors ( OKMS ) or Oct4 ( O ) served as control . The expression of pluripotency markers was determined with qPCR on day 6 during reprogramming ( E ) . During reprogramming with mES medium , Bmi1 , Ctcf , Ezh2 , Kdm2b , and Wdr5 were overexpressed simultaneously with the four Yamanaka factors ( OKMS + 5F ) or Oct4 ( O + 5F ) . All factors were delivered simultaneously via a retrovirus system on day 0 and 1 . Reprogramming with only Yamanaka factors ( OKMS ) or Oct4 ( O ) served as control . The numbers of Oct4GFP + colonies were determined on day 15 ( F ) . During reprogramming with mES medium , Bmi1 , Ctcf , Ezh2 , Kdm2b , and Wdr5 were overexpressed simultaneously with the four Yamanaka factors ( OKMS + 5F ) or Oct4 ( O + 5F ) . All factors were delivered simultaneously via a retrovirus system on day 0 and 1 . Reprogramming with only Yamanaka factors ( OKMS ) or Oct4 ( O ) served as control . The histone methylation on core pluripotency loci was determined on day 6 with ChIP - qPCR ( G ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A-B) 5C and mES medium were used during reprogramming. Representative alkaline phosphatase (AP) staining on day 15 was provided in (A). AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15 (B).(C) Thirty-two epigenetic factors were selected because they had higher expression in 5C-Oct4GFP- than in mES-Oct4GFP-. Their expression in ESCs, iPSCs, and pre-iPSCs in two previous reported assay (GSE14012 and GSE10871) was also listed.(D) The expression of Bmi1, Ctcf, Ezh2, Kdm2b, or Wdr5 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming. AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15.During reprogramming with mES medium, Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 were overexpressed simultaneously with the four Yamanaka factors (OKMS+5F) or Oct4 (O+5F). All factors were delivered simultaneously via a retrovirus system on day 0 and 1. Reprogramming with only Yamanaka factors (OKMS) or Oct4 (O) served as control. The expression of pluripotency markers was determined with qPCR on day 6 during reprogramming (E).During reprogramming with mES medium, Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 were overexpressed simultaneously with the four Yamanaka factors (OKMS+5F) or Oct4 (O+5F). All factors were delivered simultaneously via a retrovirus system on day 0 and 1. Reprogramming with only Yamanaka factors (OKMS) or Oct4 (O) served as control. The numbers of Oct4GFP+ colonies were determined on day 15 (F).During reprogramming with mES medium, Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 were overexpressed simultaneously with the four Yamanaka factors (OKMS+5F) or Oct4 (O+5F). All factors were delivered simultaneously via a retrovirus system on day 0 and 1. Reprogramming with only Yamanaka factors (OKMS) or Oct4 (O) served as control. The histone methylation on core pluripotency loci was determined on day 6 with ChIP-qPCR (G).


Figure 3 ( A ) The enrichment of HIF1α , SNAI2 , TWIST1 / 2 , and ZEB1 binding sites on the promoters of Bmi1 , Ctcf , Ezh2 , Kdm2b , and Wdr5 was determined by Pscan Zambelli et al , 2009 ( ) . The resulted Z - scores were listed . " Max . M " suggested the maximum Z - score generated with SNAI2 , TWIST1 / 2 , and ZEB1 binding sites on one indicated promoter . " Average " suggested the average Z - score of all transcriptional factors tested in the Pscan software on one indicated promoter . The expression of H1f1α , Snai2 , Twist1 / 2 , and Zeb1 was modulated with overexpression or sh - RNA - mediated knockdown via a retrovirus system during reprogramming with mES ( B ) The expression of Bmi1 , Ctcf , Ezh2 , Kdm2b , Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs . The comparisons were performed between all groups and corresponding Flag groups . The expression of H1f1α , Snai2 , Twist1 / 2 , and Zeb1 was modulated with overexpression or sh - RNA - mediated knockdown via a retrovirus system during reprogramming with 5C medium ( C ) . The expression of Bmi1 , Ctcf , Ezh2 , Kdm2b , Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs . The comparisons were performed between all groups and corresponding Flag groups . pre - iPSCs were isolated during reprogramming with mES - Vc , mES or 5C medium . These pre - iPSCs were further cultured with a different medium ( mES - Vc and mES medium ) for seven days . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . The numbers of Oct4GFP + colonies were determined on day 7 ( E ) . Reprogramming was also performed with mES - Vc or mES medium . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . AP + Oct4GFP - and AP + Oct4GFP + colonies were counted on day 15 ( G ) . Reprogramming was also performed with mES - Vc or mES medium . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . In addition , energy metabolism was determined on day 6 with Seahorse instrumentReprogramming was also performed with mES - Vc or mES medium . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . In addition , energy metabolism was determined on day 6 with Seahorse instrumentReprogramming was also performed with mES - Vc or mES medium . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . Cell migration was determined by measuring the migration rate with live - cell imaging on day 6 ( J ) . Reprogramming was also performed with mES - Vc or mES medium . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . The expression of Bmi1 , Ctcf , Ezh2 , Kdm2b , or Wdr5 was determined on day 6 with qPCR ( K ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) The enrichment of HIF1α, SNAI2, TWIST1/2, and ZEB1 binding sites on the promoters of Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 was determined by Pscan Zambelli et al, 2009(). The resulted Z-scores were listed. "Max. M" suggested the maximum Z-score generated with SNAI2, TWIST1/2, and ZEB1 binding sites on one indicated promoter. "Average" suggested the average Z-score of all transcriptional factors tested in the Pscan software on one indicated promoter.The expression of H1f1α, Snai2, Twist1/2, and Zeb1 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming with mES (B) The expression of Bmi1, Ctcf, Ezh2, Kdm2b, Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs. The comparisons were performed between all groups and corresponding Flag groups.The expression of H1f1α, Snai2, Twist1/2, and Zeb1 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming with 5C medium (C). The expression of Bmi1, Ctcf, Ezh2, Kdm2b, Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs. The comparisons were performed between all groups and corresponding Flag groups.pre-iPSCs were isolated during reprogramming with mES-Vc, mES or 5C medium. These pre-iPSCs were further cultured with a different medium (mES-Vc and mES medium) for seven days. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. The numbers of Oct4GFP+ colonies were determined on day 7 (E).Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15 (G).Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. In addition, energy metabolism was determined on day 6 with Seahorse instrumentReprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. In addition, energy metabolism was determined on day 6 with Seahorse instrumentReprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. Cell migration was determined by measuring the migration rate with live-cell imaging on day 6 (J).Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. The expression of Bmi1, Ctcf, Ezh2, Kdm2b, or Wdr5 was determined on day 6 with qPCR (K).


Figure 4Expression of Hif1α was modulated via a retrovirus system or small - molecule compounds , oligomycin ( 1 µM ) and 2 - DG ( 5 mM ) , during reprogramming . The expression of mesenchymal markers ( A ) Expression of Hif1α was modulated via a retrovirus system or small - molecule compounds , oligomycin ( 1 µM ) and 2 - DG ( 5 mM ) , during reprogramming . The expression of mesenchymal markers cell migration ( B ) was determined with qPCR and live - cell imaging , respectively , on day 6 . Repsox ( 1 μM ) was used on day 2 - 7 to inhibit early EMT during reprogramming with 5C medium . Except for OK + M + S group , the four Yamanaka factors were delivered simultaneously . Energy metabolism was determined on day 6 with Seahorse instrument ( E - F ) . Repsox ( 1 μM ) was used on day 2 - 7 to inhibit early EMT during reprogramming with 5C medium . The expression of glycolysis markers was determined on day 6 with qPCR ( G ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Expression of Hif1α was modulated via a retrovirus system or small-molecule compounds, oligomycin (1 µM) and 2-DG (5 mM), during reprogramming. The expression of mesenchymal markers (A)Expression of Hif1α was modulated via a retrovirus system or small-molecule compounds, oligomycin (1 µM) and 2-DG (5 mM), during reprogramming. The expression of mesenchymal markers cell migration (B) was determined with qPCR and live-cell imaging, respectively, on day 6.Repsox (1 μM) was used on day 2-7 to inhibit early EMT during reprogramming with 5C medium. Except for OK+M+S group, the four Yamanaka factors were delivered simultaneously. Energy metabolism was determined on day 6 with Seahorse instrument (E-F).Repsox (1 μM) was used on day 2-7 to inhibit early EMT during reprogramming with 5C medium. The expression of glycolysis markers was determined on day 6 with qPCR (G).


Figure 5On day 15 during reprogramming , the expression of E - cadherin ( E - CAD ) and N - cadherin ( N - CAD ) , was determined with immunofluorescence together with the morphology in Oct4GFP + colonies ( A , Scale bar , 25 μM ) . After sorting Oct4GFP + cells out ( 5C - Oct4GFP + and mES - Oct4GFP + cells ) , the expression of markers of different pluripotent states ( B ) After sorting Oct4GFP + cells out ( 5C - Oct4GFP + and mES - Oct4GFP + cells ) , the expression of markers of Xist ( C ) After sorting Oct4GFP + cells out ( 5C - Oct4GFP + and mES - Oct4GFP + cells ) their abilities to form chimeras ( D ) were determined . ( F ) The isolated 5C - Oct4GFP + cells were cultured in naïve or primed medium for three days , and the morphology changed towards R1 ESCs ( naïve PSCs ) and R1 ESCs - primed ( primed PSCs ) , respectively . The obtained cells were maintained for additional 20 passages . The 5C - Oct4GFP + cells cultured in primed medium lost GFP fluorescence . R1 ESCs - primed were primed PSCs generated from R1 ESCs . Scale bar , 100 μM . The isolated 5C - Oct4GFP + cells were cultured in naïve or primed medium for three days , and the morphology changed towards R1 ESCs ( naïve PSCs ) and R1 ESCs - primed ( primed PSCs ) , respectively . ( G ) PSCs mentioned in ( F ) were used for RNA - Seq analysis . Similarity among gene expression profiles was determined . The isolated 5C - Oct4GFP + cells were cultured in naïve or primed medium for three days to generate 5C - Oct4GFP + ( naïve ) and 5C - Oct4GFP + ( primed ) cells . The expression of Xist ( H ) The isolated 5C - Oct4GFP + cells were cultured in naïve or primed medium for three days to generate 5C - Oct4GFP + ( naïve ) and 5C - Oct4GFP + ( primed ) cells . the abilities to form chimeras ( I - J ) were determined .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5On day 15 during reprogramming, the expression of E-cadherin (E-CAD) and N-cadherin (N-CAD), was determined with immunofluorescence together with the morphology in Oct4GFP+ colonies (A, Scale bar, 25 μM).After sorting Oct4GFP+ cells out (5C-Oct4GFP+ and mES-Oct4GFP+ cells), the expression of markers of different pluripotent states (B)After sorting Oct4GFP+ cells out (5C-Oct4GFP+ and mES-Oct4GFP+ cells), the expression of markers of Xist (C)After sorting Oct4GFP+ cells out (5C-Oct4GFP+ and mES-Oct4GFP+ cells) their abilities to form chimeras (D) were determined.(F) The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days, and the morphology changed towards R1 ESCs (naïve PSCs) and R1 ESCs-primed (primed PSCs), respectively. The obtained cells were maintained for additional 20 passages. The 5C-Oct4GFP+ cells cultured in primed medium lost GFP fluorescence. R1 ESCs-primed were primed PSCs generated from R1 ESCs. Scale bar, 100 μM.The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days, and the morphology changed towards R1 ESCs (naïve PSCs) and R1 ESCs-primed (primed PSCs), respectively. (G) PSCs mentioned in (F) were used for RNA-Seq analysis. Similarity among gene expression profiles was determined.The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days to generate 5C-Oct4GFP+ (naïve) and 5C-Oct4GFP+ (primed) cells. The expression of Xist (H)The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days to generate 5C-Oct4GFP+ (naïve) and 5C-Oct4GFP+ (primed) cells. the abilities to form chimeras (I-J) were determined.


Figure 6The expression Oct4GFP and E - cadherin was determined with FACS on day 15 during reprogramming ( A ) . Based on the FACS results , the expression of Oct4GFP and E - cadherin was summarized in Oct4GFP + E - cadherin + cells ( B ) and in Oct4GFP - E - cadherin - cells ( C ) . The percentage of E - cadherin + cells in Oct4GFP + cells was listed in ( D ) , while the percentage of Oct4GFP + cells in E - cadherin + cells was listed in ( E ) . ( F - G ) Energy metabolism was analyzed in 5C - Oct4GFP + and mES - Oct4GFP + cells with Seahorse instrument . The numbers of three types of Oct4GFP + colonies ( J ) the expression of four epithelial markers in Oct4GFP + cells ( K ) were determined on day 15 . " Average " in ( K ) suggested the average expression of four epithelia markers .

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Figure 6The expression Oct4GFP and E-cadherin was determined with FACS on day 15 during reprogramming (A).Based on the FACS results, the expression of Oct4GFP and E-cadherin was summarized in Oct4GFP+E-cadherin+ cells (B) and in Oct4GFP-E-cadherin- cells (C).The percentage of E-cadherin+ cells in Oct4GFP+ cells was listed in (D), while the percentage of Oct4GFP+ cells in E-cadherin+ cells was listed in (E).(F-G) Energy metabolism was analyzed in 5C-Oct4GFP+ and mES-Oct4GFP+ cells with Seahorse instrument.The numbers of three types of Oct4GFP+ colonies (J)the expression of four epithelial markers in Oct4GFP+ cells (K) were determined on day 15. "Average" in (K) suggested the average expression of four epithelia markers.


Figure 7 ( A - B ) Energy metabolism was analyzed with 5C - Oct4GFP + , R1 ESCs ( naïve PSCs ) , and R1 ESCs - primed ( primed PSCs ) with Seahorse instrument . ( C ) Expression of epithelial , mesenchymal , glycolysis and oxidative phosphorylation ( OXPHOS ) markers ( listed in Dataset EV4 ) was summarized in R1 ESCs , 5C - Oct4GFP + ( naïve ) , 5C - Oct4GFP + , 5C - Oct4GFP + ( primed ) , and R1 ESCs - primed , based on the current RNA - Seq . For each marker , Log2 values of FKPM were normalized by subtracting the average of five groups . Log2 values of FKPM of each group of markers were then averaged and plotted . 5C - Oct4GFP + ( naïve ) and 5C - Oct4GFP + ( primed ) were 5C - Oct4GFP + cultured in naïve and primed medium , respectively , for three days . ( D ) Genes with higher expression at different developmental stages were selected out from previously reported RNA - seq ( Data ref : Bertone , 2015 ) . " Mor " , " ICM " , " pre - EPI " and " post - EPI " represented genes that have high expression only in morula , inner cell mass , pre - implantation epiblast and post - implantation epiblast , respectively . " Mor + ICM " , " ICM + pre " , and " EPI " indicated genes that have high expression in both morula and inner cell mass , in both inner cell mass and pre - implantation epiblast , and in both pre - implantation and post - implantation epiblast , respectively . The results of previously reported RNA - seq were summarized in the left panel , while the expression of these genes in current RNA - seq was summarized in the right panel . Log2 values of FKPM were presented similarly as in ( C ) . ( F ) Markers of 5C , naïve and primed states ( Dataset EV4 ) were used to analyze expression profiles of epiblast during the early development of mouse embryos ( Data ref : Mohammed et al , 2017a ) ( F ) . Single - cell was plotted based on the average expression of each group of markers . ( G ) The average expression of both naïve and primed markers was calculated based on GSE100597 . The top - 2 cells from E4 . 5 epiblast and top - 5 cells from E5 . 5 epiblast were selected . The expression of several naïve markers ( Dppa3 , Esrrb , Klf4 , Klf5 , Tbx3 , and Zfp42 ) and several mesenchymal markers ( Fgf5 , Lef1 , Nodal , and Pou3f1 ) in these selected cells were listed ( upper part ) . The average expression of these genes in E3 . 5 , 4 . 5 , 5 . 5 and 6 . 5 epiblast were also listed ( lower part ) . These results were all normalized against the average expression of these genes in all tested cells . ( H ) The expression of representative markers of 5C , naïve and primed states was summarized in E3 . 5 , 4 . 5 , 5 . 5 and 6 . 5 epiblast . Cells from E5 . 5 epiblast were separated into 8 groups ( I - VIII ) basing on the average expression of epithelial , mesenchymal , and glycolysis markers ( H ) . The average expression of three groups of markers were summarized in ( J ) . The expression of six important markers of 5C state was summarized in these eight groups of cells ( K )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O

Figure 7(A-B) Energy metabolism was analyzed with 5C-Oct4GFP+, R1 ESCs (naïve PSCs), and R1 ESCs-primed (primed PSCs) with Seahorse instrument.(C) Expression of epithelial, mesenchymal, glycolysis and oxidative phosphorylation (OXPHOS) markers (listed in Dataset EV4) was summarized in R1 ESCs, 5C-Oct4GFP+ (naïve), 5C-Oct4GFP+, 5C-Oct4GFP+ (primed), and R1 ESCs-primed, based on the current RNA-Seq. For each marker, Log2 values of FKPM were normalized by subtracting the average of five groups. Log2 values of FKPM of each group of markers were then averaged and plotted. 5C-Oct4GFP+ (naïve) and 5C-Oct4GFP+ (primed) were 5C-Oct4GFP+ cultured in naïve and primed medium, respectively, for three days.(D) Genes with higher expression at different developmental stages were selected out from previously reported RNA-seq (Data ref: Bertone, 2015). "Mor", "ICM", "pre-EPI" and "post-EPI" represented genes that have high expression only in morula, inner cell mass, pre-implantation epiblast and post-implantation epiblast, respectively. "Mor+ICM", "ICM+pre", and "EPI" indicated genes that have high expression in both morula and inner cell mass, in both inner cell mass and pre-implantation epiblast, and in both pre-implantation and post-implantation epiblast, respectively. The results of previously reported RNA-seq were summarized in the left panel, while the expression of these genes in current RNA-seq was summarized in the right panel. Log2 values of FKPM were presented similarly as in (C).(F) Markers of 5C, naïve and primed states (Dataset EV4) were used to analyze expression profiles of epiblast during the early development of mouse embryos (Data ref: Mohammed et al, 2017a) (F). Single-cell was plotted based on the average expression of each group of markers.(G) The average expression of both naïve and primed markers was calculated based on GSE100597. The top-2 cells from E4.5 epiblast and top-5 cells from E5.5 epiblast were selected. The expression of several naïve markers (Dppa3, Esrrb, Klf4, Klf5, Tbx3, and Zfp42) and several mesenchymal markers (Fgf5, Lef1, Nodal, and Pou3f1) in these selected cells were listed (upper part). The average expression of these genes in E3.5, 4.5, 5.5 and 6.5 epiblast were also listed (lower part). These results were all normalized against the average expression of these genes in all tested cells.(H) The expression of representative markers of 5C, naïve and primed states was summarized in E3.5, 4.5, 5.5 and 6.5 epiblast.Cells from E5.5 epiblast were separated into 8 groups (I-VIII) basing on the average expression of epithelial, mesenchymal, and glycolysis markers (H). The average expression of three groups of markers were summarized in (J).The expression of six important markers of 5C state was summarized in these eight groups of cells (K)


Figure 1 ( B ) Samples from ( A ) were separated on a native agarose gel and visualized by autoradiography Aberrant Replication Products ( ARPs ) are also indicated , as are the different species of catenated dimers : nicked - nicked catenanes , ' n - nCats ' ; nicked - supercoiled catenanes , ' n - scCats ' ; supercoiled - supercoiled catenanes ' sc - scCats ' . ( C ) Quantification of total DNA synthesis from ( B ) lanes 1 - 15 . Mean ± SD , n = 3 independent experiments . Total lane signal ( AU ) was normalized to the maximum recorded signal within each individual experiment . ( D ) Quantification of total DNA synthesis from ( B ) lanes 1 - 5 , 16 - 25 . Mean ± SD , n = 3 independent experiments . Total lane signal ( AU ) was normalized to the maximum recorded signal within each individual experiment . ( E ) Quantification of supercoiled circular monomers ( ' scCMs ' ) from ( B ) lanes 1 - 15 . Mean ± SD , n = 3 independent experiments . ( F ) Quantification of scCMs from ( B ) lanes 1 - 5 , 16 - 25 . Mean ± SD , n = 3 independent experiments . ( G ) Quantification of linears ( ' Lins ' ) from ( B ) lanes 1 - 15 . Mean ± SD , n = 3 independent experiments . ( H ) Quantification of Lins from ( B ) lanes 1 - 5 , 16 - 25 . Mean ± SD , n = 3 independent experiments . Data information : For ( E ) - ( H ) , abundance of the indicated species was calculated as a percentage of total lane signal ( % of total ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Samples from (A) were separated on a native agarose gel and visualized by autoradiography Aberrant Replication Products (ARPs) are also indicated, as are the different species of catenated dimers: nicked-nicked catenanes, 'n-nCats'; nicked-supercoiled catenanes, 'n-scCats'; supercoiled-supercoiled catenanes 'sc-scCats'. (C) Quantification of total DNA synthesis from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. Total lane signal (AU) was normalized to the maximum recorded signal within each individual experiment. (D) Quantification of total DNA synthesis from (B) lanes 1-5, 16-25. Mean ± SD, n=3 independent experiments. Total lane signal (AU) was normalized to the maximum recorded signal within each individual experiment. (E) Quantification of supercoiled circular monomers ('scCMs') from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. (F) Quantification of scCMs from (B) lanes 1-5, 16-25. Mean ± SD, n=3 independent experiments. (G) Quantification of linears ('Lins') from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. (H) Quantification of Lins from (B) lanes 1-5, 16-25. Mean ± SD, n=3 independent experiments. Data information: For (E)-(H), abundance of the indicated species was calculated as a percentage of total lane signal (% of total).


Figure 2 ( A ) Plasmid DNA was replicated in the presence of [ α - 32P ] dATP and vehicle control , etoposide , or doxorubicin . Purified DNA intermediates were digested with XmnI , which cuts the plasmid template once , to allow for identification of DNA breaks . ( B ) Samples from ( A ) were separated on a native agarose gel and visualized by autoradiography . ( C ) Quantification of DNA breaks from ( B ) . Mean ± SD , n = 3 independent experiments . Abundance of breaks was calculated as a percentage of total lane signal ( % of total ) . ( D ) Quantification of linears ( ' Lins ' ) from ( B ) . Mean ± SD , n = 3 independent experiments . Abundance of linears ( AU ) was normalized to the maximum recorded signal within each individual experiment . ( F ) Samples from ( E ) were separated on native agarose gels and visualized by autoradiography . ( G ) Quantification of DPC - containing molecules from ( F ) . Mean ± SD , n = 3 independent experiments . Abundance of the DPC - containing molecules was calculated as a percentage of total lane signal ( % of total ) . ( H ) Quantification of the amount of supercoiled circular monomers ( ' scCMs ' ) from ' + ProK ' samples from ( F ) . Mean ± SD , n = 3 independent experiments . Abundance of supercoiled monomers ( AU ) was normalized to the maximum recorded signal within each individual experiment .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Plasmid DNA was replicated in the presence of [α-32P]dATP and vehicle control, etoposide, or doxorubicin. Purified DNA intermediates were digested with XmnI, which cuts the plasmid template once, to allow for identification of DNA breaks. (B) Samples from (A) were separated on a native agarose gel and visualized by autoradiography. (C) Quantification of DNA breaks from (B). Mean ± SD, n=3 independent experiments. Abundance of breaks was calculated as a percentage of total lane signal (% of total). (D) Quantification of linears ('Lins') from (B). Mean ± SD, n=3 independent experiments. Abundance of linears (AU) was normalized to the maximum recorded signal within each individual experiment.(F) Samples from (E) were separated on native agarose gels and visualized by autoradiography. (G) Quantification of DPC-containing molecules from (F). Mean ± SD, n=3 independent experiments. Abundance of the DPC-containing molecules was calculated as a percentage of total lane signal (% of total). (H) Quantification of the amount of supercoiled circular monomers ('scCMs') from '+ProK' samples from (F). Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers (AU) was normalized to the maximum recorded signal within each individual experiment.


Figure 3 ( A ) Plasmid DNA was replicated in Mock - or TOP2α - immunodepleted extracts in the presence or absence of vehicle control , etoposide , or doxorubicin . In mock - depleted extracts ( ΔMock ) , the final replication product are supercoiled circular monomers ( ' scCMs ' ) . In TOP2α - depleted extracts ( ΔTOP2α ) , the final replication product are catenanes ( ' Cats ' ) because TOP2α is required for DNA unlinking . ( B ) Samples from ( A ) were separated on a native agarose gel and visualized by autoradiography . Linear molecules , arising from DNA breaks , are indicated with a red arrow migrate at a similar molecular weight to a subset of catenanes . ( C ) Quantification of scCMs from ( B ) lanes 1 - 15 . Mean ± SD , n = 3 independent experiments . Abundance of supercoiled monomers was calculated as a percentage of total lane signal ( % of total ) . ( D ) Quantification of Cats from ( B ) lanes 16 - 30 . Mean ± SD , n = 3 independent experiments . Abundance of catenanes was calculated as a percentage of total lane signal ( % of total ) . ( F ) Samples from ( E ) were separated on native agarose gels and visualized by autoradiography . Linear molecules are indicated with a red arrow . ( G ) Quantification of scCMs from + ProK gel in ( F ) lanes 6 - 20 . Mean ± SD , n = 3 independent experiments . Abundance of supercoiled monomers was calculated as a percentage of total lane signal ( % of total ) . ( H ) Quantification of DPC Containing Molecules from ( F ) lanes 6 - 20 . Mean ± SD , n = 3 independent experiments . Abundance of DPC - containing molecules was calculated as a percentage of total lane signal ( % of total ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Plasmid DNA was replicated in Mock- or TOP2α-immunodepleted extracts in the presence or absence of vehicle control, etoposide, or doxorubicin. In mock-depleted extracts (ΔMock), the final replication product are supercoiled circular monomers ('scCMs'). In TOP2α-depleted extracts (ΔTOP2α), the final replication product are catenanes ('Cats') because TOP2α is required for DNA unlinking. (B) Samples from (A) were separated on a native agarose gel and visualized by autoradiography. Linear molecules, arising from DNA breaks, are indicated with a red arrow migrate at a similar molecular weight to a subset of catenanes. (C) Quantification of scCMs from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers was calculated as a percentage of total lane signal (% of total). (D) Quantification of Cats from (B) lanes 16-30. Mean ± SD, n=3 independent experiments. Abundance of catenanes was calculated as a percentage of total lane signal (% of total).(F) Samples from (E) were separated on native agarose gels and visualized by autoradiography. Linear molecules are indicated with a red arrow. (G) Quantification of scCMs from + ProK gel in (F) lanes 6-20. Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers was calculated as a percentage of total lane signal (% of total). (H) Quantification of DPC Containing Molecules from (F) lanes 6-20. Mean ± SD, n=3 independent experiments. Abundance of DPC-containing molecules was calculated as a percentage of total lane signal (% of total).


Figure 4 ( A ) Plasmid DNA was replicated in the presence or absence of etoposide and p97 - i , which inhibits removal of replisomes ( green ) from DNA . At different timepoints chromatin - bound proteins were recovered via plasmid pulldowns . ( B ) Proteins from ( A ) were recovered and analyzed via Western blotting . Input corresponds to 1 % of total reaction . Modified TOP2α species are also indicated . ( C ) Quantification of TOP2α ( both modified and unmodified species ) from ( B ) lanes 1 - 8 . ( D ) Quantification of TOP2α ( both modified and unmodified species ) from ( B ) lanes 9 - 16 . ( E ) Quantification of CDC45 from ( B ) lanes 1 - 8 . ( F ) Quantification of CDC45 from ( B ) lanes 9 - 16 . Data information : ( C - F Mean ± SD , n = 3 independent experiments . Protein abundance ( AU ) was normalized to the maximum recorded signal within each individual experiment . ( G ) Plasmid DNA was replicated in the presence or absence of etoposide and aphidicolin ( Aph ) , which prevents DNA synthesis but still permits unwinding by replisomes . At different timepoints chromatin - bound proteins were recovered via plasmid pulldowns . ( H ) Proteins from ( G ) were recovered and analyzed via Western blotting . Input corresponds to 1 % of total reaction . ( I ) Quantification of RPA30 from ( H ) lanes 9 - 16 . ( J ) Quantification of RPA30 from ( H ) lanes 1 - 8 . ( K ) Quantification of TOP2α ( both modified and unmodified species ) from ( H ) lanes 1 - 4 and 9 - 12 . ( L ) Quantification of TOP2α ( both modified and unmodified species ) from ( H ) lanes 5 - 8 and 13 - 16 . Data information : I - L ) Mean ± SD , n = 3 independent experiments . Protein abundance ( AU ) was normalized to the maximum recorded signal within each individual experiment .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Plasmid DNA was replicated in the presence or absence of etoposide and p97-i, which inhibits removal of replisomes (green) from DNA. At different timepoints chromatin-bound proteins were recovered via plasmid pulldowns. (B) Proteins from (A) were recovered and analyzed via Western blotting. Input corresponds to 1% of total reaction. Modified TOP2α species are also indicated. (C) Quantification of TOP2α (both modified and unmodified species) from (B) lanes 1-8. (D) Quantification of TOP2α (both modified and unmodified species) from (B) lanes 9-16. (E) Quantification of CDC45 from (B) lanes 1-8. (F) Quantification of CDC45 from (B) lanes 9-16. Data information: (C-F Mean ± SD, n=3 independent experiments. Protein abundance (AU) was normalized to the maximum recorded signal within each individual experiment.(G) Plasmid DNA was replicated in the presence or absence of etoposide and aphidicolin (Aph), which prevents DNA synthesis but still permits unwinding by replisomes. At different timepoints chromatin-bound proteins were recovered via plasmid pulldowns. (H) Proteins from (G) were recovered and analyzed via Western blotting. Input corresponds to 1% of total reaction. (I) Quantification of RPA30 from (H) lanes 9-16. (J) Quantification of RPA30 from (H) lanes 1-8. (K) Quantification of TOP2α (both modified and unmodified species) from (H) lanes 1-4 and 9-12. (L) Quantification of TOP2α (both modified and unmodified species) from (H) lanes 5-8 and 13-16. Data information: I-L) Mean ± SD, n=3 independent experiments. Protein abundance (AU) was normalized to the maximum recorded signal within each individual experiment.


Figure 5 ( A ) Plasmid DNA was replicated in the presence of [ α - 32P ] dATP and vehicle control , aclarubicin , or doxorubicin . ( B ) Samples from ( A ) were separated on a native agarose gel and visualized by autoradiography . ( C ) Quantification of supercoiled circular monomers from ( B ) . Mean ± SD , n = 3 independent experiments . Abundance of supercoiled monomers was calculated as a percentage of total lane signal ( % of total ) . ( E ) Samples from ( D ) were separated on a native agarose gel and visualized by autoradiography . ( F ) Quantification of fork merger from ( E ) lanes 1 - 8 . Mean ± SD , n = 3 independent experiments . ( G ) Quantification of fork merger from ( E ) lanes 9 - 17 . Mean ± SD , n = 3 independent experiments . Fork merger was calculated as a percentage of total lane signal ( % of total ) for ( F ) - ( G ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Plasmid DNA was replicated in the presence of [α-32P]dATP and vehicle control, aclarubicin, or doxorubicin. (B) Samples from (A) were separated on a native agarose gel and visualized by autoradiography. (C) Quantification of supercoiled circular monomers from (B). Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers was calculated as a percentage of total lane signal (% of total).(E) Samples from (D) were separated on a native agarose gel and visualized by autoradiography. (F) Quantification of fork merger from (E) lanes 1-8. Mean ± SD, n=3 independent experiments. (G) Quantification of fork merger from (E) lanes 9-17. Mean ± SD, n=3 independent experiments. Fork merger was calculated as a percentage of total lane signal (% of total) for (F)-(G).


Figure 1B . DCC and netrin - 1 expression ( FACS analysis ) in B - cells lymphocytes isolated from DCC + / + and DCCmut / mut mice spleen and lymph nodes . Results are presented as expression ratios between means expression levels in mutant animals ( n = 3 ) and control ones ( n = 3 ) . No significant difference was observed between wild - type and mutant animals . C . DCCmut / mut mouse with splenomegaly and systemic lymphadenopathy . Left panel : black and white arrows indicate respectively the inguinal and mesenteric lymph nodes . Right panel : enlarged spleen in a lymphoma - bearing mutant mouse ( left ) as compared to control organ ( right ) . D . Incidence of lymphoid proliferations and lymphoma in DCC + / + ( n = 19 ) and DCCmut / mut ( n = 29 ) mice . Tumors classification was performed blinded to genotype according to anatomopathologists consensus ( 29 ) . * : p = 0 . 036 ; one - tailed Fisher ' s exact test . E . Histology analysis and immunophenotyping of lymphoid proliferations in DCC control and mutant mice . Sections from control spleen , low - grade FL and high - grade DLBCL were stained with hematoxylin - eosin - safran or with antibodies to B220 or Ki67 . w : white pulp , r : red pulp , cr : compressed red pulp .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. DCC and netrin-1 expression (FACS analysis) in B-cells lymphocytes isolated from DCC+/+ and DCCmut/mut mice spleen and lymph nodes. Results are presented as expression ratios between means expression levels in mutant animals (n=3) and control ones (n=3). No significant difference was observed between wild-type and mutant animals.C. DCCmut/mut mouse with splenomegaly and systemic lymphadenopathy. Left panel: black and white arrows indicate respectively the inguinal and mesenteric lymph nodes. Right panel: enlarged spleen in a lymphoma-bearing mutant mouse (left) as compared to control organ (right).D. Incidence of lymphoid proliferations and lymphoma in DCC+/+ (n=19) and DCCmut/mut (n=29) mice. Tumors classification was performed blinded to genotype according to anatomopathologists consensus (29). *: p=0.036; one-tailed Fisher's exact test.E. Histology analysis and immunophenotyping of lymphoid proliferations in DCC control and mutant mice. Sections from control spleen, low-grade FL and high-grade DLBCL were stained with hematoxylin-eosin-safran or with antibodies to B220 or Ki67. w: white pulp, r: red pulp, cr: compressed red pulp.


Figure 2A - B . Analysis of DCC ( A ) and netrin - 1 ( B ) expression in a cohort of normal tonsils ( n = 9 ) , Germinal - Center - B - Cell - like - DLBCL ( n = 13 ) , Activated - B - Cell - like - DLBCL ( n = 18 ) and MCL ( n = 36 ) . Quantification of genes expression was performed by Q - RT - PCR , relatively to HPRT housekeeping gene . * * : p < 0 . 005 ; * * * * : p < 0 . 0001 ; two - sided Mann - Whitney U - test . C - D . Analysis of netrin - 1 expression in DLBCL ( C ) and MCL ( D ) by immunohistochemistry . C . 21 DLBCL patients biopsies were used to build a TMA . Normal tissues ( normal tonsils , reactive lymph nodes , muscle , liver ) are included ( the left - most spot , upper panel , corresponds to liver ) . Enlarged representative image of netrin - 1 expression in DLBCL is shown ( lower panel ) . D . Representative images of netrin - 1 expression in paraffin - embedded biopsies of MCL ( n = 9 ) . Five tumors were positive for netrin - 1 ( left panel ) and four were negative ( right panel ) .

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Figure 2A-B. Analysis of DCC (A) and netrin-1 (B) expression in a cohort of normal tonsils (n=9), Germinal-Center-B-Cell-like-DLBCL (n=13), Activated-B-Cell-like-DLBCL (n=18) and MCL (n=36). Quantification of genes expression was performed by Q-RT-PCR, relatively to HPRT housekeeping gene. **: p<0.005; ****: p<0.0001; two-sided Mann-Whitney U-test.C-D. Analysis of netrin-1 expression in DLBCL (C) and MCL (D) by immunohistochemistry. C. 21 DLBCL patients biopsies were used to build a TMA. Normal tissues (normal tonsils, reactive lymph nodes, muscle, liver) are included (the left-most spot, upper panel, corresponds to liver). Enlarged representative image of netrin-1 expression in DLBCL is shown (lower panel). D. Representative images of netrin-1 expression in paraffin-embedded biopsies of MCL (n=9). Five tumors were positive for netrin-1 (left panel) and four were negative (right panel).


Figure 3A . Expression of netrin - 1 and DCC in lymphoma cell lines . Quantification was performed by Q - RT - PCR in 21 lymphoma cell lines . HPRT housekeeping gene was used as a standardization control . Netrin - 1 and DCC levels are indicated as follow : − , not detectable ; + to + + + + , moderate to very high expression . ABC and GC - DLBCL cell lines are highlighted respectively in blue and green , MCL ones are in grey . Expression of netrin - 1 ( in green ) by immunofluorescence using netrin - 1 antibody on OCI - Ly3 and Granta - 519 cell lines is shown on the right panel . Nuclei are counterstained in blue by Hoechst . B . Caspase - 3 activity in Granta - 519 cells after transfection of scrambled siRNA ( si - Ctl ) or netrin - 1 siRNA ( si - netrin - 1 ) , with or without DCC siRNA ( si - DCC ) . Results are means + / - std of four independent experiments . $ : p = 0 . 03 ; * : p = 0 . 02 ; two - sided Mann - Whitney U - test . D . Effect of net - 1 mAb on Granta - 519 ( left panel ) and OCI - Ly3 ( right panel ) cell density . Results are means + / - std indexed to control of four independent experiments . $ : p = 0 . 05 ; * : p = 0 . 03 ; two - sided Mann - Whitney U - test . E . Effect of net - 1 mAb on induction of Granta - 519 cells apoptosis , detected by TUNEL staining . Left panel : representative images are shown . TUNEL positive cells are labelled in red . Nuclei are counterstained in blue by Hoechst staining . Right panel : quantification of one representative experiment out of three performed is exposed . Results are presented as percentage of TUNEL - positive cells per field . * : p = 0 . 04 ; two - sided Mann - Whitney U - test . F . Caspase - 3 activity in Granta - 519 cells treated with net - 1 mAb antibody or with an unrelated Ig - G1 antibody ( Ctl ) , with or without silencing of DCC via siRNA ( siDCC ) . Results are means + / - std of four independent experiments . $ : p = 0 . 05 ; * : p = 0 . 02 ; two - sided Mann - Whitney U - test . G - H . Effect of netrin - 1 interfering antibody net - 1 mAb on OCI - Ly3 ( G ) and Granta - 519 ( H ) xenograft tumors growth . Results are presented as means + / - 95 % CIs of tumor growth rates from the beginning of treatment . * : p = 0 . 02 ; * * : p = 0 . 01 ; $ : p = 0 . 007 ; $ $ : p = 0 . 002 ; * * * : p = 0 . 0002 ; * * * * : p < 0 . 0001 ; two - sided Student t - test ( Granta - 519 ) or Welch t - test ( OCI - Ly3 ) . I . Caspase - 3 activity in tumors from control ( n = 4 ) or netrin - 1 - interfering antibody ( n = 4 ) treated mice . Tumors resection was performed at the end of the treatment . Results are means + / - std of caspase - 3 activity . * : p = 0 . 02 ; two - sided Mann - Whitney test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Expression of netrin-1 and DCC in lymphoma cell lines. Quantification was performed by Q-RT-PCR in 21 lymphoma cell lines. HPRT housekeeping gene was used as a standardization control. Netrin-1 and DCC levels are indicated as follow: −, not detectable; + to ++++, moderate to very high expression. ABC and GC-DLBCL cell lines are highlighted respectively in blue and green, MCL ones are in grey. Expression of netrin-1 (in green) by immunofluorescence using netrin-1 antibody on OCI-Ly3 and Granta-519 cell lines is shown on the right panel. Nuclei are counterstained in blue by Hoechst.B. Caspase-3 activity in Granta-519 cells after transfection of scrambled siRNA (si-Ctl) or netrin-1 siRNA (si-netrin-1), with or without DCC siRNA (si-DCC). Results are means+/-std of four independent experiments. $: p=0.03; *: p=0.02; two-sided Mann-Whitney U-test.D. Effect of net-1 mAb on Granta-519 (left panel) and OCI-Ly3 (right panel) cell density. Results are means+/-std indexed to control of four independent experiments. $: p=0.05; *: p=0.03; two-sided Mann-Whitney U-test.E. Effect of net-1 mAb on induction of Granta-519 cells apoptosis, detected by TUNEL staining. Left panel: representative images are shown. TUNEL positive cells are labelled in red. Nuclei are counterstained in blue by Hoechst staining. Right panel: quantification of one representative experiment out of three performed is exposed. Results are presented as percentage of TUNEL-positive cells per field. *: p=0.04; two-sided Mann-Whitney U-test.F. Caspase-3 activity in Granta-519 cells treated with net-1 mAb antibody or with an unrelated Ig-G1 antibody (Ctl), with or without silencing of DCC via siRNA (siDCC). Results are means+/-std of four independent experiments. $: p=0.05; *: p=0.02; two-sided Mann-Whitney U-test.G-H. Effect of netrin-1 interfering antibody net-1 mAb on OCI-Ly3 (G) and Granta-519 (H) xenograft tumors growth. Results are presented as means+/-95% CIs of tumor growth rates from the beginning of treatment. *:p=0.02; **:p=0.01; $: p=0.007; $$:p=0.002; ***: p=0.0002; ****:p<0.0001; two-sided Student t-test (Granta-519) or Welch t-test (OCI-Ly3).I. Caspase-3 activity in tumors from control (n=4) or netrin-1-interfering antibody (n=4) treated mice. Tumors resection was performed at the end of the treatment. Results are means +/-std of caspase-3 activity. *:p=0.02; two-sided Mann-Whitney test.


Figure 1Electron microscopy showing two adjacent mitochondria ( one intact and one ruptured ) in a DM platelet . a . Lower powered view of the single DM platelet . b . The enlarged inset highlights the ruptured mitochondria ( with loss of cristae morphology ) next to the intact mitochondria . c . Black dash line indicates mitochondria membrane and red dash line indicate phagophore formation . ( M - mitochondria ) Immunoprecipitation ( Meng et al ) of MsrB2 in HC and DM human platelets followed by MsrB2 and LC3I / II western . LC3 and MsrB2 immuno - EM analysis of HC and DM platelets . 15 nm dots indicate immunogold - labeled LC3 clusters and 5nm dots indicate immunogold - labeled MsrB2 clusters . No significant clusters were found in HC ( a ) platelets . Representative areas of clusters of gold labeling in DM patients ( b - d ) are presented . The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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Figure 1Electron microscopy showing two adjacent mitochondria (one intact and one ruptured) in a DM platelet. a. Lower powered view of the single DM platelet. b. The enlarged inset highlights the ruptured mitochondria (with loss of cristae morphology) next to the intact mitochondria. c. Black dash line indicates mitochondria membrane and red dash line indicate phagophore formation. (M- mitochondria)Immunoprecipitation (Meng et al) of MsrB2 in HC and DM human platelets followed by MsrB2 and LC3I/II western.LC3 and MsrB2 immuno-EM analysis of HC and DM platelets. 15 nm dots indicate immunogold-labeled LC3 clusters and 5nm dots indicate immunogold-labeled MsrB2 clusters. No significant clusters were found in HC (a) platelets. Representative areas of clusters of gold labeling in DM patients (b-d) are presented. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Figure 2Western blot analysis of MsrB2 and LC3 I / II in Meg - 01 cell after shMsrB2 transfection ( 72 hrs ) . Cells were then treated with H2O2 ( 1 mM for 1 hr ) alone or with NAC ( 100 μM for 30 min ) . GAPDH was used as the loading control . Quantification and analysis of individual groups . GAPDH served as the loading control . ( shMsrB2 vs . shCon ; * p = 0 . 024 , shMsrB2 / H2O2 vs . shCon / H2O2 ; * p = 0 . 0295 , shMsrB2 / H2O2 / NAC vs . shCon / H2O2 / NAC * * p = 0 . 0100 , n = 3 ) Western blot analysis of MsrB2 , pp53 ( S15 ) and LC3 I / II in Meg - 01 cell after 25 mM HG treatment for 24 hrs . GAPDH was used as the loading control . Quantification analysis on individual groups . GAPDH served as the loading control . ( MsrB2 : shCon / HG vs . shCon ; * * p = 0 . 0017 , shMsrB2 / HG vs . shCon / HG ; * p = 0 . 0189 , pp53 : shMsrB2 / HG vs . shCon / HG ; * p = 0 . 0317 , LC3II : shCon / HG vs . shCon ; * p = 0 . 0215 , shMsrB2 / HG vs . shCon / HG ; * p = 0 . 0158 , n = 3 ) The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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Figure 2Western blot analysis of MsrB2 and LC3 I/II in Meg-01 cell after shMsrB2 transfection (72 hrs). Cells were then treated with H2O2 (1 mM for 1 hr) alone or with NAC (100 μM for 30 min). GAPDH was used as the loading control. Quantification and analysis of individual groups. GAPDH served as the loading control. (shMsrB2 vs. shCon; *p=0.024, shMsrB2/H2O2 vs. shCon/H2O2; *p=0.0295, shMsrB2/H2O2/NAC vs. shCon/H2O2/NAC **p=0.0100, n=3)Western blot analysis of MsrB2, pp53(S15) and LC3 I/II in Meg-01 cell after 25 mM HG treatment for 24 hrs. GAPDH was used as the loading control. Quantification analysis on individual groups. GAPDH served as the loading control. (MsrB2: shCon/HG vs. shCon; **p=0.0017, shMsrB2/HG vs. shCon/HG; *p=0.0189, pp53: shMsrB2/HG vs. shCon/HG; *p=0.0317, LC3II: shCon/HG vs. shCon; *p=0.0215, shMsrB2/HG vs. shCon/HG; *p=0.0158, n=3) The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Figure 3Levels of reactive oxygen species ( ROS ) quantified in the platelets using the fluorescent dye DCFH - DA . ( ROS ; * p = 0 . 0139 , MsrB2 fl / fl n = 5 , MsrB2 - / - n = 6 ) C . Representative image and quantification of mitochondrial apoptosis , measured in freshly isolated platelets from the MsrB2 knockouts and age matched floxed mice . Results are expressed as % apoptotic cells . ( TMRE ; * p = 0 . 0118 , MsrB2 fl / fl n = 5 , MsrB2 - / - n = 5 ) Representative images showing the intraplatelet expression of LC3 and active caspase - 3 in CD41 stained platelets from the KO mice using fluorescent labelled antibodies against the respective proteins of interest . Magnification : 100X . Rightmost panel represents zoom in on an individual platelet showing the colocalization and expression of LC3 and active caspase - 3 . Graph indicates platelets positive for active caspase - 3 signal . The y ‐ axis indicates percentage of caspase - 3 positive cells from total cells calculated from n = 10 independent images per sample . ( active caspase - 3 signal ; * * p = 0 . 0003Intracellular caspase - 3 activity measured in freshly isolated platelets using the chromophore Ac - DEVD - pNA . Experiments performed in a minimum of n = 6 samples per group . Values expressed as Mean ± SEM . ( caspase - 3 activity ; * p = 0 . 0157 ) and H . Western blot analysis of Parkin , LC3I / II , MsrB2 and active caspasee3 in platelet after cell penetration peptide injection in chow and HFD mice . Control peptide ( CP ) , LIF peptide ( Foroud et al ) . Quantification analysis of individual band intensity . ( Parkin ; * p = 0 . 02964 , LC3II ; * p = 0 . 03200 , MsrB2 ; * * p = 0 . 0059 , Active caspase3 ; * p = 0 . 0368 vs . chow or HFD group , each group n = 3 ) . Actin served as the loading control . The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Levels of reactive oxygen species (ROS) quantified in the platelets using the fluorescent dye DCFH-DA. (ROS; *p=0.0139, MsrB2 fl/fl n=5, MsrB2 -/- n=6)C. Representative image and quantification of mitochondrial apoptosis, measured in freshly isolated platelets from the MsrB2 knockouts and age matched floxed mice. Results are expressed as % apoptotic cells. (TMRE; *p=0.0118, MsrB2 fl/fl n=5, MsrB2 -/- n=5)Representative images showing the intraplatelet expression of LC3 and active caspase-3 in CD41 stained platelets from the KO mice using fluorescent labelled antibodies against the respective proteins of interest. Magnification: 100X. Rightmost panel represents zoom in on an individual platelet showing the colocalization and expression of LC3 and active caspase-3.Graph indicates platelets positive for active caspase-3 signal. The y‐axis indicates percentage of caspase-3 positive cells from total cells calculated from n = 10 independent images per sample. (active caspase-3 signal ; **p=0.0003Intracellular caspase-3 activity measured in freshly isolated platelets using the chromophore Ac-DEVD-pNA. Experiments performed in a minimum of n=6 samples per group. Values expressed as Mean ± SEM. (caspase-3 activity ; *p=0.0157)and H. Western blot analysis of Parkin, LC3I/II, MsrB2 and active caspasee3 in platelet after cell penetration peptide injection in chow and HFD mice. Control peptide (CP), LIF peptide (Foroud et al). Quantification analysis of individual band intensity. (Parkin; *p=0.02964, LC3II; *p=0.03200, MsrB2; **p=0.0059, Active caspase3; *p=0.0368 vs. chow or HFD group, each group n=3). Actin served as the loading control. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Figure 4Immunoprecipitation ( Meng et al ) of MsrB2 in HC and DM platelets , followed by western blot analysis of precipitated Parkin and modified Parkin . Immunoprecipitation ( Meng et al ) of Parkin in HC and DM platelets , followed by Western blot analysis of precipitated Parkin and MsrB2 . Confocal microscopy was used to corroborate the Western analysis using triple staining for Parkin , LC3 and MsrB2 in HC and DM platelets . Arrows indicate sites of colocalization of Parkin , LC3 and MsrB2 as determined by Volocity software ( PerkinElmer , USA ) . Graphical quantification of colocalization between Parkin , LC3 and MsrB2 signal . Signal intensity of each group was converted to fold and compared with HC values ( colocalization of parkin , LC3 and MsrB2 ; * * p = 0 . 0006 ) . Immunoprecipitation ( Meng et al ) of Parkin in HC and DM platelets . Western blot analysis of MetO Parkin using specific Parkin and MetO antibodies . The detection of MetO Parkin , was performed w / o β - mercaptoethanol ( NonRe ) SDS sample buffer . In vitro assay . Parkin oxidation was induced with 1mM H2O2 then incubated with MsrB2 protein at 37ºC for 2hrs . Western blot analysis of Parkin and MsrB2 was performed . ( red arrow , low MsrB2 expression ; blue arrow , high MsrB2 expression ) . The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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Figure 4Immunoprecipitation (Meng et al) of MsrB2 in HC and DM platelets, followed by western blot analysis of precipitated Parkin and modified Parkin.Immunoprecipitation (Meng et al) of Parkin in HC and DM platelets, followed by Western blot analysis of precipitated Parkin and MsrB2.Confocal microscopy was used to corroborate the Western analysis using triple staining for Parkin, LC3 and MsrB2 in HC and DM platelets. Arrows indicate sites of colocalization of Parkin, LC3 and MsrB2 as determined by Volocity software (PerkinElmer, USA). Graphical quantification of colocalization between Parkin, LC3 and MsrB2 signal.Signal intensity of each group was converted to fold and compared with HC values (colocalization of parkin, LC3 and MsrB2; **p=0.0006).Immunoprecipitation (Meng et al) of Parkin in HC and DM platelets. Western blot analysis of MetO Parkin using specific Parkin and MetO antibodies. The detection of MetO Parkin, was performed w/o β-mercaptoethanol (NonRe) SDS sample buffer.In vitro assay. Parkin oxidation was induced with 1mM H2O2 then incubated with MsrB2 protein at 37ºC for 2hrs. Western blot analysis of Parkin and MsrB2 was performed. (red arrow, low MsrB2 expression; blue arrow, high MsrB2 expression). The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.