D ) Frequency of new fired origins for Mybl2 + / + and Mybl2Δ / Δ ESCs with or without ATM inhibitor treatment . Error bars represent SEM . Statistical analysis was carried out using unpaired t - test . At least 500 replication structures were counted per treatment group from 4 separate repeats .

D) Frequency of new fired origins for Mybl2+/+ and Mybl2Δ/Δ ESCs with or without ATM inhibitor treatment. Error bars represent SEM. Statistical analysis was carried out using unpaired t-test. At least 500 replication structures were counted per treatment group from 4 separate repeats.


E ) Frequency of anaphases positive for UFB positive cells ( based on ERCC / PICH positive immunostaining ) for Mybl2 + / + , the Mybl2Δ / Δ and the Mybl2 + / + treated for 2 hours with ATM inhibitor ( KU60019 ) . Data represents at least 100 anaphases from each group from 2 experimental repeats . Statistical analysis was performed using an unpaired two - tailed t - test .

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E) Frequency of anaphases positive for UFB positive cells (based on ERCC/PICH positive immunostaining) for Mybl2+/+, the Mybl2Δ/Δ and the Mybl2+/+ treated for 2 hours with ATM inhibitor (KU60019). Data represents at least 100 anaphases from each group from 2 experimental repeats. Statistical analysis was performed using an unpaired two-tailed t-test.


F ) Immunoblot showing the levels of phosphorylated ATM ( Ser1987 ) and ATM in the Mybl2 + / + and Mybl2Δ / Δ ESCs with or without CPT treatment ( 10µM CPT for 2 hours ) . Bar graph ( lower panel ) represents average band density of P - ATM in Mybl2Δ / Δ ESCs treated with CPT relative to ATM and relative to wild type CPT treated cells from three independent repeats . Error bars represent SD . Statistical analysis was carried out using a two - tailed unpaired t - test .

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F) Immunoblot showing the levels of phosphorylated ATM (Ser1987) and ATM in the Mybl2+/+ and Mybl2Δ/Δ ESCs with or without CPT treatment (10µM CPT for 2 hours). Bar graph (lower panel) represents average band density of P-ATM in Mybl2Δ/Δ ESCs treated with CPT relative to ATM and relative to wild type CPT treated cells from three independent repeats. Error bars represent SD. Statistical analysis was carried out using a two-tailed unpaired t-test.


G ) Distribution plot of replication speed in Mybl2 + / + and ATM - / - ESC treated or not for 90 minutes with 10µM KU60019 ( ATMi ) , before sequential addition of IdU and CldU for 20 minutes each . Statistical analysis was carried out using the Mann Whitney U test . At least 200 replication forks were counted from 3 separate experiments for wild type and ATM - / - and two separate experiments for ATM - / - treated with ATM inhibitor . ( * * * * p < 0 . 0001 )

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G) Distribution plot of replication speed in Mybl2+/+and ATM-/- ESC treated or not for 90 minutes with 10µM KU60019 (ATMi), before sequential addition of IdU and CldU for 20 minutes each. Statistical analysis was carried out using the Mann Whitney U test. At least 200 replication forks were counted from 3 separate experiments for wild type and ATM-/- and two separate experiments for ATM-/- treated with ATM inhibitor . ( **** p< 0.0001)


H ) Quantification of the number of replication factories per cell for the different genotypes and treatments . A minimum of 65 early S - phase nuclei were counted per condition from two independent repeats . Statistical analysis was performed using a two - tailed unpaired unequal variance t - test . ( * * * * p < 0 . 0001 )

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H) Quantification of the number of replication factories per cell for the different genotypes and treatments. A minimum of 65 early S-phase nuclei were counted per condition from two independent repeats. Statistical analysis was performed using a two-tailed unpaired unequal variance t-test.( **** p< 0.0001)


I ) Distribution plot of replication speed in human iPSC and MEFs ( primary and immortal ) . Statistical analysis was carried out using the Mann Whitney U test . At least 400 replication forks for iPSC , 200 for primary MEFS and 250 for immortal MEFs were counted from 3 independent experiments . ( * * * * p < 0 . 0001 )

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I) Distribution plot of replication speed in human iPSC and MEFs (primary and immortal). Statistical analysis was carried out using the Mann Whitney U test. At least 400 replication forks for iPSC,200 for primary MEFS and 250 for immortal MEFs were counted from 3 independent experiments. ( **** p< 0.0001)


B and C ) Distribution curve of replication fork rates for Mybl2 + / + and Mybl2Δ / Δ ESCs treated with mirin . Statistical analysis was performed using the Mann Whitney U test . n = 3 experimental replicates . The value of the mean fork length is indicated above arrow ( * * * * p < 0 . 0001 )

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B and C) Distribution curve of replication fork rates for Mybl2+/+ and Mybl2Δ/Δ ESCs treated with mirin. Statistical analysis was performed using the Mann Whitney U test. n=3 experimental replicates. The value of the mean fork length is indicated above arrow ( **** p< 0.0001)


D ) Representative images of new firing origins , and elongating forks showing stable replication and fork instability .

D) Representative images of new firing origins, and elongating forks showing stable replication and fork instability.


E ) Frequency of new firing origins ( relative to total structures counted ) from Mybl2 + / + and Mybl2Δ / Δ ESCs treated or not with Mirin . At least 450 replication structures were counted per treatment from 3 independent repeats . Error bars represent SEM . Statistical analysis using two - tailed unpaired t - test .

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E) Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with Mirin. At least 450 replication structures were counted per treatment from 3 independent repeats. Error bars represent SEM. Statistical analysis using two- tailed unpaired t-test.


F ) Distribution and the average IdU / CldU ratio in Mybl2 + / + and Mybl2Δ / Δ ESCs with or without MRE11 inhibitor treatment . The length of both incorporated labels for each elongating fork was measured in µm and the positive ratio was calculated . 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 260 ratios were calculated per treatment group from 3 separate experiments . ( * * * * p < 0 . 0001 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) Distribution and the average IdU/CldU ratio in Mybl2+/+ and Mybl2Δ/Δ ESCs with or without MRE11 inhibitor treatment. The length of both incorporated labels for each elongating fork was measured in µm and the positive ratio was calculated. 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 260 ratios were calculated per treatment group from 3 separate experiments.( **** p< 0.0001)


C ) Frequency of new firing origins ( relative to total structures counted ) from Mybl2 + / + and Mybl2Δ / Δ ESCs treated or not with the indicated inhibitors : CDC7 inhibitor ( PHA - 767491 ) and / or ATM inhibitor ( Ku60019 ) . At least 300 replication structures were counted per treatment . Error bars represent SEM . Statistical analysis using two - tailed unpaired t - test ( * p < 0 . 05 ) .

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C ) Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with the indicated inhibitors: CDC7 inhibitor (PHA-767491) and/or ATM inhibitor (Ku60019). At least 300 replication structures were counted per treatment. Error bars represent SEM. Statistical analysis using two- tailed unpaired t-test (*p < 0.05).


D ) Replication rate ( kb / min ) of Mybl2 + / + and Mybl2Δ / Δ ESCs treated with the indicated inhibitors . Statistical analysis was carried out using an unpaired Mann Whitney U test ( * * * * p < 0 . 0001 ) . Minimum three independent experiments . At least 270 forks were scored for non - ATM inhibitor treated groups , and a minimum of 130 for ATM inhibitor treated groups .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D ) Replication rate (kb/min) of Mybl2+/+ and Mybl2Δ/Δ ESCs treated with the indicated inhibitors. Statistical analysis was carried out using an unpaired Mann Whitney U test (**** p< 0.0001). Minimum three independent experiments. At least 270 forks were scored for non-ATM inhibitor treated groups, and a minimum of 130 for ATM inhibitor treated groups.


E ) Fork stability ( average IdU / CldU ratio ) of Mybl2 + / + ESC and Mybl2Δ / Δ ESCs treated with the indicated inhibitors alone or in combination . At least 240 ratios were calculated for non - ATM inhibitor treated groups , and a minimm of 130 for ATM inhibitor treated groups . Y - axis was cut to change scale and display large values . Statistical analysis was carried out using Mann Whitney U test ( * * * * p < 0 . 0001 ) . Data for Mybl2 + / + ESCs treated with ATM inhibitor alone or in combination with CDC7 inhibitor , was collected from two independent experiments . For the rest of the conditions , a minimum of three independent repeats was performed .

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E ) Fork stability (average IdU/CldU ratio) of Mybl2+/+ ESC and Mybl2Δ/Δ ESCs treated with the indicated inhibitors alone or in combination. At least 240 ratios were calculated for non-ATM inhibitor treated groups, and a minimm of 130 for ATM inhibitor treated groups. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using Mann Whitney U test (**** p< 0.0001). Data for Mybl2+/+ ESCs treated with ATM inhibitor alone or in combination with CDC7 inhibitor, was collected from two independent experiments. For the rest of the conditions, a minimum of three independent repeats was performed.


F ) Immunoblot showing the levels of CDC7 and actin in the Mybl2 + / + and Mybl2Δ / Δ ESCs . Bar graph ( lower panel ) represents average band density of CDC7 in Mybl2Δ / Δ ESCs relative to actin and relative to Mybl2 + / + ESCs from three independent repeats . Error bars represent SD . Statistical analysis was carried out using a two - tailed unpaired t - test ( ns = no significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) Immunoblot showing the levels of CDC7 and actin in the Mybl2+/+ and Mybl2Δ/Δ ESCs. Bar graph (lower panel) represents average band density of CDC7 in Mybl2Δ/Δ ESCs relative to actin and relative to Mybl2+/+ ESCs from three independent repeats. Error bars represent SD. Statistical analysis was carried out using a two-tailed unpaired t-test (ns=no significant).


a ) QF - FRL of unilamellar liposomes containing 95 mol % of phosphatidylcholine and 5 mol % of a phosphoinositide , labelled with GST - PX . Only liposomes containing PtdIns ( 3 ) P were labelled intensely . GST - PXR58A , a PX - domain mutant that does not bind PtdIns ( 3 ) P , yielded virtually no labelling in PtdIns ( 3 ) P - containing liposomes . Scale bar , 0 . 2 μm .

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a) QF-FRL of unilamellar liposomes containing 95 mol% of phosphatidylcholine and 5 mol% of a phosphoinositide, labelled with GST-PX. Only liposomes containing PtdIns(3)P were labelled intensely. GST-PXR58A, a PX-domain mutant that does not bind PtdIns(3)P, yielded virtually no labelling in PtdIns(3)P-containing liposomes. Scale bar, 0.2 μm.


( b ) QF - FRL of unilamellar liposomes containing 0 - 5 mol % of PtdIns ( 3 ) P . Scale bar , 0 . 2 μm . The labelling density increased roughly in proportion to the PtdIns ( 3 ) P content of the liposomes . The experiment was repeated twice and the number of colloidal gold particles per μm2 was measured in more than 75 randomly selected liposomes . Values for all concentrations are statistically different from those in the adjacent conditions ( for example , 1 versus 2 mol % ; mean ± s . e . m . ; Student ' s t - test ; P0 . 05 ) .

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(b) QF-FRL of unilamellar liposomes containing 0-5 mol % of PtdIns(3)P. Scale bar, 0.2 μm. The labelling density increased roughly in proportion to the PtdIns(3)P content of the liposomes. The experiment was repeated twice and the number of colloidal gold particles per μm2 was measured in more than 75 randomly selected liposomes. Values for all concentrations are statistically different from those in the adjacent conditions (for example, 1 versus 2 mol%; mean±s.e.m.; Student's t-test; P0.05).


( b ) QF - FRL in wild - type yeast in log phase . PtdIns ( 3 ) P was labelled with GFP - PX and marked with colloidal gold ( 10 nm ) . The vacuolar membrane PF was labelled intensely , whereas other organellar membranes did not exhibit significant labelling . Scale bar , 0 . 5 μm .

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(b) QF-FRL in wild-type yeast in log phase. PtdIns(3)P was labelled with GFP-PX and marked with colloidal gold (10 nm). The vacuolar membrane PF was labelled intensely, whereas other organellar membranes did not exhibit significant labelling. Scale bar, 0.5 μm.


c ) Quantification of the labelling intensity of PtdIns ( 3 ) P ( mean ± s . e . m . ) . The experiment was repeated three times and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images .

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c) Quantification of the labelling intensity of PtdIns(3)P (mean±s.e.m.). The experiment was repeated three times and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images.


( d , e ) Negative controls . Both wild - type yeast incubated with GST - PXR58A ( d ) and vps34Δ yeast incubated with GST - PX ( e ) were virtually unlabelled . Scale bar , 0 . 5 μm .

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(d,e) Negative controls. Both wild-type yeast incubated with GST-PXR58A (d) and vps34Δ yeast incubated with GST-PX (e) were virtually unlabelled. Scale bar, 0.5 μm.


( a ) Wild - type yeast cultured in S ( - NC ) for 4 h . PtdIns ( 3 ) P was labelled intensely in the EF of autophagosomal membranes . A lower but still significant amount of labelling was also seen in the PF of autophagosome membranes as well as in the EF of the vacuolar membrane . Scale bar , 0 . 5 μm .

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(a) Wild-type yeast cultured in S(-NC) for 4 h. PtdIns(3)P was labelled intensely in the EF of autophagosomal membranes. A lower but still significant amount of labelling was also seen in the PF of autophagosome membranes as well as in the EF of the vacuolar membrane. Scale bar, 0.5 μm.


( b ) Autophagosomes in wild - type yeast cultured in S ( - NC ) for 4 h . The autophagosome was identified as a smooth - surfaced double - membrane structure , and seen in two different patterns , as shown in the diagrams in c . Upper panel : convex structures displaying the outer membrane EF and the inner membrane PF . Lower panel : concave structures displaying the outer membrane PF and the inner membrane EF . In both cases , PtdIns ( 3 ) P was labelled more intensely in the EF than in the PF . Scale bars , 0 . 2 μm .

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(b) Autophagosomes in wild-type yeast cultured in S(-NC) for 4 h. The autophagosome was identified as a smooth-surfaced double-membrane structure, and seen in two different patterns, as shown in the diagrams in c. Upper panel: convex structures displaying the outer membrane EF and the inner membrane PF. Lower panel: concave structures displaying the outer membrane PF and the inner membrane EF. In both cases, PtdIns(3)P was labelled more intensely in the EF than in the PF. Scale bars, 0.2 μm.


( a ) Wild - type yeast cultured in S ( - NC ) for 4 h . PtdIns ( 3 ) P was labelled intensely in the EF of autophagic body membranes in the vacuolar lumen . A lower amount of labelling was also seen in the PF of autophagic body membranes . Scale bar , 0 . 5 μm . (

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(a) Wild-type yeast cultured in S(-NC) for 4 h. PtdIns(3)P was labelled intensely in the EF of autophagic body membranes in the vacuolar lumen. A lower amount of labelling was also seen in the PF of autophagic body membranes. Scale bar, 0.5 μm. (


( c ) Labelling intensity of PtdIns ( 3 ) P in wild - type yeast cultured in S ( - NC ) for 4 h ( average ± s . e . m . ) . PtdIns ( 3 ) P was found in both leaflets of the autophagosome and autophagic body membrane , and the labelling was much stronger in the EF than in the PF . In the vacuolar membrane , significant labelling was found in the EF , albeit at much lower levels than in the PF . The experiment was repeated three times , and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images .

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(c) Labelling intensity of PtdIns(3)P in wild-type yeast cultured in S(-NC) for 4 h (average±s.e.m.). PtdIns(3)P was found in both leaflets of the autophagosome and autophagic body membrane, and the labelling was much stronger in the EF than in the PF. In the vacuolar membrane, significant labelling was found in the EF, albeit at much lower levels than in the PF. The experiment was repeated three times, and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images.


( a ) Huh7 cells expressing GFP - LC3 treated with 0 . 25 μM Torin1 and 0 . 4 μM bafilomycin A1 for 1 h . The autophagosome was identified as the IMP - poor double - membrane structure labelled with anti - GFP antibody ( large gold , 10 nm ) . Both PtdIns ( 3 ) P ( small gold , 6 nm ) and GFP - LC3 were seen exclusively in the PF in both convex and concave structures . The small and large gold particles are shown as green and red dots , respectively , in the figures at right . Scale bars , 0 . 1 μm . Figures displaying wider areas , as well as additional examples , are presented in Supplementary Fig . 4c . (

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(a) Huh7 cells expressing GFP-LC3 treated with 0.25 μM Torin1 and 0.4 μM bafilomycin A1 for 1 h. The autophagosome was identified as the IMP-poor double-membrane structure labelled with anti-GFP antibody (large gold, 10 nm). Both PtdIns(3)P (small gold, 6 nm) and GFP-LC3 were seen exclusively in the PF in both convex and concave structures. The small and large gold particles are shown as green and red dots, respectively, in the figures at right. Scale bars, 0.1 μm. Figures displaying wider areas, as well as additional examples, are presented in Supplementary Fig. 4c. (


( b ) NIH3T3 cells expressing Atg4C78A were incubated in Earle ' s balanced salt solution for 1 h . The isolation membrane was observed as parallel membranes with few IMPs . PtdIns ( 3 ) P was labelled only in the PF . Scale bar , 0 . 1 μm . Other examples are presented in Supplementary Fig . 5 .

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(b) NIH3T3 cells expressing Atg4C78A were incubated in Earle's balanced salt solution for 1 h. The isolation membrane was observed as parallel membranes with few IMPs. PtdIns(3)P was labelled only in the PF. Scale bar, 0.1 μm. Other examples are presented in Supplementary Fig. 5.


( c ) Labelling density of PtdIns ( 3 ) P in mammalian autophagosomal membrane ( mean ± s . e . m . ) . The experiment was repeated three times and the number of colloidal goldparticles per μm2 was measured in more than ten randomly selected images . Some labelling was observed in the EF but the majority of gold particles in the EF occurred near the EF - PF boundary ( white circles ; see Supplementary Fig . 6c for more examples ) , suggesting that many of them actually represent PtdIns ( 3 ) P in the PF .

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(c) Labelling density of PtdIns(3)P in mammalian autophagosomal membrane (mean±s.e.m.). The experiment was repeated three times and the number of colloidal goldparticles per μm2 was measured in more than ten randomly selected images. Some labelling was observed in the EF but the majority of gold particles in the EF occurred near the EF-PF boundary (white circles; see Supplementary Fig. 6c for more examples), suggesting that many of them actually represent PtdIns(3)P in the PF.


( a ) ymr1Δsjl3Δ cells cultured in S ( - NC ) for 4 h . PtdIns ( 3 ) P was labelled in comparable densities in PF and EF of autophagosomal membranes . Scale bar , 0 . 2 μm .

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(a) ymr1Δsjl3Δ cells cultured in S(-NC) for 4 h. PtdIns(3)P was labelled in comparable densities in PF and EF of autophagosomal membranes. Scale bar, 0.2 μm.


( b ) The PF / EF ratio of the PtdIns ( 3 ) P labelling density in autophagosomal membrane ( mean ± s . e . m . ) . The ratio was small ( that is , PF ) in WT , but close to 1 ( that is , PF ≅ EF ) in ymr1Δsjl3Δ . The experiment was repeated three times and the number of colloidal goldparticles per μm2 was measured in more than ten randomly selected images .

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(b) The PF/EF ratio of the PtdIns(3)P labelling density in autophagosomal membrane (mean±s.e.m.). The ratio was small (that is, PF) in WT, but close to 1 (that is, PF≅EF) in ymr1Δsjl3Δ. The experiment was repeated three times and the number of colloidal goldparticles per μm2 was measured in more than ten randomly selected images.


A - C . PTEN mRNA levels in LCM normal ovarian surface epithelium versus high - grade serous ovarian cancer ( HGSOC ) epithelium or normal ovarian stroma versus ovarian cancer - associated stroma . Datasets ; ( A ) GSE40595 , ( B ) GSE38666 , ( C ) Epithelium only , GSE14407 . Sample size ( n ) and p - values , ( Mann - Whitney ) annotated , whiskers Min - Max , line at median .

A-C. PTEN mRNA levels in LCM normal ovarian surface epithelium versus high-grade serous ovarian cancer (HGSOC) epithelium or normal ovarian stroma versus ovarian cancer-associated stroma. Datasets; (A) GSE40595, (B) GSE38666, (C) Epithelium only, GSE14407. Sample size (n) and p-values, (Mann-Whitney) annotated, whiskers Min-Max, line at median.


F . Overall survival ( % patients , months ) of OC patients . Highest quartile ( Q4 ) versus combination of quartiles 1 - 3 ( Q1 + 2 + 3 ) , PTEN mRNA ( TCGA , OV ) . Median survival ( 40 and 44 months ) , sample size ( n ) and p - value , Log - rank test ( Mantel - Cox ) annotated . G . Overall survival ( % patients , months ) of OC patients . Highest quartile ( Q4 ) versus combination of quartiles 1 - 3 ( Q1 + 2 + 3 ) , PTEN protein . Reverse Phase Protein Array Data , TCGA OV . Median survival ( 46 and 57 months ) , sample size ( n ) and p - value , Log - rank test ( Mantel - Cox ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Overall survival (% patients, months) of OC patients. Highest quartile (Q4) versus combination of quartiles 1-3 (Q1+2+3), PTEN mRNA (TCGA, OV). Median survival (40 and 44 months), sample size (n) and p-value, Log-rank test (Mantel-Cox) annotated. G. Overall survival (% patients, months) of OC patients. Highest quartile (Q4) versus combination of quartiles 1-3 (Q1+2+3), PTEN protein. Reverse Phase Protein Array Data, TCGA OV. Median survival (46 and 57 months), sample size (n) and p-value, Log-rank test (Mantel-Cox).


H . Differential abundance ( x , Log Ratio between conditions ; y , - Log10 q - values ) of Reverse Phase Protein Array data ( TCGA , OV ) in patient grouped by PTEN mRNA , High ( Q4 ) vs Low ( Q1 ) . Significant , blue ( - Log10 q - values > 1 . 3 ) ; AKT signalling pathway , labelled . I . Differential abundance ( x , Log Ratio between conditions ; y , - Log10 q - values ) of proteins in PTEN High ( Q4 ) vs PTEN Low ( Q1 ) protein samples . Reverse Phase Protein Array Data , TCGA OV . Significantly altered components in AKT signalling pathway labelled ( - Log10 q - value > 1 . 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Differential abundance (x, Log Ratio between conditions; y, -Log10 q-values) of Reverse Phase Protein Array data (TCGA, OV) in patient grouped by PTEN mRNA, High (Q4) vs Low (Q1). Significant, blue (-Log10 q-values >1.3); AKT signalling pathway, labelled. I. Differential abundance (x, Log Ratio between conditions; y, -Log10 q-values) of proteins in PTEN High (Q4) vs PTEN Low (Q1) protein samples. Reverse Phase Protein Array Data, TCGA OV. Significantly altered components in AKT signalling pathway labelled (-Log10 q-value> 1.3).


B . Time series , showing a representative spheroid for each genotype , 12 hr intervals . Arrowheads , protrusions into ECM . Scale bar , 20μm . Right , cartoon of phenotype .

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B. Time series, showing a representative spheroid for each genotype, 12 hr intervals. Arrowheads, protrusions into ECM. Scale bar, 20μm. Right, cartoon of phenotype.


E . Representative phase contrast images of spheroids described in ( D ) . Outlines pseudocoloured for classification ( Spherical , green ; Hyper - protrusive , blue ) . Scale bars , 400μm or 17μm , as indicated . Magnified individual spheroids from boxed regions at indicated timepoints . Arrowheads , protrusions into ECM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Representative phase contrast images of spheroids described in (D). Outlines pseudocoloured for classification (Spherical, green; Hyper-protrusive, blue). Scale bars, 400μm or 17μm, as indicated. Magnified individual spheroids from boxed regions at indicated timepoints. Arrowheads, protrusions into ECM.


A . Confocal images ( single slice ) of Trp53 - / - or Trp53 - / - ; Pten - / - ( 1 . 15 ) spheroids expressing mNeonGreen - tagged ( mNG ) biosensors for PI ( 4 , 5 ) P2 ( PH - PLCδ1 ) or PIP3 ( CYTH32G / GRP1 ) . Magnified images from boxed regions , max projection of 8 ( PH - PLCδ1 ) or 3 ( CYTH32G / GRP1 ) z - slices , pseudocoloured in FIRE LUT . Arrowheads : red , cell - cell contact ; yellow , nucleus ; green , protrusion tip . Scale bar , 7μm . Representative of 8 ( Trp53 - / - ) or 10 ( Trp53 - / - ; Pten - / - ) spheroids imaged across n = 2 independent experiments ( PH - PLCδ1 ) and 22 ( Trp53 - / - ) or 23 ( Trp53 - / - ; Pten - / - ) spheroids imaged across n = 4 independent experiments ( CYTH32G / GRP1 ) . B . Intensity profiles for PH - PLCδ1 and PH - CYTH3 from spheroids shown in ( A ) . Protrusions measured are annotated on images in upper panels , yellow lines . Arrowheads : red , protrusion tips .

O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Confocal images (single slice) of Trp53-/- or Trp53-/-;Pten-/- (1.15) spheroids expressing mNeonGreen-tagged (mNG) biosensors for PI(4,5)P2 (PH-PLCδ1) or PIP3 (CYTH32G/GRP1). Magnified images from boxed regions, max projection of 8 (PH-PLCδ1) or 3 (CYTH32G/GRP1) z-slices, pseudocoloured in FIRE LUT. Arrowheads: red, cell-cell contact; yellow, nucleus; green, protrusion tip. Scale bar, 7μm. Representative of 8 (Trp53-/-) or 10 (Trp53-/-;Pten-/-) spheroids imaged across n=2 independent experiments (PH-PLCδ1) and 22 (Trp53-/-) or 23 (Trp53-/-;Pten-/-) spheroids imaged across n=4 independent experiments (CYTH32G/GRP1). B. Intensity profiles for PH-PLCδ1 and PH-CYTH3 from spheroids shown in (A). Protrusions measured are annotated on images in upper panels, yellow lines. Arrowheads: red, protrusion tips.


D . Western blotting and quantitation for S6RP pS235 / 236 , S6RP , GAPDH ( sample integrity control ) in Trp53 - / - ; Pten - / - 1 . 15 spheroids treated with DMSO or inhibitors annotated in ( B ) for 2 days . Representative of n = 3 independent lysate preparations . Data , mean ± SD of pS235 / 236 : total S6RP ratio , normalised to DMSO . P - values , unpaired , 2 - tailed t tests , as annotated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Western blotting and quantitation for S6RP pS235/236, S6RP, GAPDH (sample integrity control) in Trp53-/-;Pten-/- 1.15 spheroids treated with DMSO or inhibitors annotated in (B) for 2 days. Representative of n=3 independent lysate preparations. Data, mean ± SD of pS235/236:total S6RP ratio, normalised to DMSO. P-values, unpaired, 2-tailed t tests, as annotated.


Quantitation of ID8 Trp53 - / - ; Pten - / - spheroids treated with PI3K isoform specific inhibitors : A66 ( PI3Kα ) , AZD8186 ( PI3Kβ ) , AS605240 ( PI3Kγ ) or CAL - 101 ( PI3Kδ ) , 6hr time intervals over 72 hrs . ( I ) Frequency of Spherical and Hyper - protrusive phenotypes . Heatmap ( grayscale ) - phenotype proportion ( z - score ) in control . Heatmap ( blue - red ) - log2 fold change from control . P - values , bubble size ( Cochran - Mantel - Haenszel test with Bonferroni adjustment ) . Black dot , homogenous effect across independent experiments ( Breslow - Day test , Bonferroni adjustment , non - significant ) . N = 2 independent experiments , 3 - 5 technical replicates / experiment .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitation of ID8 Trp53-/-;Pten-/- spheroids treated with PI3K isoform specific inhibitors: A66 (PI3Kα), AZD8186 (PI3Kβ), AS605240 (PI3Kγ) or CAL-101 (PI3Kδ), 6hr time intervals over 72 hrs. (I) Frequency of Spherical and Hyper-protrusive phenotypes. Heatmap (grayscale) - phenotype proportion (z-score) in control. Heatmap (blue-red) - log2 fold change from control. P-values, bubble size (Cochran-Mantel-Haenszel test with Bonferroni adjustment). Black dot, homogenous effect across independent experiments (Breslow-Day test, Bonferroni adjustment, non-significant). N=2 independent experiments, 3-5 technical replicates/experiment.


K . Confocal image of Trp53 - / - ; Pten - / - ( 1 . 15 ) spheroids stained for PI3Kβ ( green ) , F - actin ( magenta ) and Hoechst ( grey ) . Magnified images from boxed regions , pseudocoloured in inverted grayscale ( F - actin ) or FIRE LUT ( PI3Kβ ) . Yellow or white / black arrowheads , enrichment of F - actin or PI3Kβ at protrusion tips respectively . Scale bar , 5μm . Representative of n = 5 spheroids .

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K. Confocal image of Trp53-/-;Pten-/- (1.15) spheroids stained for PI3Kβ (green), F-actin (magenta) and Hoechst (grey). Magnified images from boxed regions, pseudocoloured in inverted grayscale (F-actin) or FIRE LUT (PI3Kβ). Yellow or white/black arrowheads, enrichment of F-actin or PI3Kβ at protrusion tips respectively. Scale bar, 5μm. Representative of n=5 spheroids.


L . Intensity profiles for PI3Kβ ( green ) and F - Actin ( magenta ) from spheroid in ( K ) . Tip measured is annotated , ECM to body , yellow arrow , tip , arrowhead .

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L. Intensity profiles for PI3Kβ (green) and F-Actin (magenta) from spheroid in (K). Tip measured is annotated, ECM to body, yellow arrow, tip, arrowhead.


A - B . Quantitation of ID8 Trp53 - / - ; Pten - / - 1 . 15 spheroids expressing shScramble , shArf5 or shArf6 , 6hr time intervals over 72 hrs . ( A ) Heatmap ( viridis ) - area presented as mean of Z - score values , normalised to control ( shScramble ) . ( B ) Frequency of Spherical and Hyper - protrusive phenotypes . Heatmap ( grayscale ) - phenotype proportion ( z - score ) in control . Heatmap ( blue - red ) - log2 fold change from control . P - values , bubble size ( Cochran - Mantel - Haenszel test with Bonferroni adjustment ) . Black dot , homogenous effect across independent experiments ( Breslow - Day test , Bonferroni adjustment , non - significant ) . N = 3 independent experiments , 4 - 5 technical replicates / experiment . C . Representative phase contrast images of spheroids described in ( A - B ) . Outlines pseudocoloured for classification ( Spherical , green ; Hyper - protrusive , blue ) . Magnified individual spheroids from boxed regions at indicated timepoints . Arrowheads , protrusions into ECM . Scale bars , 400μm or 17μm , as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-B. Quantitation of ID8 Trp53-/-;Pten-/- 1.15 spheroids expressing shScramble, shArf5 or shArf6, 6hr time intervals over 72 hrs. (A) Heatmap (viridis) - area presented as mean of Z-score values, normalised to control (shScramble). (B) Frequency of Spherical and Hyper-protrusive phenotypes. Heatmap (grayscale) - phenotype proportion (z-score) in control. Heatmap (blue-red) - log2 fold change from control. P-values, bubble size (Cochran-Mantel-Haenszel test with Bonferroni adjustment). Black dot, homogenous effect across independent experiments (Breslow-Day test, Bonferroni adjustment, non-significant). N=3 independent experiments, 4-5 technical replicates/experiment. C. Representative phase contrast images of spheroids described in (A-B). Outlines pseudocoloured for classification (Spherical, green; Hyper-protrusive, blue). Magnified individual spheroids from boxed regions at indicated timepoints. Arrowheads, protrusions into ECM. Scale bars, 400μm or 17μm, as indicated.


Representative confocal images and intensity profiles of ID8 Trp53 - / - and Trp53 - / - ; Pten - / - 1 . 15 cells expressing mNeonGreen ( mNG ) - tagged ARF6 ( green ) and F - actin ( magenta ) at the invasive front of wounded monolayers Scale bar , 5μm . Tips for which ARF6 - mNG and F - Actin intensity profiles were measured are annotated , ECM to body , yellow arrow .

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Representative confocal images and intensity profiles of ID8 Trp53-/- and Trp53-/-;Pten-/- 1.15 cells expressing mNeonGreen (mNG)-tagged ARF6 (green) and F-actin (magenta) at the invasive front of wounded monolayers Scale bar, 5μm. Tips for which ARF6-mNG and F-Actin intensity profiles were measured are annotated, ECM to body, yellow arrow.


Representative confocal images and intensity profiles of ID8 Trp53 - / - and Trp53 - / - ; Pten - / - 1 . 15 cells expressing mNeonGreen ( mNG ) - tagged ARF6 ( green ) and F - actin ( magenta ) in spheroids ( max . projection ~ 10 Z - slices ) Scale bar , 5μm . Tips for which ARF6 - mNG and F - Actin intensity profiles were measured are annotated , ECM to body , yellow arrow .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal images and intensity profiles of ID8 Trp53-/- and Trp53-/-;Pten-/- 1.15 cells expressing mNeonGreen (mNG)-tagged ARF6 (green) and F-actin (magenta) in spheroids (max. projection ~ 10 Z-slices) Scale bar, 5μm. Tips for which ARF6-mNG and F-Actin intensity profiles were measured are annotated, ECM to body, yellow arrow.


Heatmap , ( J ) ARF6 interactors across genotypes . White to blue colour or blue to red , ARF6 interaction score , Log2Fold Student ' s t - test Difference in LFQ intensity compared to control ID8 Trp53 - / - ; Pten - / - 1 . 15 TurboID alone . Interactors , sorted , descending order of mean interaction . N = 4 independent lysate preparations from each sub - line .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Heatmap, (J) ARF6 interactors across genotypes. White to blue colour or blue to red, ARF6 interaction score, Log2Fold Student's t-test Difference in LFQ intensity compared to control ID8 Trp53-/-;Pten-/- 1.15 TurboID alone. Interactors, sorted, descending order of mean interaction. N=4 independent lysate preparations from each sub-line.


C . Western blot , β1 - integrin ( ITGB1 ) , pS473 - AKT , AKT , ARF6 from deconvolved ITGB1 sgRNA - expressing cells . VCL , loading control for ITGB1 , sample integrity control for other blots . Representative blots of n = 3 independent lysate preparations . D . Quantitation of ( C ) . Data , mean ± SD for pS473 - AKT : total AKT band intensity ratio , total AKT or ARF6 intensity , normalised to control ( sgNT ID8 Trp53 - / - ; Pten - / - clone 1 . 15 ) cells . P - values , unpaired , two - tailed t - test .

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C. Western blot, β1-integrin (ITGB1), pS473-AKT, AKT, ARF6 from deconvolved ITGB1 sgRNA-expressing cells. VCL, loading control for ITGB1, sample integrity control for other blots. Representative blots of n=3 independent lysate preparations. D. Quantitation of (C). Data, mean ± SD for pS473-AKT:total AKT band intensity ratio, total AKT or ARF6 intensity, normalised to control (sgNT ID8 Trp53-/-;Pten-/- clone 1.15) cells. P-values, unpaired, two-tailed t-test.


E - F . Frequency of Spherical and Hyper - protrusive phenotypes in ID8 Trp53 - / - ; Pten - / - 1 . 15spheroids upon CRISPR - mediated KO of ( E ) Itgβ1 or ( F ) Agap1 , 6hr time intervals over 72 hrs . Heatmap ( grayscale ) - phenotype proportion ( z - score ) in control ( sgNT ) . Heatmap ( blue - red ) - log2 fold change from control . P - values , bubble size ( Cochran - Mantel - Haenszel test with Bonferroni adjustment ) . Black dot , homogenous effect across independent experiments ( Breslow - Day test , Bonferroni adjustment , non - significant ) . N = 3 independent experiments , 1 - 5 technical replicates / experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E-F. Frequency of Spherical and Hyper-protrusive phenotypes in ID8 Trp53-/-;Pten-/- 1.15spheroids upon CRISPR-mediated KO of (E) Itgβ1 or (F) Agap1, 6hr time intervals over 72 hrs. Heatmap (grayscale) - phenotype proportion (z-score) in control (sgNT). Heatmap (blue-red) - log2 fold change from control. P-values, bubble size (Cochran-Mantel-Haenszel test with Bonferroni adjustment). Black dot, homogenous effect across independent experiments (Breslow-Day test, Bonferroni adjustment, non-significant). N=3 independent experiments, 1-5 technical replicates/experiment.


H . Representative confocal images of Trp53 - / - and Trp53 - / - ; Pten - / - clone 1 . 15 spheroids expressing sgNT , sgAgap1 ( sg3 ) or sgItgb1 ( sg4 ) , stained for collagen IV ( grayscale ) and F - Actin ( magenta ) . Boxed areas , basement membrane region in higher magnification . Arrowheads , Collagen IV labelling that is : well - defined , green ; fragmented , yellow ; absent , navy . Scale bar , 53μm . I . Quantitation of ( H ) . Collagen IV basement membrane staining as Defined , Fragmented , or Absent in spheroids set up across n = 3 independent experiments , 1 technical replicate / experiment , 5 - 9 fields imaged per technical replicate , 365 spheroids scored in total . Data , mean ± SD of % of spheroids in each phenotype for independent experiments , with circles representing technical replicates . Unpaired t - test , p values annotated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Representative confocal images of Trp53-/- and Trp53-/-;Pten-/- clone 1.15 spheroids expressing sgNT, sgAgap1 (sg3) or sgItgb1 (sg4), stained for collagen IV (grayscale) and F-Actin (magenta). Boxed areas, basement membrane region in higher magnification. Arrowheads, Collagen IV labelling that is: well-defined, green; fragmented, yellow; absent, navy. Scale bar, 53μm. I. Quantitation of (H). Collagen IV basement membrane staining as Defined, Fragmented, or Absent in spheroids set up across n=3 independent experiments, 1 technical replicate/experiment, 5-9 fields imaged per technical replicate, 365 spheroids scored in total. Data, mean ± SD of % of spheroids in each phenotype for independent experiments, with circles representing technical replicates. Unpaired t-test, p values annotated.


C . Western blots of ID8 Trp53 - / - ; Pten - / - 1 . 15 cells expressing either sgNT or sgAgap1 ( sg3 ) and either mNeonGreen ( mNG ) or CRISPR - resistant mNG - Agap1 _ S or - L isoforms . Blotted for ARF6 , pS473 - AKT , AKT , mNG , and VCL . VCL is loading control for AKT , pS473 - AKT and ARF6 and sample integrity control for others . n = 3 independent lysate preparations . D . Quantitation of ( C ) . Data , mean ± SD for ARF6 and pS473 / AKT band intensity ratio , normalised to sgNT . P - values , unpaired two - tailed t - test , annotated when significant .

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C. Western blots of ID8 Trp53-/-;Pten-/- 1.15 cells expressing either sgNT or sgAgap1 (sg3) and either mNeonGreen (mNG) or CRISPR-resistant mNG-Agap1_S or -L isoforms. Blotted for ARF6, pS473-AKT, AKT, mNG, and VCL. VCL is loading control for AKT, pS473-AKT and ARF6 and sample integrity control for others. n=3 independent lysate preparations. D. Quantitation of (C). Data, mean ± SD for ARF6 and pS473/AKT band intensity ratio, normalised to sgNT. P-values, unpaired two-tailed t-test, annotated when significant.


E - F . Quantitation of ID8 Trp53 - / - ; Pten - / - 1 . 15 spheroids treated with sgNT or AGAP1 - targeting sg3 and expressing either mNG or mNG - fusion with either isoform of AGAP1 , 6 hr time intervals over 72 hrs . ( E ) Heatmap ( viridis ) - area presented as mean of Z - score values , normalised to control ( sgNT ) . ( F ) Frequency of Spherical and Hyper - protrusive phenotypes . Heatmap ( grayscale ) - phenotype proportion ( z - score ) in control . Heatmap ( blue - red ) - log2 fold change from control . P - values , bubble size ( Cochran - Mantel - Haenszel test with Bonferroni adjustment ) . Black dot , homogenous effect across independent experiments ( Breslow - Day test , Bonferroni adjustment , non - significant ) . N = 3 independent experiments , 5 - 6 technical replicates / experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E-F. Quantitation of ID8 Trp53-/-;Pten-/- 1.15 spheroids treated with sgNT or AGAP1-targeting sg3 and expressing either mNG or mNG-fusion with either isoform of AGAP1, 6 hr time intervals over 72 hrs. (E) Heatmap (viridis) - area presented as mean of Z-score values, normalised to control (sgNT). (F) Frequency of Spherical and Hyper-protrusive phenotypes. Heatmap (grayscale) - phenotype proportion (z-score) in control. Heatmap (blue-red) - log2 fold change from control. P-values, bubble size (Cochran-Mantel-Haenszel test with Bonferroni adjustment). Black dot, homogenous effect across independent experiments (Breslow-Day test, Bonferroni adjustment, non-significant). N=3 independent experiments, 5-6 technical replicates/experiment.


G . Representative phase contrast images of spheroids described in ( E - F ) . Outlines pseudocoloured for classification ( Spherical , green ; Hyper - protrusive , blue ) . Magnified individual spheroids from boxed regions at indicated timepoints . Arrowheads , protrusions into ECM . Scale bars , 400μm or 17μm , as indicated .

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G. Representative phase contrast images of spheroids described in (E-F). Outlines pseudocoloured for classification (Spherical, green; Hyper-protrusive, blue). Magnified individual spheroids from boxed regions at indicated timepoints. Arrowheads, protrusions into ECM. Scale bars, 400μm or 17μm, as indicated.


ARF6 and AGAP1 mRNA levels in LCM normal ovarian surface epithelium versus HGSOC epithelium or normal ovarian stroma versus OC - associated stroma . Specific datasets , sample size ( n ) and p - values ( Mann - Whitney ) annotated , whiskers Min - Max , line at median .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ARF6 and AGAP1 mRNA levels in LCM normal ovarian surface epithelium versus HGSOC epithelium or normal ovarian stroma versus OC-associated stroma. Specific datasets, sample size (n) and p-values (Mann-Whitney) annotated, whiskers Min-Max, line at median.


K - O . Overall survival ( % patients , months ; TCGA OV dataset ) , of patients grouped by low ( M1 ) versus high ( M2 ) levels , based on a median split , of ( K ) ARF6 mRNA , ( L ) AGAP1 mRNA , ( M ) AGAP1 Exon 14 percentage spliced in ratio ( PSI ) , ( N ) combination of ARF6 and AGAP1 mRNA , or ( O ) combination of ARF6 mRNA and AGAP1 Ex14 PSI . Median survival , sample size ( n ) and p - value , Log - rank test ( Mantel - Cox ) annotated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K-O. Overall survival (% patients, months; TCGA OV dataset), of patients grouped by low (M1) versus high (M2) levels, based on a median split, of (K) ARF6 mRNA, (L) AGAP1 mRNA, (M) AGAP1 Exon 14 percentage spliced in ratio (PSI), (N) combination of ARF6 and AGAP1 mRNA, or (O) combination of ARF6 mRNA and AGAP1 Ex14 PSI. Median survival, sample size (n) and p-value, Log-rank test (Mantel-Cox) annotated.


A - B . Immunofluorescence and confocal imaging of Trp53 - / - ; Pten - / - 1 . 15 spheroids stained for α5 - integrin or β1 - integrin ( grey or FIRE LUT ) , Hoechst ( blue ) and F - actin ( magenta ) . Magnified images from boxed regions shown . Arrowheads , labelling at protrusion tips . Scale bars , 5 μm . Representative of n = 3 spheroids imaged . ( B ) Intensity profiles for integrins ( grey ) and F - actin ( magenta ) from spheroids in ( A ) . Tip measured is annotated , ECM to body , yellow arrow , tip , white arrowhead .

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A-B. Immunofluorescence and confocal imaging of Trp53-/-;Pten-/- 1.15 spheroids stained for α5-integrin or β1-integrin (grey or FIRE LUT), Hoechst (blue) and F-actin (magenta). Magnified images from boxed regions shown. Arrowheads, labelling at protrusion tips. Scale bars, 5 μm. Representative of n=3 spheroids imaged. (B) Intensity profiles for integrins (grey) and F-actin (magenta) from spheroids in (A). Tip measured is annotated, ECM to body, yellow arrow, tip, white arrowhead.


C - D . Immunofluorescence and confocal imaging of Trp53 - / - ; Pten - / - 1 . 15 spheroids stained for pFAK ( Y379 ) or pSRC Family Kinases ( SFK pY416 ) ( grey or FIRE LUT ) , Hoechst ( blue ) and F - actin ( magenta ) . Magnified images from boxed regions shown . Arrowheads , positive staining . Scale bars , 5 μm . Representative of n = 5 spheroids imaged . ( D ) Intensity profiles for active FAK and Src ( grey ) and F - actin ( magenta ) from spheroids in ( C ) . Tip measured is annotated , ECM to body , yellow arrow , tip , white arrowhead .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-D. Immunofluorescence and confocal imaging of Trp53-/-;Pten-/- 1.15 spheroids stained for pFAK (Y379) or pSRC Family Kinases (SFK pY416) (grey or FIRE LUT), Hoechst (blue) and F-actin (magenta). Magnified images from boxed regions shown. Arrowheads, positive staining. Scale bars, 5 μm. Representative of n=5 spheroids imaged. (D) Intensity profiles for active FAK and Src (grey) and F-actin (magenta) from spheroids in (C). Tip measured is annotated, ECM to body, yellow arrow, tip, white arrowhead.


E - H . Representative capture ELISA graphs ( E , G ) and associated quantitation ( F , H ) for recycling of internalized cargoes between Trp53 - / - versus Trp53 - / - ; Pten - / - cells or Trp53 - / - ; Pten - / - cells expressing shScramble versus shArf6 for active β1 - integrin . Graphs shown are representative of n = 2 ( E ) or n = 3 ( G ) independent replicates . Data , mean ( black square ) ± SD for repeated experiments ( large circles ) , 1 - 3 technical replicates / experiment / timepoint ( small circles ) , two - tailed t - test , p - values are annotated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E-H. Representative capture ELISA graphs (E,G) and associated quantitation (F,H) for recycling of internalized cargoes between Trp53-/- versus Trp53-/-;Pten-/- cells or Trp53-/-;Pten-/- cells expressing shScramble versus shArf6 for active β1-integrin. Graphs shown are representative of n=2 (E) or n=3 (G) independent replicates. Data, mean (black square) ± SD for repeated experiments (large circles), 1-3 technical replicates/experiment/timepoint (small circles), two-tailed t-test, p-values are annotated.


I - K . Overall survival ( % patients , months ; TCGA OV dataset ) of patients grouped into combined expression based on median mRNA split . ( I ) Low ( red line , M1 ) or high ( blue line , M2 ) expression for all mRNA , control , remaining patients ( green line ) , ( J ) , same as ( I ) , but CYTH2 Ex9 PSI , rather than total CYTH2 . ( K ) , as for ( I ) , but PTEN protein levels split by quantiles ( red and blue , Q1 + Q2 , Q3 , low PTEN , green Q4 , high PTEN ) . Median survival , sample size ( n ) and p - value , Log - rank test ( Mantel - Cox ) annotated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I-K. Overall survival (% patients, months; TCGA OV dataset) of patients grouped into combined expression based on median mRNA split. (I) Low (red line, M1) or high (blue line, M2) expression for all mRNA, control, remaining patients (green line), (J), same as (I), but CYTH2 Ex9 PSI, rather than total CYTH2. (K), as for (I), but PTEN protein levels split by quantiles (red and blue, Q1+Q2,Q3, low PTEN, green Q4, high PTEN). Median survival, sample size (n) and p-value, Log-rank test (Mantel-Cox) annotated.


A DNA content of mCFU - E and BaF3 - EpoR cells in response to different Epo concentrations . [ 3H ] - Thymidine incorporation was measured after 14 h ( mCFU - E ) or 38 h ( BaF3 - EpoR ) . Data represented as mean ± standard deviation , N = 3 . Lines represent sigmoidal regression . EC50 values are given .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A DNA content of mCFU-E and BaF3-EpoR cells in response to different Epo concentrations. [3H]-Thymidine incorporation was measured after 14 h (mCFU-E) or 38 h (BaF3-EpoR). Data represented as mean ± standard deviation, N=3. Lines represent sigmoidal regression. EC50 values are given.


B Cell doubling with time in response to 50 U / ml Epo . Cell numbers were determined by manual counting using trypan blue exclusion assay . Data represented as mean ± standard deviation , N = 3 . Lines represent exponential regression . Doubling time is indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 Cell doubling with time in response to 50 U/ml Epo. Cell numbers were determined by manual counting using trypan blue exclusion assay. Data represented as mean ± standard deviation, N=3. Lines represent exponential regression. Doubling time is indicated.


C Size determinations of mCFU - E and BaF3 - EpoR cells . Exemplary fluorescence microscopy pictures upon Hoechst staining for nucleus visualization with 60x objective . The bar respresents 10 µm distance ( upper panel ) . Cell diameter was measured by Imaging flow cytometry . Cytoplasm was stained with Calcein , nuclei were stained with DRAQ5 . Probability density function of size distribution with indicated mean diameter of mCFU - E and BaF3EpoR cells . All cells were growth - factor deprived and unstimulated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Size determinations of mCFU-E and BaF3-EpoR cells. Exemplary fluorescence microscopy pictures upon Hoechst staining for nucleus visualization with 60x objective. The bar respresents 10 µm distance (upper panel). Cell diameter was measured by Imaging flow cytometry. Cytoplasm was stained with Calcein, nuclei were stained with DRAQ5. Probability density function of size distribution with indicated mean diameter of mCFU-E and BaF3EpoR cells. All cells were growth-factor deprived and unstimulated.


D Epo - induced phosphorylation of EpoR , AKT and ERK of mCFU - E and BaF3 - EpoR cells . Above each panel the number of growth - factor deprived cells examined per time point is indicated as well as the concentration of Epo applied for stimulation . To the left the position of the molecular weight marker is indicated in kDa and arrowheads indicate the position of the protein of interest . For the detection of the EpoR , 5x106 mCFU - E cells were stimulated with 2 . 5 U / ml Epo and 5x106 BaF3 - EpoR cells were stimulated with 5 U / ml Epo . Cells were lysed , subjected to immunoprecipitation with anti - EpoR and were analyzed by immunoblotting using either anti - pTyr ( pEpoR ) or anti - EpoR ( EpoR ) antibodies . For the detection of AKT , mCFU - E cells were stimulated with 2 . 5 U / ml Epo and BaF3 - EpoR cells were stimulated with 5 U / ml Epo . Per time point cellular lysates equivalent to 5x105 mCFU - E cells and 1x106 BaF3 - EpoR cells were analyzed by immunoblotting using anti - pAKT and anti - AKT antibodies . For the detection of ERK , cells were stimulated with 50 U / ml Epo . Per time point cellular lysates equivalent to 8x105 cells were analyzed by immunoblotting . Immunoblot detection was performed with chemiluminescence utilizing a CCD camera device ( ImageQuant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Epo-induced phosphorylation of EpoR, AKT and ERK of mCFU-E and BaF3-EpoR cells. Above each panel the number of growth-factor deprived cells examined per time point is indicated as well as the concentration of Epo applied for stimulation. To the left the position of the molecular weight marker is indicated in kDa and arrowheads indicate the position of the protein of interest. For the detection of the EpoR, 5x106 mCFU-E cells were stimulated with 2.5 U/ml Epo and 5x106 BaF3-EpoR cells were stimulated with 5 U/ml Epo. Cells were lysed, subjected to immunoprecipitation with anti-EpoR and were analyzed by immunoblotting using either anti-pTyr (pEpoR) or anti-EpoR (EpoR) antibodies. For the detection of AKT, mCFU-E cells were stimulated with 2.5 U/ml Epo and BaF3-EpoR cells were stimulated with 5 U/ml Epo. Per time point cellular lysates equivalent to 5x105 mCFU-E cells and 1x106 BaF3-EpoR cells were analyzed by immunoblotting using anti-pAKT and anti-AKT antibodies. For the detection of ERK, cells were stimulated with 50 U/ml Epo. Per time point cellular lysates equivalent to 8x105 cells were analyzed by immunoblotting. Immunoblot detection was performed with chemiluminescence utilizing a CCD camera device (ImageQuant).


E Absolute concentrations of pEpoR , pAKT and ppERK in mCFU - E and BaF3 - EpoR cells with time in response to 50 U / ml Epo . Experimental data of a representative experiment is depicted with filled circles , dashed lines represent splines . Error bars indicate standard deviation estimated by an error model .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Absolute concentrations of pEpoR, pAKT and ppERK in mCFU-E and BaF3-EpoR cells with time in response to 50 U/ml Epo. Experimental data of a representative experiment is depicted with filled circles, dashed lines represent splines. Error bars indicate standard deviation estimated by an error model.


F Expression of cell - cycle indicator genes of mCFU - E and BaF3 - EpoR cells in response to 5 U / ml Epo . Genes were selected based on microarray analysis . Experimental data is shown as fold change to unstimulated cells with mean ± standard deviation , N = 3 . Welch Modified Two - Sample t - Test , * p < 0 . 05 .

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F Expression of cell-cycle indicator genes of mCFU-E and BaF3-EpoR cells in response to 5 U/ml Epo. Genes were selected based on microarray analysis. Experimental data is shown as fold change to unstimulated cells with mean ± standard deviation, N=3. Welch Modified Two-Sample t-Test, * p<0.05.


G Fold change of fractions of cells in S / G2 / M phase of the cell cycle with respect to 0 U / ml Epo . Growth - factor deprived cells were stimulated with indicated Epo doses for given time . Fractions in sub - G1 , G1 , S , G2 / M were determined by propidium iodide ( PI ) staining for DNA content . N = 3 . Welch Modified Two - Sample t - Test , n . s . = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . The percentage of cells in S / G2 / M phase in unstimulated cells is additionally indicated .

G Fold change of fractions of cells in S/G2/M phase of the cell cycle with respect to 0 U/ml Epo. Growth-factor deprived cells were stimulated with indicated Epo doses for given time. Fractions in sub-G1, G1, S, G2/M were determined by propidium iodide (PI) staining for DNA content. N=3. Welch Modified Two-Sample t-Test, n.s. = not significant, * p < 0.05 , ** p < 0.01 , *** p < 0.005. The percentage of cells in S/G2/M phase in unstimulated cells is additionally indicated.


B - G Model calibration with time - resolved quantitative immunoblot data of mCFU - E cells in blue and BaF3 - EpoR cells in black . Growth - factor deprived mCFU - E cells ( 5x106 cells per condition ) and BaF3 - EpoR cells ( 1x107 cells per condition ) were stimulated with different Epo doses and absolute concentrations were determined for pEpoR ( B ) , pAKT ( C ) , ppERK ( D ) . The scale for pS6 ( E ) was estimated in arbitrary units .

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B-G Model calibration with time-resolved quantitative immunoblot data of mCFU-E cells in blue and BaF3-EpoR cells in black. Growth-factor deprived mCFU-E cells (5x106 cells per condition) and BaF3-EpoR cells (1x107 cells per condition) were stimulated with different Epo doses and absolute concentrations were determined for pEpoR (B), pAKT (C), ppERK (D). The scale for pS6 (E) was estimated in arbitrary units.


GTP - Ras ( F ) and ppERK were determined upon stimulation with indicated , color - coded Epo doses . pEpoR was analyzed by immunoprecipitation followed by immunoblotting , GTP - Ras was analyzed after pulldown using a fusion protein harboring GST fused to the Ras binding domain of Raf1 followed by detection by quantitative immunoblotting . For pAkt and ppERK , cellular lysates were subjected to quantitative immunoblotting . Calibrator proteins were used for EpoR , AKT , GTP - Ras and ERK to facilitate the conversion to nM concentrations . Experimental data represented by filled circles . Error bars represent standard deviation estimated by an error model . Solid lines represent model trajectories .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

GTP-Ras (F) and ppERK were determined upon stimulation with indicated, color-coded Epo doses. pEpoR was analyzed by immunoprecipitation followed by immunoblotting, GTP-Ras was analyzed after pulldown using a fusion protein harboring GST fused to the Ras binding domain of Raf1 followed by detection by quantitative immunoblotting. For pAkt and ppERK, cellular lysates were subjected to quantitative immunoblotting. Calibrator proteins were used for EpoR, AKT, GTP-Ras and ERK to facilitate the conversion to nM concentrations. Experimental data represented by filled circles. Error bars represent standard deviation estimated by an error model. Solid lines represent model trajectories.


A Model calibration with mCFU - E wild - type cells , and mCFU - E cells overexpressing SHIP1 or PTEN , and BaF3 - EpoR wild - type cells . Experimental data represented by filled circles . Error bars represent standard deviation estimated by an error model . Solid lines represent model trajectories .

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A Model calibration with mCFU-E wild-type cells, and mCFU-E cells overexpressing SHIP1 or PTEN, and BaF3-EpoR wild-type cells. Experimental data represented by filled circles. Error bars represent standard deviation estimated by an error model. Solid lines represent model trajectories.


B Model prediction of pAKT dynamics in BaF3 - EpoR cells overexpressing PTEN or SHIP1 . Model predictions are represented by solid lines . Experimental validation data obtained by quantitative immunoblotting represented by filled circles . Error bars represent standard deviation estimated by an error model .

O O O O O O O O O O O O O O O O O O O O O O O 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 Model prediction of pAKT dynamics in BaF3-EpoR cells overexpressing PTEN or SHIP1. Model predictions are represented by solid lines. Experimental validation data obtained by quantitative immunoblotting represented by filled circles. Error bars represent standard deviation estimated by an error model.


D Experimental validation of basal DUSP expression in mCFU - E and BaF3 - EpoR . Quantitative RT - PCR was performed with all samples on the same plate , allowing a direct comparison of mRNA levels between mCFU - E and BaF3 - EpoR cells . Data is normalized to the Rpl32 gene . Ratios of expression in BaF3 - EpoR cells compared to mCFU - E cells are shown as box plots . N = 10 . For details , see Appendix K .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Experimental validation of basal DUSP expression in mCFU-E and BaF3-EpoR. Quantitative RT-PCR was performed with all samples on the same plate, allowing a direct comparison of mRNA levels between mCFU-E and BaF3-EpoR cells. Data is normalized to the Rpl32 gene. Ratios of expression in BaF3-EpoR cells compared to mCFU-E cells are shown as box plots. N=10. For details, see Appendix K.


A Model prediction and experimental validation for pAKT , ppERK and pS6 dynamics of 32D - EpoR cells in response to 50 U / ml Epo . Simulations are based on measured , cell - type - specific protein abundance of 32D - EpoR cells and global kinetic rates estimated from mCFUE and BaF3 - EpoR cells . Model predictions are represented by solid lines . Experimental validation data obtained by quantitative immunoblotting represented by filled circles . Error bars represent standard deviation estimated by an error model .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Model prediction and experimental validation for pAKT, ppERK and pS6 dynamics of 32D-EpoR cells in response to 50 U/ml Epo. Simulations are based on measured, cell-type-specific protein abundance of 32D-EpoR cells and global kinetic rates estimated from mCFUE and BaF3-EpoR cells. Model predictions are represented by solid lines. Experimental validation data obtained by quantitative immunoblotting represented by filled circles. Error bars represent standard deviation estimated by an error model.


C Model prediction and experimental validation for RSK , AKT , ERK and S6 activation upon RSK overexpression ( oe ) and 5 U / ml Epo stimulation in 32DEpoR cells . Simulations are based on measured , cell - type - specific protein abundance of 32D - EpoR cells and global kinetic rates estimated from mCFUE and BaF3 - EpoR cells . Model predictions are represented by solid lines . Experimental validation data obtained by quantitative immunoblotting represented by filled circles . Error bars represent standard deviation estimated by an error model . Integrated pS6 was significantly higher upon RSK overexpression as compared to 32D - EpoR wild - type ( wt ) cells , N = 4 ( lower right panel ) . Two - Sample t - Test , * * * p < 0 . 005 .

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C Model prediction and experimental validation for RSK, AKT, ERK and S6 activation upon RSK overexpression (oe) and 5 U/ml Epo stimulation in 32DEpoR cells. Simulations are based on measured, cell-type-specific protein abundance of 32D-EpoR cells and global kinetic rates estimated from mCFUE and BaF3-EpoR cells. Model predictions are represented by solid lines. Experimental validation data obtained by quantitative immunoblotting represented by filled circles. Error bars represent standard deviation estimated by an error model. Integrated pS6 was significantly higher upon RSK overexpression as compared to 32D-EpoR wild-type (wt) cells, N=4 (lower right panel). Two-Sample t-Test, *** p<0.005.


D Impact of AKT , Ras or PTEN overexpression on BaF3 - induced pS6 dynamics in EpoR and BaF3 - PTEN cells . Quantitative AKT upon overexpression of PTEN , Ras , a constitutive active BaF3 protein , or the empty vector control in EpoR cells or BaF3 - mCFU - E cells . BaF3 - factor deprived mCFU - E cells ( 5x106 cells per condition ) and Epo - immunoblotting cells ( 1x107 cells per condition ) were stimulated with 5 U / ml Epo for indicated time points . Cellular lysates were analyzed by S6 employing sequential reprobing anti - pAKT , anti - ppERK , anti - pS6 , anti - Epo and to ensure equal loading with anti - beta - actin antibodies . Detection was performed with chemiluminescence using a CCD camera device ( ImageQuant ) . Quantification of pS6 on the right is depicted as fold change to wild - type samples at 30 minutes after Epo stimulation . Error bars represent standard deviation . oe : overexpression . N = 3 . Welch Modified Two - Sample t - Test , n . s . = not significant , * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Impact of AKT, Ras or PTEN overexpression on BaF3-induced pS6 dynamics in EpoR and BaF3-PTEN cells. Quantitative AKT upon overexpression of PTEN, Ras, a constitutive active BaF3 protein, or the empty vector control in EpoR cells or BaF3-mCFU-E cells. BaF3-factor deprived mCFU-E cells (5x106 cells per condition) and Epo-immunoblotting cells (1x107 cells per condition) were stimulated with 5 U/ml Epo for indicated time points. Cellular lysates were analyzed by S6 employing sequential reprobing anti-pAKT, anti-ppERK, anti-pS6, anti-Epo and to ensure equal loading with anti-beta-actin antibodies. Detection was performed with chemiluminescence using a CCD camera device (ImageQuant). Quantification of pS6 on the right is depicted as fold change to wild-type samples at 30 minutes after Epo stimulation. Error bars represent standard deviation. oe: overexpression. N=3. Welch Modified Two-Sample t-Test, n.s. = not significant, * p < 0.05


A Epo - induced signaling upon AKT or MEK inhibitor treatment . Growth - factor deprived cells were pretreated for half an hour with AKT VIII or U0126 , respectively , and subsequently stimulated with 5 U / ml Epo . PDI served as loading control .

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-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

A Epo-induced signaling upon AKT or MEK inhibitor treatment. Growth-factor deprived cells were pretreated for half an hour with AKT VIII or U0126, respectively, and subsequently stimulated with 5 U/ml Epo. PDI served as loading control.


B Model calibration with integrated pAKT , ppERK and pS6 data upon inhibitor treatment and 5 U / ml Epo stimulation of mCFU - E , 32D - EpoR and BaF3 - EpoR cells . Area under curve from time - resolved , quantitative immunoblotting data was calculated and is represented by filled circles . Error bars represent standard deviation estimated by an error model . Solid lines represent model trajectories .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Model calibration with integrated pAKT, ppERK and pS6 data upon inhibitor treatment and 5 U/ml Epo stimulation of mCFU-E, 32D-EpoR and BaF3-EpoR cells. Area under curve from time-resolved, quantitative immunoblotting data was calculated and is represented by filled circles. Error bars represent standard deviation estimated by an error model. Solid lines represent model trajectories.


C Analysis of cell - cycle indicator genes upon AKT VIII and U0126 treatment . Growth - factor deprived cells were pretreated for half an hour indicated doses of a single inhibitor , followed by stimulation with 5 U / ml Epo for 0 h and 3 h . The expression of cyclinD2 , cyclinG2 and p27 was measured by quantitative RT - PCR and normalized to the Rpl32 gene . Genes were selected based on microarray analysis . Experimental data is shown as fold change to unstimulated cells with mean ± standard deviation , N = 3 . Welch Modified Two - Sample t - Test , n . s . not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Analysis of cell-cycle indicator genes upon AKT VIII and U0126 treatment. Growth-factor deprived cells were pretreated for half an hour indicated doses of a single inhibitor, followed by stimulation with 5 U/ml Epo for 0 h and 3 h. The expression of cyclinD2, cyclinG2 and p27 was measured by quantitative RT-PCR and normalized to the Rpl32 gene. Genes were selected based on microarray analysis. Experimental data is shown as fold change to unstimulated cells with mean ± standard deviation, N=3. Welch Modified Two-Sample t-Test, n.s. not significant, * p<0.05, ** p<0.01, *** p<0.005.


B Linear regression fit for the respective cell - cycle indicator of experimental data . Data points represent mean ± standard deviation , N = 3 . Solid lines represent the linear regression fit .

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B Linear regression fit for the respective cell-cycle indicator of experimental data. Data points represent mean ± standard deviation, N=3. Solid lines represent the linear regression fit.


Lower panel : Experimental validation of PTEN and SHIP1 overexpression effects on Epo - dependent proliferation . Proliferation was assessed using [ 3H ] - Thymidine incorporation 14 h ( mCFU - E ) or 38 h ( BaF3 - EpoR ) after retroviral transduction with PTEN or SHIP1 construct for overexpression . Data represented as mean ± standard deviation , N = 3 . EC50 values are given .

Lower panel: Experimental validation of PTEN and SHIP1 overexpression effects on Epo-dependent proliferation. Proliferation was assessed using [3H]-Thymidine incorporation 14 h (mCFU-E) or 38 h (BaF3-EpoR) after retroviral transduction with PTEN or SHIP1 construct for overexpression. Data represented as mean ± standard deviation, N=3. EC50 values are given.


Lower panel : Experimental validation of proliferation upon combined inhibitor treatment and 5 U / ml Epo stimulation . Proliferation was measured as cell numbers after 14 h ( mCFU - E ) or 38 h ( BaF3 - EpoR , 32D - EpoR ) with Coulter Counter . Maximum was scaled to

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Lower panel: Experimental validation of proliferation upon combined inhibitor treatment and 5 U/ml Epo stimulation. Proliferation was measured as cell numbers after 14 h (mCFU-E) or 38 h (BaF3-EpoR, 32D-EpoR) with Coulter Counter. Maximum was scaled to 1


A Venn diagramm representing overlap of hCFU - E proteome from three independent healthy donors . In total 6 , 925 proteins were

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A Venn diagramm representing overlap of hCFU-E proteome from three independent healthy donors. In total 6,925 proteins were quantified


B Size determinations of hCFU - E compared to mCFU - E cells . Cell diameter was measured by Imaging flow cytometry . Cytoplasm was stained with Calcein , nuclei were stained with DRAQ5 . Probability density function of size distribution with indicated mean diameter of hCFU - E and mCFU - E cells . All cells were growth - factor deprived and unstimulated .

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B Size determinations of hCFU-E compared to mCFU-E cells. Cell diameter was measured by Imaging flow cytometry. Cytoplasm was stained with Calcein, nuclei were stained with DRAQ5. Probability density function of size distribution with indicated mean diameter of hCFU-E and mCFU-E cells. All cells were growth-factor deprived and unstimulated.


C Cytoplasmic volumes of BaF3 - EpoR , 32D - EpoR , mCFU - E and the hCFU - E cells . All volumes were determined using imaging flow cytometry as described in ( B ) .

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C Cytoplasmic volumes of BaF3-EpoR, 32D-EpoR, mCFU-E and the hCFU-E cells. All volumes were determined using imaging flow cytometry as described in (B).


D Correlation between protein molecules per cell for key signaling components determined by quantiative immunoblotting or quantitative mass spectrometry and the " proteomic ruler " method ( Wiśniewski et al , 2014 ) in BaF3 - EpoR and mCFU - E cells . Diagonal line as guide to the eye . Spearman ' s rank - based coefficient of correlation ρ = 0 . 88 .

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D Correlation between protein molecules per cell for key signaling components determined by quantiative immunoblotting or quantitative mass spectrometry and the "proteomic ruler" method (Wiśniewski et al, 2014) in BaF3-EpoR and mCFU-E cells. Diagonal line as guide to the eye. Spearman's rank-based coefficient of correlation ρ=0.88.


E Protein abundance of key signaling components were determined by mass spectrometry of whole - cell lysates of hCFU - E cells . Copy numbers of proteins per cell were obtained by the " proteomic ruler " method and converted to cytoplasmic concentrations with the volumes calculated from ( B ) and shown in ( C ) .

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E Protein abundance of key signaling components were determined by mass spectrometry of whole-cell lysates of hCFU-E cells. Copy numbers of proteins per cell were obtained by the "proteomic ruler" method and converted to cytoplasmic concentrations with the volumes calculated from (B) and shown in (C).


F Model prediction and experimental validation of proliferation upon single or combined inhibitor treatment with AKT VIII and U0126 upon 5 U / ml Epo stimulation . Maximum proliferation was scaled to 1 . Proliferation was measured as cell numbers , counted with a hemocytometer after 96 hours by trypan blue exclusion assay . Solid lines represent model trajectories . Experimental data is represented as mean ± standard deviation . N =

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Model prediction and experimental validation of proliferation upon single or combined inhibitor treatment with AKT VIII and U0126 upon 5 U/ml Epo stimulation. Maximum proliferation was scaled to 1. Proliferation was measured as cell numbers, counted with a hemocytometer after 96 hours by trypan blue exclusion assay. Solid lines represent model trajectories. Experimental data is represented as mean ± standard deviation. N=3


( a ) RT - PCR expression and quantification of IGF1 , IGF1Rβ and IGF2 levels in untreated C2C12 and shCD20 C2C12 myoblasts and 10 nM IGF1 - treated C2C12 and over - expressing CD20 C2C12 myoblasts . Each experiment was replicated independently four times .

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(a) RT-PCR expression and quantification of IGF1, IGF1Rβ and IGF2 levels in untreated C2C12 and shCD20 C2C12 myoblasts and 10 nM IGF1-treated C2C12 and over-expressing CD20 C2C12 myoblasts. Each experiment was replicated independently four times.


( b ) Immunofluorescence for IGF2 ( in green ) in untreated and 10 nM IGF1 - treated C2C12 myoblasts . Scale bars = 75 μm .

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(b) Immunofluorescence for IGF2 (in green) in untreated and 10 nM IGF1-treated C2C12 myoblasts. Scale bars = 75 μm.


Representative CD20 immunoprecipitation using anti - pSer + pThr in ( c ) C2C12 cells treated with IGF1 for two hours ( 2 h ) or overnight ( ON ) as indicated . Densitometric analysis of data are expressed as the ratio of CD20 / vinculin or pSer + pThr / CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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Representative CD20 immunoprecipitation using anti-pSer+pThr in (c) C2C12 cells treated with IGF1 for two hours (2 h) or overnight (ON) as indicated. Densitometric analysis of data are expressed as the ratio of CD20/vinculin or pSer+pThr/CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM.


Representative CD20 immunoprecipitation using anti - pSer + pThr in ( d ) sh - empty ( shCTR ) - and shCD20 - treated C2C12 cells treated with anti - Flag and 10 nM anti - IGF2R , as indicated . Densitometric analysis of data are expressed as the ratio of CD20 / vinculin or pSer + pThr / CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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Representative CD20 immunoprecipitation using anti-pSer+pThr in (d) sh-empty (shCTR)- and shCD20-treated C2C12 cells treated with anti-Flag and 10 nM anti-IGF2R, as indicated. Densitometric analysis of data are expressed as the ratio of CD20/vinculin or pSer+pThr/CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM.


Representative CD20 immunoprecipitation using anti - pSer + pThr in ( e ) C2C12 cells treated with IGF1 and IGF2 , as indicated . Densitometric analysis of data are expressed as the ratio of CD20 / vinculin or pSer + pThr / CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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Representative CD20 immunoprecipitation using anti-pSer+pThr in (e) C2C12 cells treated with IGF1 and IGF2, as indicated. Densitometric analysis of data are expressed as the ratio of CD20/vinculin or pSer+pThr/CD20 and are shown normalized to vinculin in arbitrary units in the lower panels shown Two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM.


( f ) Representative WB of IGF2R and phosphorylated IGF2R ( pIGF2R ) in untreated , shCD20 - treated and anti - IGF2R - treated C2C12 cells . Densitometric analysis of data are expressed as the ratio of IGF2R / actin or pIGF2R / IGF2R in arbitrary units in the lower panels . One - way ANOVA . * p < 0 . 05 ; Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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(f) Representative WB of IGF2R and phosphorylated IGF2R (pIGF2R) in untreated, shCD20-treated and anti-IGF2R-treated C2C12 cells. Densitometric analysis of data are expressed as the ratio of IGF2R/actin or pIGF2R/IGF2R in arbitrary units in the lower panels. One-way ANOVA. *p<0.05; Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM.


( g ) IGF2R immunoprecipitation products were immunoblotted for Gαi2 and WB expression of IGF2R in untreated , IGF2 - treated , anti - IGF2R - treated and IGF2 + anti - IGF2R treated C2C12 cells . Densitometric analysis of data are expressed as the ratio of Gαi2 / IGF2R in arbitrary units in the lower panel .

(g) IGF2R immunoprecipitation products were immunoblotted for Gαi2 and WB expression of IGF2R in untreated, IGF2-treated, anti-IGF2R-treated and IGF2+anti-IGF2R treated C2C12 cells. Densitometric analysis of data are expressed as the ratio of Gαi2/IGF2R in arbitrary units in the lower panel.


( h ) Representative WB of IGF1Rβ and IGF1Rβ immunoprecipitation products immunoblotted for p - Tyr in untreated , IGF2 - treated and IGF2 + anti - IGF2R - treated ( 1 : 500 , 1 : 1000 and 1 : 2000 dilutions of anti - IGF2R ) C2C12 cells ( cells were treated for 2 and 24 h ) . Densitometric analysis of data are expressed as the ratio of pTYR / IGF1Rβ in arbitrary units in the lower panel . Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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(h) Representative WB of IGF1Rβ and IGF1Rβ immunoprecipitation products immunoblotted for p-Tyr in untreated, IGF2-treated and IGF2+anti-IGF2R-treated (1:500, 1:1000 and 1:2000 dilutions of anti-IGF2R) C2C12 cells (cells were treated for 2 and 24 h). Densitometric analysis of data are expressed as the ratio of pTYR/IGF1Rβ in arbitrary units in the lower panel. Each experiment was performed in triplicate wells. All values are expressed as the mean±SEM.


( d ) Over - expressing CD20 in C2C12 myoblasts that co - expressed CD20 ( in green ) and IGF2R ( in red ) . DAPI - labelled nuclei are shown in blue . Scale bars = 75 μm .

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(d) Over-expressing CD20 in C2C12 myoblasts that co-expressed CD20 (in green) and IGF2R (in red). DAPI-labelled nuclei are shown in blue. Scale bars=75 μm.


( e ) Representative CD20 and IGF2R immunoprecipitation products immunoblotted for IGF2R and CD20 , respectively , in UNTREATED and shCD20 - treated C2C12 cell membranes and whole lysates of proteins . The immunoprecipitation output is shown as IP neg .

(e) Representative CD20 and IGF2R immunoprecipitation products immunoblotted for IGF2R and CD20, respectively, in UNTREATED and shCD20-treated C2C12 cell membranes and whole lysates of proteins. The immunoprecipitation output is shown as IP neg.


( f ) Myotubes immunofluorescence of cells in proliferation medium ( P . M . ) and after 2 , 4 and 6 days of myogenic differentiation in serum free medium . Control ( untreated ) , shCD20 - treated C2C12 cells , C2C12 and HSkM myoblasts pre - treated with anti - IGF2R for 24 hours were stained . Desmin - positive myotubes are shown in green . Scale bars = 75 μm .

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(f) Myotubes immunofluorescence of cells in proliferation medium (P.M.) and after 2, 4 and 6 days of myogenic differentiation in serum free medium. Control (untreated), shCD20-treated C2C12 cells, C2C12 and HSkM myoblasts pre-treated with anti-IGF2R for 24 hours were stained. Desmin-positive myotubes are shown in green. Scale bars=75 μm.


( g ) Fusion index quantification after 6 days of differentiation . One - way ANOVA . * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . Each experiment was performed in triplicate wells . All values are expressed as mean ± SEM .

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(g) Fusion index quantification after 6 days of differentiation. One-way ANOVA. **p<0.01; ***p<0.001; ****p<0.0001. Each experiment was performed in triplicate wells. All values are expressed as mean±SEM.


( h ) Representative WB of anti - myogenin , anti - Myf5 , anti - MyHC and anti - β - actin in total protein lysates obtained from untreated , shCD20 - treated C2C12 cells , and C2C12 and HSkM myoblasts pre - treated with anti - IGF2R for 24 hours ; cells were collected under P . M . and after 2 , 4 and 6 days of myogenic differentiation . Densitometric analysis of WB data expressed as the ratio of the indicated antibody / β - actin in arbitrary units . Two - way ANOVA test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 in comparison to the results obtained in untreated cells at the corresponding time point . # # p < 0 . 01 ; # # # p < 0 . 001 ; # # # # p < 0 . 0001 in comparison to the results obtained in shCD20 - treated C2C12 cells at the corresponding time point . Each experiment was performed in triplicate wells . All values are expressed as the mean ± SEM .

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(h) Representative WB of anti-myogenin, anti-Myf5, anti-MyHC and anti-β-actin in total protein lysates obtained from untreated, shCD20-treated C2C12 cells, and C2C12 and HSkM myoblasts pre-treated with anti-IGF2R for 24 hours; cells were collected under P.M. and after 2, 4 and 6 days of myogenic differentiation. Densitometric analysis of WB data expressed as the ratio of the indicated antibody/β-actin in arbitrary units. Two-way ANOVA test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 in comparison to the results obtained in untreated cells at the corresponding time point. ##p<0.01; ###p<0.001; ####p<0.0001 in comparison to the results obtained in shCD20-treated C2C12 cells at the corresponding time point. Each experiment was performed in triplicate wells. All values are expressed as the mean±SEM.


Immunofluorescence analysis of CD20 and IGF2R expression ( in red ) in the TA muscles of three - month - old C57Bl6 / J and mdx mice . Scale bars = 75μm . ( a ) .

Immunofluorescence analysis of CD20 and IGF2R expression (in red) in the TA muscles of three-month-old C57Bl6/J and mdx mice. Scale bars=75μm. (a).


IGF2R ( green ) and sarcomeric actin ( magenta ) expression in the VM muscles of two DMD patients . Scale bars = 25μm . ( b ) .

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IGF2R (green) and sarcomeric actin (magenta) expression in the VM muscles of two DMD patients. Scale bars=25μm. (b).


Representative WB analysis of CD20 and β - actin ( c ) expression in the TA and VM muscles of C57Bl6 / J and mdx mice ( n = 10 mice per group ) . Densitometry analysis of WB data are expressed as the CD20 / β - actin ratio , in arbitrary units in the lower panels One - way ANOVA . * * * * p < 0 . 0001 .

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Representative WB analysis of CD20 and β-actin (c) expression in the TA and VM muscles of C57Bl6/J and mdx mice (n=10 mice per group). Densitometry analysis of WB data are expressed as the CD20/β-actin ratio, in arbitrary units in the lower panels One-way ANOVA. ****p<0.0001.


Representative WB analysis of pSer + pThr and GADPH ( d ) expression in the TA and VM muscles of C57Bl6 / J and mdx mice ( n = 10 mice per group ) . Densitometry analysis of WB data are expressed as the pSer + pThr / CD20 ratio in arbitrary units in the lower panels One - way ANOVA . * * * * p < 0 . 0001 .

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Representative WB analysis of pSer+pThr and GADPH (d) expression in the TA and VM muscles of C57Bl6/J and mdx mice (n=10 mice per group). Densitometry analysis of WB data are expressed as the pSer+pThr/CD20 ratio in arbitrary units in the lower panels One-way ANOVA. ****p<0.0001.