Figure 2A . Representative H & amp ; amp ; E staining of whole lungs from EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - and EGFRL858R / Rhob + / + mice treated or not with erlotinib ( 12 . 5 mg / kg / day ) for four days . Scale bar : 5 mm . B . Quantification of the tumor / lung ratio . n = 7 for each group except for EGFRL858R / Rhob + / - placebo ( n = 6 ) . C . Representative Ki67 immunostaining of EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - and EGFRL858R / Rhob + / + mice treated or not with erlotinib ( 12 . 5 mg / kg / day ) for four days ( scale bar : 50 µm ) , and the corresponding quantification ( D ) . Three independent zones / mice lung were used for quantification . n = 21 ( 7 mice ) for each group except for EGFRL858R / Rhob + / - placebo ( n = 18 ; 6 mice ) . E . Immunostaining of cleaved caspase - 3 in lung tumors from EGFRL858R / Rhob + / + or EGFRL858R / Rhob - / - mice treated for 24 h with erlotinib at 12 . 5 mg / kg . Scale bar : 50 µm . ( * * : p < 0 . 001 vs . placebo ; * * * : p < 0 . 0001 vs . placebo ) .

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

Figure 2A. Representative H&amp;E staining of whole lungs from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days. Scale bar: 5 mm. B. Quantification of the tumor/lung ratio. n=7 for each group except for EGFRL858R/Rhob+/- placebo (n=6).C. Representative Ki67 immunostaining of EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days (scale bar: 50 µm), and the corresponding quantification (D). Three independent zones/mice lung were used for quantification. n=21 (7 mice) for each group except for EGFRL858R/Rhob+/- placebo (n=18; 6 mice).E. Immunostaining of cleaved caspase-3 in lung tumors from EGFRL858R/Rhob+/+ or EGFRL858R/Rhob-/- mice treated for 24 h with erlotinib at 12.5 mg/kg. Scale bar: 50 µm. (**: p<0.001 vs. placebo; ***: p<0.0001 vs. placebo).


Figure 3HCC4006 cells were ( A ) transfected with two siRNA against RHOB ( siB1 , siB2 ) then treated with increasing doses of erlotinib . The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells . HCC4006 cells were ( A ) transfected with two siRNA against RHOB ( siB1 , siB2 ) . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control cells ) . Data are representative of at least three independent experiments . HCC4006 cells were ( B ) transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control cells ) . Data are representative of at least three independent experiments . HCC4006 cells were ( B ) transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) then treated with increasing doses of erlotinib . The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells . HCC4006 cells were ( C ) both transfected with siRNA and transduced by adenoviruses ; then treated with increasing doses of erlotinib . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control cells ) . Data are representative of at least three independent experiments . HCC4006 cells were ( C ) both transfected with siRNA and transduced by adenoviruses ; then treated with increasing doses of erlotinib . The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells . Erlotinib IC50 values were quantified in RHOB - overexpressing or RHOB - depleted ( D ) HCC827 cells , ( E ) HCC2935 cells and ( F ) H3255 cells , as determined by an MTS assay after 72 h treatment . RHOB overexpression or inhibition was monitored by western blotting for each condition . G . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) at an increasing multiplicity of infection ( MOI ) , then erlotinib IC50 values were determined after 72 h by an MTS assay and a correlation analysis was performed . G . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) at an increasing multiplicity of infection ( MOI ) . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control cells ) . Data are representative of at least three independent experiments .

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

Figure 3HCC4006 cells were (A) transfected with two siRNA against RHOB (siB1, siB2) then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.HCC4006 cells were (A) transfected with two siRNA against RHOB (siB1, siB2). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.HCC4006 cells were (B) transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.HCC4006 cells were (B) transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.HCC4006 cells were (C) both transfected with siRNA and transduced by adenoviruses; then treated with increasing doses of erlotinib. RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.HCC4006 cells were (C) both transfected with siRNA and transduced by adenoviruses; then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.Erlotinib IC50 values were quantified in RHOB-overexpressing or RHOB-depleted (D) HCC827 cells, (E) HCC2935 cells and (F) H3255 cells, as determined by an MTS assay after 72 h treatment. RHOB overexpression or inhibition was monitored by western blotting for each condition.G. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) at an increasing multiplicity of infection (MOI), then erlotinib IC50 values were determined after 72 h by an MTS assay and a correlation analysis was performed.G. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) at an increasing multiplicity of infection (MOI). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.


Figure 4A . HCC4006 , HCC827 , HCC2935 and H3255 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for four hours with erlotinib at concentrations corresponding to the respective IC50 values determined for each control cell line . The phosphorylation status of AKT , ERK1 / 2 and EGFR was assessed by western blotting and normalized according to total protein levels . RHOB overexpression was also monitored by western blotting . B . Representative immunostaining of phospho - AKT ( Ser473 ) , phospho ERK1 / 2 and their total protein amounts in lung tumors from EGFRL858R / Rhob - / - or EGFRL858R / Rhob + / + mice treated or not with erlotinib ( 12 . 5 mg / kg / day ) for four days . The remaining hyperplastic areas were selected in erlotinib - treated mice to efficiently characterize the effect of erlotinib on ERK and AKT pathways in both Rhob genotypes . Scale bar : 100 µm . C . HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant ( AKTmyr , myristoylated ) or an empty vector ( ø ) , and treated for 72 h with increasing concentrations of erlotinib . The surviving cell fraction was determined by an MTS assay . Data are representative of at least three independent experiments . C . HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant ( AKTmyr , myristoylated ) or an empty vector ( ø ) , and treated for 72 h with increasing concentrations of erlotinib . AKT overexpression and phosphorylation at Ser473 were assessed by western blotting . Data are representative of at least three independent experiments .

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

Figure 4A. HCC4006, HCC827, HCC2935 and H3255 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for four hours with erlotinib at concentrations corresponding to the respective IC50 values determined for each control cell line. The phosphorylation status of AKT, ERK1/2 and EGFR was assessed by western blotting and normalized according to total protein levels. RHOB overexpression was also monitored by western blotting.B. Representative immunostaining of phospho-AKT (Ser473), phospho ERK1/2 and their total protein amounts in lung tumors from EGFRL858R/Rhob-/- or EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days. The remaining hyperplastic areas were selected in erlotinib-treated mice to efficiently characterize the effect of erlotinib on ERK and AKT pathways in both Rhob genotypes. Scale bar: 100 µm.C. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKTmyr, myristoylated) or an empty vector (ø), and treated for 72 h with increasing concentrations of erlotinib. The surviving cell fraction was determined by an MTS assay. Data are representative of at least three independent experiments.C. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKTmyr, myristoylated) or an empty vector (ø), and treated for 72 h with increasing concentrations of erlotinib. AKT overexpression and phosphorylation at Ser473 were assessed by western blotting. Data are representative of at least three independent experiments.


Figure 5A . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for four hours with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs . The phosphorylation status of GSK3β ( Ser9 ) , ERK1 / 2 and EGFR ( Tyr1173 ) was assessed by western blotting and normalized according to the total protein levels . RHOB overexpression was also monitored by western blotting . B . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for 72 h with erlotinib alone ( black and red curves ) or in combination with the AKT inhibitor G594 at 100 nM ( green and blue curves ) . The surviving cell fraction was determined by an MTS assay . C . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) and treated for 72 h with increasing concentrations of erlotinib in the absence or presence of increasing doses of G594 . The surviving cell fraction was determined by an MTS assay , and erlotinib IC50 values were determined for each condition . ( * * : p < 0 . 001 vs . AdCont cells ; * * * : p < 0 . 0001 vs . AdCont cells ) . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for 48 h with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for 48 h with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs . Apoptosis was then determined by quantification of the subG1 cell population ( D ) . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for 48 h with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs . Apoptosis was then determined by detection of cleaved PARP and caspase - 3 ( E ) .

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

Figure 5A. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for four hours with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. The phosphorylation status of GSK3β (Ser9), ERK1/2 and EGFR (Tyr1173) was assessed by western blotting and normalized according to the total protein levels. RHOB overexpression was also monitored by western blotting.B. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 72 h with erlotinib alone (black and red curves) or in combination with the AKT inhibitor G594 at 100 nM (green and blue curves). The surviving cell fraction was determined by an MTS assay.C. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) and treated for 72 h with increasing concentrations of erlotinib in the absence or presence of increasing doses of G594. The surviving cell fraction was determined by an MTS assay, and erlotinib IC50 values were determined for each condition. (**: p<0.001 vs. AdCont cells; ***: p<0.0001 vs. AdCont cells). HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs.HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. Apoptosis was then determined by quantification of the subG1 cell population (D).HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. Apoptosis was then determined by detection of cleaved PARP and caspase-3 (E).


Figure 6A . Representative H & amp ; amp ; E staining of lung tumors from EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - and EGFRL858R / Rhob + / + mice treated or not during four days with erlotinib ( 12 . 5 mg / kg / day ) , with the AKT inhibitor G594 ( 25 mg / kg / day ) , or in combination with both drugs . Scale bar : 500 µm . B . Quantification of the tumor / lung ratio of mice treated or not with the individual drugs or with a combination of both . C . Representative Ki67 immunostaining of lung tumors from EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - and EGFRL858R / Rhob + / + mice treated or not with erlotinib alone ( 12 . 5 mg / kg / day ) or in combination with the AKT inhibitor G594 ( 25 mg / kg / day ) for four days ( scale bar : 50 µm ) , and the corresponding quantification ( D ) .

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

Figure 6A. Representative H&amp;E staining of lung tumors from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not during four days with erlotinib (12.5 mg/kg/day), with the AKT inhibitor G594 (25 mg/kg/day), or in combination with both drugs. Scale bar: 500 µm. B. Quantification of the tumor/lung ratio of mice treated or not with the individual drugs or with a combination of both.C. Representative Ki67 immunostaining of lung tumors from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib alone (12.5 mg/kg/day) or in combination with the AKT inhibitor G594 (25 mg/kg/day) for four days (scale bar: 50 µm), and the corresponding quantification (D).


Figure 1A ) Distribution curve of replication fork rates for Mybl2 + / + and Mybl2Δ / Δ ESCs . Statistical analysis was performed using the Mann Whitney U test . n = 6 experimental replicates . A minimum of 490 replication forks were counted . ( * * * * p < 0 . 0001 ) . B ) Left panel : representative elongating replication forks ( stable and unstable ) . Quantification of the average IdU / CldU ratio in the Mybl2 + / + and Mybl2Δ / Δ ESCs . Percentage of highly unstable forks ( ratio above 2 ) are indicated . Y - axis was cut to change scale and display large values . Statistical analysis was performed using the Mann Whitney U test . ( * * * * p < 0 . 0001 ) . At least 450 ratios were recorded from 6 experimental replicates . C ) The length of both CldU labels surrounding IdU tracks ( newly fired origins ) was measured before calculating a ratio representing symmetry around new origins . Quantification of the average positive ratio in the Mybl2 + / + and Mybl2Δ / Δ ESCs . Y - axis was cut to change scale and display large values . Statistical analysis was performed using the Mann Whitney U test . 55 and 75 ratios were recorded in the Mybl2 + / + and Mybl2Δ / Δ respectively from 3 replicates ( * * * * p < 0 . 0001 ) . D ) Representative image showing the presence of UFB by positive Immunostaining for PICH . ( Scale bar 10μm ) . Frequency of anaphases positive for UFBs relative to all anaphases in the Mybl2 + / + and Mybl2Δ / Δ ESCs . At least 75 anaphases were counted per experimental group from 3 independent experiments . Statistical analysis was performed using an unpaired two - tailed t - test ( * * * * p < 0 . 0001 ) . E ) Representative images for comets in Mybl2 + / + and Mybl2Δ / Δ untreated and Mybl2 + / + and Mybl2Δ / Δ plus CPT ( 20x magnification ) . ( Scale bar 50μm ) . F ) Quantification of the mean and distributions of olive tail moments in Mybl2 + / + and Mybl2Δ / Δ untreated and CPT - treated . Y - axis was cut to change scale and display large values . Statistical analysis using Mann Whitney U tests . At least 300 comets were measured for each group from 5 experimental replicates ( * * * * p < 0 . 0001 ) . G ) Frequency of EdU positive cells with over 6 53BP1 foci in Mybl2 + / + , Mybl2Δ / Δ untreated and Mybl2 + / + and Mybl2Δ / Δ plus CPT . Error bars represent SEM . Statistical analysis using unpaired two - tailed t - test . At least 200 EdU positive nuclei were analysed from 4 experimental repeats .

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

Figure 1A) Distribution curve of replication fork rates for Mybl2+/+ and Mybl2Δ/Δ ESCs. Statistical analysis was performed using the Mann Whitney U test. n=6 experimental replicates. A minimum of 490 replication forks were counted.( **** p< 0.0001).B) Left panel: representative elongating replication forks (stable and unstable). Quantification of the average IdU/CldU ratio in the Mybl2+/+ and Mybl2Δ/Δ ESCs. Percentage of highly unstable forks (ratio above 2) are indicated. Y-axis was cut to change scale and display large values. Statistical analysis was performed using the Mann Whitney U test. (**** p< 0.0001). At least 450 ratios were recorded from 6 experimental replicates.C) The length of both CldU labels surrounding IdU tracks (newly fired origins) was measured before calculating a ratio representing symmetry around new origins. Quantification of the average positive ratio in the Mybl2+/+ and Mybl2Δ/Δ ESCs. Y-axis was cut to change scale and display large values. Statistical analysis was performed using the Mann Whitney U test. 55 and 75 ratios were recorded in the Mybl2+/+ and Mybl2Δ/Δ respectively from 3 replicates ( **** p< 0.0001).D) Representative image showing the presence of UFB by positive Immunostaining for PICH. (Scale bar 10μm). Frequency of anaphases positive for UFBs relative to all anaphases in the Mybl2+/+ and Mybl2Δ/Δ ESCs. At least 75 anaphases were counted per experimental group from 3 independent experiments. Statistical analysis was performed using an unpaired two-tailed t-test (**** p< 0.0001).E) Representative images for comets in Mybl2+/+ and Mybl2Δ/Δ untreated and Mybl2+/+ and Mybl2Δ/Δ plus CPT (20x magnification). (Scale bar 50μm).F) Quantification of the mean and distributions of olive tail moments in Mybl2+/+ and Mybl2Δ/Δ untreated and CPT-treated. Y-axis was cut to change scale and display large values. Statistical analysis using Mann Whitney U tests. At least 300 comets were measured for each group from 5 experimental replicates (**** p< 0.0001).G) Frequency of EdU positive cells with over 6 53BP1 foci in Mybl2+/+, Mybl2Δ/Δ untreated and Mybl2+/+ and Mybl2Δ/Δ plus CPT. Error bars represent SEM. Statistical analysis using unpaired two-tailed t-test. At least 200 EdU positive nuclei were analysed from 4 experimental repeats.


Figure 2A ) Cells were treated for 1 . 5 hours with 5µM CPT , before sequential addition of IdU and CldU for 20 minutes each . Frequency of new firing origins ( relative to total structures counted ) from Mybl2 + / + and Mybl2Δ / Δ ESCs treated or not with CPT ( n = 3 independent experiments ; Error bars indicate SEM ) . Statistical analysis using two - tailed unpaired t - test . ( B ) Distribution of replication rate in Mybl2 + / + and Mybl2Δ / Δ ESC treated or not with CPT as above . Statistical analysis was carried out using unpaired Mann Whitney U tests . A minimum of 200 forks were quantified for each of the genotypes and conditions shown from 3 independent repeats ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . C ) ESCs were treated with or without CPT for 4 hours before 0 . 1ug / ml colcemid was added during the final 3 . 5 hours of treatment . Representative image of H3 - pS10 positive cells ( turquoise ) . ( Scale bar 10μm ) . Frequency of H3 - pS10 positive cells in Mybl2 + / + and Mybl2Δ / Δ ESCs . Data from three independent experiments . Error bars represent SEM . Statistical analysis was carried out using a two - tailed unpaired t - test ( * p < 0 . 05 ; * * p < 0 . 01 ; ns = no significant ) . D ) P - CDK1 ( Tyr15 ) , CDK1 and Beta - actin expression levels of Mybl2 + / + and Mybl2Δ / Δ ESCs with or without CPT treatment analyzed by immunoblotting . Bar graph represents average band density of P - CDK1 in Mybl2Δ / Δ ESCs relative to loading control and relative to the MYBL2 + / + from 6 repeats . Error bars represent SEM . Statistical analysis was carried out using a two - tailed unpaired t - test ( * * p < 0 . 01 ) .

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

Figure 2A) Cells were treated for 1.5 hours with 5µM CPT, before sequential addition of IdU and CldU for 20 minutes each. Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with CPT (n=3 independent experiments; Error bars indicate SEM). Statistical analysis using two- tailed unpaired t-test.(B) Distribution of replication rate in Mybl2+/+ and Mybl2Δ/Δ ESC treated or not with CPT as above. Statistical analysis was carried out using unpaired Mann Whitney U tests. A minimum of 200 forks were quantified for each of the genotypes and conditions shown from 3 independent repeats (*p < 0.05; **p< 0.01; ***p< 0.001; **** p< 0.0001).C) ESCs were treated with or without CPT for 4 hours before 0.1ug/ml colcemid was added during the final 3.5 hours of treatment. Representative image of H3-pS10 positive cells (turquoise). (Scale bar 10μm). Frequency of H3-pS10 positive cells in Mybl2+/+ and Mybl2Δ/Δ ESCs. Data from three independent experiments. Error bars represent SEM. Statistical analysis was carried out using a two-tailed unpaired t-test (*p < 0.05; **p< 0.01; ns= no significant).D) P-CDK1 (Tyr15), CDK1 and Beta-actin expression levels of Mybl2+/+ and Mybl2Δ/Δ ESCs with or without CPT treatment analyzed by immunoblotting. Bar graph represents average band density of P-CDK1 in Mybl2Δ/Δ ESCs relative to loading control and relative to the MYBL2+/+ from 6 repeats. Error bars represent SEM. Statistical analysis was carried out using a two-tailed unpaired t-test (**p< 0.01).


Figure 3A ) Immunoblot showing the levels of phosphorylated CHK1 ( Ser345 ) and GAPDH in the Mybl2 + / + and Mybl2Δ / Δ ESCs with or without CPT treatment ( 2 . 5µM CPT for 4 hours ) . B ) Immunoblot showing the levels of P - CHK1 ( Ser345 ) and CHK1 in irradiated Mybl2 + / + , and Mybl2Δ / Δ ESCs with or without 3 hours inhibitor treatment : ATR ( AZ20 , 5µM ) and ATM ( Ku60019 , 10µM ) . Cells were also exposed to 5Gy irradiation before the final hour of inhibitor treatment . Bar graph ( lower panel ) represents average band density of P - CHK1 in Mybl2Δ / Δ ESCs relative to CHK1 and relative to untreated cells . Mybl2 + / + from two independent repeats and Mybl2Δ / Δ ESCs from three independent repeats . Error bars represent SD . Statistical analysis was carried out using a two - tailed unpaired t - test ( * p < 0 . 05 ) . C ) Cells were treated for 1 . 5 hours with 5µM AZ20 , before sequential addition of IdU and CldU for 20 minutes each . Dot plot representing the effect of ATR inhibition upon replication rate in Mybl2 + / + and Mybl2Δ / Δ ESC . Statistical analysis of distributions was carried out using unpaired Mann Whitney U tests . A minimum of 240 forks were quantified for each of the genotypes and conditions shown from 2 independent repeats ( * * * * p < 0 . 0001 ) D ) Frequency of new firing origins ( relative to total structures counted ) from Mybl2 + / + and Mybl2Δ / Δ ESCs treated or not with ATR inhibitor AZ20 . At least 500 replication structures were counted per treatment from two independent repeats . Statistical analysis using two - tailed unpaired t - test . ( * p < 0 . 05 ; ns = no significant ) . E ) Distribution and average of IdU / CldU fork ratio in Mybl2 + / + and Mybl2Δ / Δ ESCs with or without ATR inhibitor treatment . Percentage of highly unstable forks ( ratio above 2 ) are indicated . Dotted line at 1 indicates positive ratio . Y - axis was cut to change scale and display large values . Statistical analysis was carried out using the Mann Whitney U test . At least 150 ratios were calculated per treatment group from two independent repeats ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

Figure 3A) Immunoblot showing the levels of phosphorylated CHK1 (Ser345) and GAPDH in the Mybl2+/+ and Mybl2Δ/Δ ESCs with or without CPT treatment (2.5µM CPT for 4 hours).B) Immunoblot showing the levels of P-CHK1 (Ser345) and CHK1 in irradiated Mybl2+/+, and Mybl2Δ/Δ ESCs with or without 3 hours inhibitor treatment: ATR (AZ20, 5µM) and ATM (Ku60019, 10µM). Cells were also exposed to 5Gy irradiation before the final hour of inhibitor treatment. Bar graph (lower panel) represents average band density of P-CHK1 in Mybl2Δ/Δ ESCs relative to CHK1 and relative to untreated cells. Mybl2+/+ from two independent repeats and Mybl2Δ/Δ ESCs from three independent repeats. Error bars represent SD. Statistical analysis was carried out using a two-tailed unpaired t-test (*p< 0.05).C) Cells were treated for 1.5 hours with 5µM AZ20, before sequential addition of IdU and CldU for 20 minutes each. Dot plot representing the effect of ATR inhibition upon replication rate in Mybl2+/+ and Mybl2Δ/Δ ESC. Statistical analysis of distributions was carried out using unpaired Mann Whitney U tests. A minimum of 240 forks were quantified for each of the genotypes and conditions shown from 2 independent repeats (**** p< 0.0001)D) Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with ATR inhibitor AZ20. At least 500 replication structures were counted per treatment from two independent repeats. Statistical analysis using two- tailed unpaired t-test.(*p< 0.05; ns= no significant).E) Distribution and average of IdU/CldU fork ratio in Mybl2+/+ and Mybl2Δ/Δ ESCs with or without ATR inhibitor treatment. Percentage of highly unstable forks (ratio above 2) are indicated. Dotted line at 1 indicates positive ratio. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using the Mann Whitney U test. At least 150 ratios were calculated per treatment group from two independent repeats (*p < 0.05; **p< 0.01; ***p< 0.001; **** p< 0.0001).


Figure 4B ) Distribution of replication rate after treatment with / without ATM inhibitor . Statistical analysis of distributions was carried out using an unpaired Mann Whitney U test . n = 4 . A minimum of 270 forks was measured for each condition ( * * * * p < 0 . 0001 ) . C ) Distribution of the average IdU / CldU ratio in Mybl2 + / + and Mybl2Δ / Δ ESCs with or without ATM inhibitor treatment . Percentage of highly unstable forks ( ratio above 2 ) are indicated . Dotted line at 1 indicates positive ratio . Y - axis was cut to change scale and display large values . Statistical analysis was carried out using the Mann Whitney U . At least 260 ratios were calculated per treatment group from 4 separate experiments ( * * * * p < 0 . 0001 ) . 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 . 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 ) 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 )

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

Figure 4B) Distribution of replication rate after treatment with/without ATM inhibitor. Statistical analysis of distributions was carried out using an unpaired Mann Whitney U test. n=4. A minimum of 270 forks was measured for each condition ( **** p< 0.0001).C) Distribution of the average IdU/CldU ratio in Mybl2+/+ and Mybl2Δ/Δ ESCs with or without ATM inhibitor treatment. Percentage of highly unstable forks (ratio above 2) are indicated. Dotted line at 1 indicates positive ratio. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using the Mann Whitney U. At least 260 ratios were calculated per treatment group from 4 separate experiments ( **** p< 0.0001).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.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)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)


Figure 5B 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B 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.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)


Figure 6C ) 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6C ) 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.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).


f1a ) 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 ) .

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

f1a) 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).


f2 ( 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 . ( 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 .

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

f2(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.(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.


f3 ( 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 .

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

f3(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.


f4 ( 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 .

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

f4(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.


f5 ( 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 . ( 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 .

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

f5(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.(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.


f6 ( 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 .

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

f6(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.


Figure 1A - 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A-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).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).


Figure 2B . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B. 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.


Figure 3A . 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 . 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 . 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 .

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

Figure 3A. 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.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.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.


Figure 4A - 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-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.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.


Figure 5C . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5C. 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.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.


Figure 6C . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6C. 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.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.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.


Figure 7A - 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A-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.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.