Figure 2Expression of Rab25 increases endogenous ATP level in ovarian cancer cells . * p < 0 . 001 Rab25 versus pcDNA . The mean cellular concentration of ATP per mg of protein ( based on the standard curve generated using a known amount of ATP ) is 2 . 36 − 10 moles / mg for IOSE29htpcDNA cells , 6 . 21 − 10 moles / mg for IOSE29htRab25 cells , 8 . 14 − 11 moles / mg for IOSE80htpcDNA cells , 1 . 05 − 10 moles / mg for IOSE80htRab25 cells , 2 . 85 − 10 moles / mg for A2780pcDNA cells , 3 . 81 − 10 moles / mg for A2780Rab25 cells , 7 . 23 − 10 moles / mg for HEYpcDNA cells and 9 . 82 − 10 moles / mg for HEYRab25 cells . Rab25 expression increases ATP synthesis and delays fall in total cellular ATP levels after glucose and serum withdrawal , p < 0 . 001 Rab25 versus control pcDNA . Down - regulation of Rab25 expression decreases ability to maintain ATP after glucose and serum withdrawal ( a ) p < 0 . 05 SF - Glu versus FBS and ( b ) p < 0 . 05 versus control . Expression of Rab25 increases total cellular glycogen content while down - regulation of Rab25 expression decreases total cellular glycogen content ( µg of glycogen / mg protein ) . Cells were cultured in complete media for 24 h before glycogen measurement . ( a ) p < 0 . 01 Rab25 versus pcDNA , ( b ) p < 0 . 05 Rab25 RNAi versus NT RNAi and ( c ) p < 0 . 05 shRAb25 versus shRNA control . Decrease in cellular glycogen content after glucose and serum withdrawal * p < 0 . 001 Rab25 versus pcDNA . Effect of GPi ( Bay U6571 ) and oligomycin on ATP production . A2780pcDNA transfected and Rab25 expressing cells were pretreated with vehicle ( control ) , 30 µM of Bay U6571 ( GPi ) or 25 µg / ml of oligomycin for 2 h . Cells were then cultured in serum - and glucose free RPMI 1640 in the presence or absence of Bay U6571 or oligomycin for the indicated times . ( a ) p < 0 . 01 oligomycin time 0 versus control time 0 and ( b ) p < 0 . 01 versus time 0 at each treatment group . Effect of Bay U6571 on cellular glycogen ( left panel ) and ATP ( right panel ) levels in HEY cells . ( a ) p < 0 . 01 versus pcDNA control time 0 at 5 % FBS and ( b ) p < 0 . 01 GPi versus control at each corresponding treatment . Down regulation of Rab25 expression and inhibition of glycogen breakdown by GPi administration increases 2DG induced cell death * p < 0 . 05 versus shRNA control .

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 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 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 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 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 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

Figure 2Expression of Rab25 increases endogenous ATP level in ovarian cancer cells. *p < 0.001 Rab25 versus pcDNA. The mean cellular concentration of ATP per mg of protein (based on the standard curve generated using a known amount of ATP) is 2.36−10 moles/mg for IOSE29htpcDNA cells, 6.21−10 moles/mg for IOSE29htRab25 cells, 8.14−11 moles/mg for IOSE80htpcDNA cells, 1.05−10 moles/mg for IOSE80htRab25 cells, 2.85−10 moles/mg for A2780pcDNA cells, 3.81−10 moles/mg for A2780Rab25 cells, 7.23−10 moles/mg for HEYpcDNA cells and 9.82−10 moles/mg for HEYRab25 cells.Rab25 expression increases ATP synthesis and delays fall in total cellular ATP levels after glucose and serum withdrawal, p < 0.001 Rab25 versus control pcDNA.Down-regulation of Rab25 expression decreases ability to maintain ATP after glucose and serum withdrawal (a) p < 0.05 SF-Glu versus FBS and (b) p < 0.05 versus control.Expression of Rab25 increases total cellular glycogen content while down-regulation of Rab25 expression decreases total cellular glycogen content (µg of glycogen/mg protein). Cells were cultured in complete media for 24 h before glycogen measurement. (a) p < 0.01 Rab25 versus pcDNA, (b) p < 0.05 Rab25 RNAi versus NT RNAi and (c) p < 0.05 shRAb25 versus shRNA control.Decrease in cellular glycogen content after glucose and serum withdrawal *p < 0.001 Rab25 versus pcDNA.Effect of GPi (Bay U6571) and oligomycin on ATP production. A2780pcDNA transfected and Rab25 expressing cells were pretreated with vehicle (control), 30 µM of Bay U6571 (GPi) or 25 µg/ml of oligomycin for 2 h. Cells were then cultured in serum- and glucose free RPMI 1640 in the presence or absence of Bay U6571 or oligomycin for the indicated times. (a) p < 0.01 oligomycin time 0 versus control time 0 and (b) p < 0.01 versus time 0 at each treatment group.Effect of Bay U6571 on cellular glycogen (left panel) and ATP (right panel) levels in HEY cells. (a) p < 0.01 versus pcDNA control time 0 at 5% FBS and (b) p < 0.01 GPi versus control at each corresponding treatment.Down regulation of Rab25 expression and inhibition of glycogen breakdown by GPi administration increases 2DG induced cell death *p < 0.05 versus shRNA control.


Figure 3A . Rab25 expression increases phospho but not total AKT and GSK3 levels in A2780 cells leading to decreased phosphorylation of GS ( pGS ) . B . WB analysis of AKT and GSK3 phosphorylation confirming the inhibitory effect of PI3K pathways inhibitors PI103 , GPi and synthase kinase 3 inhibitor ( GSK3i ) in HEY cells . C . Inhibition of GSK3 activity increases total cellular glycogen levels . pcDNA transfected and Rab25 expressing ovarian cancer cells were cultured in complete media in the presence of 10 µM GSK3i for the indicated times . Total cellular glycogen content was measured and normalized with total protein content . ( a ) p < 0 . 001 versus no GSK3i treated pcDNA cells . D - E . Effect of PI3K inhibitor PI103 on cellular glycogen content and ATP production . Ovarian cancer cells were pretreated with PI103 for at least 2 h in complete media . Cells were then subjected to 2 hglucose and FBS withdrawal to deplete endogenous glycogen followed by culturing cells in RPMI - glucose media in the presence of PI103 for 2 h ( 2 hSF ) . After 2 h of nutrient stress , complete media containing PI103 was added to the cells for 30 min ( 5 % FBS ) for recovery . A second glucose and FBS withdrawal ( 15 min SF ) followed immediately to examine the effect on glycogen and ATP levels . a , p < 0 . 001 5 % FBS versus 2 hSF , b , p < 0 . 01 15 min SF versus 5 % FBS . D . There is an increase in glycogen content during recovery phase ( 5 % FBS ) and a decrease in glycogen when cells are subject to a second nutrient stress ( 15 min SF ) . D - E . Effect of PI3K inhibitor PI103 on cellular glycogen content and ATP production . Ovarian cancer cells were pretreated with PI103 for at least 2 h in complete media . Cells were then subjected to 2 hglucose and FBS withdrawal to deplete endogenous glycogen followed by culturing cells in RPMI - glucose media in the presence of PI103 for 2 h ( 2 hSF ) . After 2 h of nutrient stress , complete media containing PI103 was added to the cells for 30 min ( 5 % FBS ) for recovery . A second glucose and FBS withdrawal ( 15 min SF ) followed immediately to examine the effect on glycogen and ATP levels . a , p < 0 . 001 5 % FBS versus 2 hSF , b , p < 0 . 01 15 min SF versus 5 % FBS . E . Utilization of glycogen to produce ATP . F . Addition of AKT inhibitor MK2206 ( AKTi ) abolishes Rab25 - dependent glycogen storage . ( a ) p < 0 . 05 versus shRNA control and ( b ) p < 0 . 01 SF + AKTi versus SF . G . Inhibiting AKT pathway and glucose metabolism decreases cell viability . Cells were pretreated with 10 µM of PI103 or AKTi , 100 µM LND or 3BrPy for 2 h before switching to SF media in the presence of inhibitors for an addition16 h before cell titre blue viability assay . * p < 0 . 001 shRNA Rab25 versus shRNA control .

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

Figure 3A. Rab25 expression increases phospho but not total AKT and GSK3 levels in A2780 cells leading to decreased phosphorylation of GS (pGS).B. WB analysis of AKT and GSK3 phosphorylation confirming the inhibitory effect of PI3K pathways inhibitors PI103, GPi and synthase kinase 3 inhibitor (GSK3i) in HEY cells.C. Inhibition of GSK3 activity increases total cellular glycogen levels. pcDNA transfected and Rab25 expressing ovarian cancer cells were cultured in complete media in the presence of 10 µM GSK3i for the indicated times. Total cellular glycogen content was measured and normalized with total protein content. (a) p < 0.001 versus no GSK3i treated pcDNA cells.D-E. Effect of PI3K inhibitor PI103 on cellular glycogen content and ATP production. Ovarian cancer cells were pretreated with PI103 for at least 2 h in complete media. Cells were then subjected to 2 hglucose and FBS withdrawal to deplete endogenous glycogen followed by culturing cells in RPMI-glucose media in the presence of PI103 for 2 h (2 hSF). After 2 h of nutrient stress, complete media containing PI103 was added to the cells for 30 min (5% FBS) for recovery. A second glucose and FBS withdrawal (15 min SF) followed immediately to examine the effect on glycogen and ATP levels. a, p < 0.001 5% FBS versus 2 hSF, b, p < 0.01 15 min SF versus 5% FBS.D. There is an increase in glycogen content during recovery phase (5% FBS) and a decrease in glycogen when cells are subject to a second nutrient stress (15 min SF).D-E. Effect of PI3K inhibitor PI103 on cellular glycogen content and ATP production. Ovarian cancer cells were pretreated with PI103 for at least 2 h in complete media. Cells were then subjected to 2 hglucose and FBS withdrawal to deplete endogenous glycogen followed by culturing cells in RPMI-glucose media in the presence of PI103 for 2 h (2 hSF). After 2 h of nutrient stress, complete media containing PI103 was added to the cells for 30 min (5% FBS) for recovery. A second glucose and FBS withdrawal (15 min SF) followed immediately to examine the effect on glycogen and ATP levels. a, p < 0.001 5% FBS versus 2 hSF, b, p < 0.01 15 min SF versus 5% FBS.E. Utilization of glycogen to produce ATP.F. Addition of AKT inhibitor MK2206 (AKTi) abolishes Rab25-dependent glycogen storage. (a) p < 0.05 versus shRNA control and (b) p < 0.01 SF + AKTi versus SF.G. Inhibiting AKT pathway and glucose metabolism decreases cell viability. Cells were pretreated with 10 µM of PI103 or AKTi, 100 µM LND or 3BrPy for 2 h before switching to SF media in the presence of inhibitors for an addition16 h before cell titre blue viability assay. *p < 0.001 shRNA Rab25 versus shRNA control.


Figure 4A . Rab25 over - expression increases H3 - labelled 2DG uptake . ( a ) p < 0 . 001 Rab25 versus pcDNA . Results are mean ± s . d . of a triplicate in one of representative experiment . B . Down - regulation of Rab25 or AKT expression , or inhibition AKT activity by MK2206 decreases glucose uptake in HEY cells ( a ) p < 0 . 001 versus NT siRNA control , ( b ) p < 0 . 05 versus siRab25 and ( c ) p < 0 . 05 versus siAKT . C . Effect of down regulation of Rab25 and AKT inhibitor MK2206 ( AKTi ) on glucose uptake . Cells were either pretreated for 2 h with AKTi before assessing glucose uptake . ( a ) p < 0 . 05 versus shRNA control and ( b ) p < 0 . 05 versus shRab25 . D . Rab25 co - localization with GLUT1 in A2780 cells . Immunofluorescence microscopy staining of GLUT1 ( red ) and Rab25 ( green ) . Co - localization of GLUT1 and Rab25 ( i . e . yellow ) is indicated by arrows . Single scale bar , 10 µM . E - G . Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K / AKT pathway . Inhibition of the PI3K / AKT pathway was achieved by addition of 10 µM of PI103 or MK2206 . E . AKT inhibition increases sensitivity to 2DG induced cell death . * p < 0 . 001 versus HEYpcDNA cells . E - G . Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K / AKT pathway . Inhibition of the PI3K / AKT pathway was achieved by addition of 10 µM of PI103 or MK2206 . F . Down - regulation of AKT by siRNA specific to AKT . E - G . Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K / AKT pathway . Inhibition of the PI3K / AKT pathway was achieved by addition of 10 µM of PI103 or MK2206 . G . Or down - regulation of Rab25 by shRNA specific to Rab25 enhances 2DG induced cell death . * p < 0 . 001 versus RNAi control cells .

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Figure 4A. Rab25 over-expression increases H3-labelled 2DG uptake. (a) p < 0.001 Rab25 versus pcDNA. Results are mean ± s.d. of a triplicate in one of representative experiment.B. Down-regulation of Rab25 or AKT expression, or inhibition AKT activity by MK2206 decreases glucose uptake in HEY cells (a) p < 0.001 versus NT siRNA control, (b) p < 0.05 versus siRab25 and (c) p < 0.05 versus siAKT.C. Effect of down regulation of Rab25 and AKT inhibitor MK2206 (AKTi) on glucose uptake. Cells were either pretreated for 2 h with AKTi before assessing glucose uptake. (a) p < 0.05 versus shRNA control and (b) p < 0.05 versus shRab25.D. Rab25 co-localization with GLUT1 in A2780 cells. Immunofluorescence microscopy staining of GLUT1 (red) and Rab25 (green). Co-localization of GLUT1 and Rab25 (i.e. yellow) is indicated by arrows. Single scale bar, 10 µM.E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.E. AKT inhibition increases sensitivity to 2DG induced cell death. *p < 0.001 versus HEYpcDNA cells.E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.F. Down-regulation of AKT by siRNA specific to AKT.E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.G. Or down-regulation of Rab25 by shRNA specific to Rab25 enhances 2DG induced cell death. *p < 0.001 versus RNAi control cells.


Figure 5Ovarian cancer HEY cells ( upper panel ) were co - transfected with AKT - IFPN and Rab25 - IFPC or with AKT - IFPC and Rab25 - IFPN . Cells become fluorescent due to an interaction between AKT and Rab25 bringing the two halves of the fluorescent protein into proximity creating a stable complex and restoring fluorescence . Hela cells stably expressing AKT - IFPN ( lower panel ) were transfected with Rab25 - IFPC or PDK - IFPC as control . A brightfield image corresponding to the fluorescent image is shown and transfected cells indicated by an arrow . Detection of Rab25 and AKT interaction by immunoprecipitation . Ovarian cells expressing HA - tagged Rab25 were IP with anti - HA antibody . The resultant complex was separated by gel electrophoresis and detected by WB using anti - AKT antibody . Generation of Rab25 deletion mutants . Expression of the deletion mutants was detected by immunofluorescence staining ( upper panel ) and WB ( middle panel ) using anti - GFP antibody which interacts with YFP . Diagrammatic representation of the structure of the mutants is shown in the lower panel . Rab25 deletion mutants do not alter cellular glycogen and ATP levels . Ovarian cancer cell lysates were collected 24 h post transfection . ( a ) p < 0 . 05 versus empty vector transfected cells .

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 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 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

Figure 5Ovarian cancer HEY cells (upper panel) were co-transfected with AKT-IFPN and Rab25-IFPC or with AKT-IFPC and Rab25-IFPN. Cells become fluorescent due to an interaction between AKT and Rab25 bringing the two halves of the fluorescent protein into proximity creating a stable complex and restoring fluorescence. Hela cells stably expressing AKT-IFPN (lower panel) were transfected with Rab25-IFPC or PDK-IFPC as control. A brightfield image corresponding to the fluorescent image is shown and transfected cells indicated by an arrow.Detection of Rab25 and AKT interaction by immunoprecipitation. Ovarian cells expressing HA-tagged Rab25 were IP with anti-HA antibody. The resultant complex was separated by gel electrophoresis and detected by WB using anti-AKT antibody.Generation of Rab25 deletion mutants. Expression of the deletion mutants was detected by immunofluorescence staining (upper panel) and WB (middle panel) using anti-GFP antibody which interacts with YFP. Diagrammatic representation of the structure of the mutants is shown in the lower panel.Rab25 deletion mutants do not alter cellular glycogen and ATP levels. Ovarian cancer cell lysates were collected 24 h post transfection. (a) p < 0.05 versus empty vector transfected cells.


Figure 6A . Rab25 expression correlates with glycogen levels in patienttumours . Total RNA and total cellular extracts , isolated from 31 ovarian cancer patient specimens , were subjected to Rab25 gene expression and glycogen content analysis using qPCR and glycogen assay , respectively . B - C . The Rab25 expression signature identifies patients with a poor prognosis . Ovarian cancer patients were classified as either mirroring the Rab25 - associated gene expression signature ( Rab25 signature ) or not ( non - Rab25 signature ) using linear discriminant analysis in BRB tools , in two independent published ovarian datasets . Progression free survival curvesfor patients with advanced disease ( Stage II to IV ) are shown . Univariate analyses were plotted using Kaplan - Meier method and Coxplots used for multivariate analyses ( co - variates included stage , grade , histology and residual disease ) . B . Tothill et al , 2008 . C . Dressman et al , 2007 . D . Prediction of breast cancer overall survival in two independent published breast datasets ( upper panel ) Pawitan et al , 2005 and ( lower panel ) Chin et al , 2006 , based on Rab25 - associated gene signature . Breast patient classification was determined by BRB tool class prediction function to identify patient with Rab25 - associated gene expression signature ( Rab25 - signature ) or without ( non - Rab25 signature ) . All patients from the datasets were included in class prediction .

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Figure 6A. Rab25 expression correlates with glycogen levels in patienttumours. Total RNA and total cellular extracts, isolated from 31 ovarian cancer patient specimens, were subjected to Rab25 gene expression and glycogen content analysis using qPCR and glycogen assay, respectively.B-C. The Rab25 expression signature identifies patients with a poor prognosis. Ovarian cancer patients were classified as either mirroring the Rab25-associated gene expression signature (Rab25 signature) or not (non-Rab25 signature) using linear discriminant analysis in BRB tools, in two independent published ovarian datasets. Progression free survival curvesfor patients with advanced disease (Stage II to IV) are shown. Univariate analyses were plotted using Kaplan-Meier method and Coxplots used for multivariate analyses (co-variates included stage, grade, histology and residual disease).B. Tothill et al, 2008.C. Dressman et al, 2007.D. Prediction of breast cancer overall survival in two independent published breast datasets (upper panel) Pawitan et al, 2005 and (lower panel) Chin et al, 2006, based on Rab25-associated gene signature. Breast patient classification was determined by BRB tool class prediction function to identify patient with Rab25-associated gene expression signature (Rab25-signature) or without (non-Rab25 signature). All patients from the datasets were included in class prediction.


Figure 1Figure 1 . Extensive identification of Mmi1 RNA targets by combining RNA - IP sequencing and computational approaches . ( A ) Box - plot of the enrichment of the RNAs identified by Mmi1 RNA - IPs coupled to high throughput sequencing . The log of the average enrichments obtained from two independent RNA - IPs is plotted . The enrichment is relative to the no antibody RNA - IPs conducted in parallel of Mmi1 RNA - IPs . The mRNAs and lncRNAs enriched at least two folds in both Mmi1 RNA - IPs are shown in blue and green , respectively . Crosses represent newly identified Mmi1 targets and dots known targets . Figure 1 . Extensive identification of Mmi1 RNA targets by combining RNA - IP sequencing and computational approaches . ( D ) RNA - IPs showing the impact of Mmi1 YTH domain point mutations on Mmi1 ' s binding to ssm4 mRNA . The lower part shows a Western blot monitoring the protein level of WT and mutant Mmi1 proteins in the cells used for the RNA - IPs . Loading was monitored using an anti - Tub1 ( tubulin ) antibody . Figure 1 . Extensive identification of Mmi1 RNA targets by combining RNA - IP sequencing and computational approaches . ( E , F ) RNA - IPs showing the YTH - dependent association of Mmi1 to the lncRNAs identified in ( A ) and ( B ) . Average fold enrichment is shown with error bars that indicate mean average deviations for three independent experiments for ( D , E and F ) . Figure 1 . Extensive identification of Mmi1 RNA targets by combining RNA - IP sequencing and computational approaches . ( E , F ) RNA - IPs showing the YTH - dependent association of Mmi1 to the lncRNAs identified in ( A ) and ( B ) . Average fold enrichment is shown with error bars that indicate mean average deviations for three independent experiments for ( D , E and F ) .

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 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

Figure 1Figure 1. Extensive identification of Mmi1 RNA targets by combining RNA-IP sequencing and computational approaches. (A) Box-plot of the enrichment of the RNAs identified by Mmi1 RNA-IPs coupled to high throughput sequencing. The log of the average enrichments obtained from two independent RNA-IPs is plotted. The enrichment is relative to the no antibody RNA-IPs conducted in parallel of Mmi1 RNA-IPs. The mRNAs and lncRNAs enriched at least two folds in both Mmi1 RNA-IPs are shown in blue and green, respectively. Crosses represent newly identified Mmi1 targets and dots known targets.Figure 1. Extensive identification of Mmi1 RNA targets by combining RNA-IP sequencing and computational approaches. (D) RNA-IPs showing the impact of Mmi1 YTH domain point mutations on Mmi1's binding to ssm4 mRNA. The lower part shows a Western blot monitoring the protein level of WT and mutant Mmi1 proteins in the cells used for the RNA-IPs. Loading was monitored using an anti-Tub1 (tubulin) antibody.Figure 1. Extensive identification of Mmi1 RNA targets by combining RNA-IP sequencing and computational approaches. (E, F) RNA-IPs showing the YTH-dependent association of Mmi1 to the lncRNAs identified in (A) and (B). Average fold enrichment is shown with error bars that indicate mean average deviations for three independent experiments for (D, E and F).Figure 1. Extensive identification of Mmi1 RNA targets by combining RNA-IP sequencing and computational approaches. (E, F) RNA-IPs showing the YTH-dependent association of Mmi1 to the lncRNAs identified in (A) and (B). Average fold enrichment is shown with error bars that indicate mean average deviations for three independent experiments for (D, E and F).


Figure 2Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( A ) Upper part , scheme of S . pombe sexual differentiation . Lower part , microscopy images showing WT and mmi1 ∆ cells , and mmi1 ∆ cells expressing Mmi1 - R351E or Mmi1 - R381E mutant proteins . Images were taken after 24 hours of induction of sexual differentiation by growth on SPAS medium . The percentage of cells that underwent differentiation and iodine vapor assays , conducted on the corresponding patches , are shown at the bottom of each image . Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( B ) Mating assay showing the percentage of zygotes forming over time in the same cells as in ( A ) . Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( E ) Mmi1 RNA - IPs showing the specific loss of binding of Mmi1 to nam1 - 1 lncRNA but not to mei4 mRNA , another target of Mmi1 . Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( F ) Mating assay showing the percentage of zygotes formed over time in WT and nam1 - 1 cells . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) ( B , E and F ) . Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( G ) Western blots showing the level of Flag - Byr2 protein over the first 4 hours of sexual differentiation in WT and nam1 - 1 cells . Tubulin ( Tub1 ) level was used as a loading control . Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( H ) Microscopy images of WT ( h90 ) cells transformed with an empty plasmid ( Control ) and nam1 - 1 cells transformed with either an empty plasmid ( Control ) or a plasmid expressing Byr2 protein ( Byr2 ) , after 24 hours of induction of sexual differentiation . Scale bar , 10 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 O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (A) Upper part, scheme of S. pombe sexual differentiation. Lower part, microscopy images showing WT and mmi1∆ cells, and mmi1∆ cells expressing Mmi1-R351E or Mmi1-R381E mutant proteins. Images were taken after 24 hours of induction of sexual differentiation by growth on SPAS medium. The percentage of cells that underwent differentiation and iodine vapor assays, conducted on the corresponding patches, are shown at the bottom of each image.Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (B) Mating assay showing the percentage of zygotes forming over time in the same cells as in (A).Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (E) Mmi1 RNA-IPs showing the specific loss of binding of Mmi1 to nam1-1 lncRNA but not to mei4 mRNA, another target of Mmi1.Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (F) Mating assay showing the percentage of zygotes formed over time in WT and nam1-1 cells. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) (B, E and F).Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (G) Western blots showing the level of Flag-Byr2 protein over the first 4 hours of sexual differentiation in WT and nam1-1 cells. Tubulin (Tub1) level was used as a loading control.Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (H) Microscopy images of WT (h90) cells transformed with an empty plasmid (Control) and nam1-1 cells transformed with either an empty plasmid (Control) or a plasmid expressing Byr2 protein (Byr2), after 24 hours of induction of sexual differentiation. Scale bar, 10 µm.


Figure 3Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( A ) Northern blots showing nam1 and byr2 RNA levels during the first 4 hours of sexual differentiation . Ribosomal RNAs ( rRNAs ) stained with ethidium bromide were used as loading controls . Black lines indicate probes used to detect nam1 and byr2 RNAs . Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( B ) ChIPs showing the occupancy of the elongating RNAPII ( RNAPII - S2P ) over nam1 - byr2 locus , in WT and nam1 - 1 cells . RNAPII - S2P was immunoprecipitated with an antibody recognizing the heptameric repeats ( present in the C - terminal domain of the polymerase ) when it is phosphorylated on its serine 2 . Black lines , genomic regions investigated . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( B , C , E and F ) . Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( C ) RT - qPCRs showing the accumulation of nam1 read - through transcripts ( RT1 - qPCR ) and nam1 lncRNAs ( RT2 - qPCR ) in cells with or without the transcription terminator Ttef inserted at the 3 ' end of nam1 ( scheme ) . Black arrow , primer used for the strand - specific reverse transcription ( RT ) ; Black line , location of the region amplified by PCR . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( B , C , E and F ) . Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( D ) Microscopy images of , respectively , WT ( h90 ) , nam1 - 1 and nam1 - 1 - Ttef cells , after induction of sexual differentiation for 24 hours . The percentage of sporulation and iodine vapor assays are shown at the bottom of the images . Scale bar , 10 µm . Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( E ) RT - qPCRs showing the accumulation of nam1 read - through transcripts in mmi1 ∆ and rrp6 ∆ cells . Black arrow and line as in ( C ) . . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( B , C , E and F ) . Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( F ) ChIPs monitoring the enrichment of H3K9me2 over nam1 - byr2 locus and mei4 gene in WT and rrp6 ∆ cells . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( B , C , E and F ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (A) Northern blots showing nam1 and byr2 RNA levels during the first 4 hours of sexual differentiation. Ribosomal RNAs (rRNAs) stained with ethidium bromide were used as loading controls. Black lines indicate probes used to detect nam1 and byr2 RNAs.Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (B) ChIPs showing the occupancy of the elongating RNAPII (RNAPII-S2P) over nam1-byr2 locus, in WT and nam1-1 cells. RNAPII-S2P was immunoprecipitated with an antibody recognizing the heptameric repeats (present in the C-terminal domain of the polymerase) when it is phosphorylated on its serine 2. Black lines, genomic regions investigated. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (B, C, E and F).Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (C) RT-qPCRs showing the accumulation of nam1 read-through transcripts (RT1-qPCR) and nam1 lncRNAs (RT2-qPCR) in cells with or without the transcription terminator Ttef inserted at the 3' end of nam1 (scheme). Black arrow, primer used for the strand-specific reverse transcription (RT); Black line, location of the region amplified by PCR. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (B, C, E and F).Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (D) Microscopy images of, respectively, WT (h90), nam1-1 and nam1-1-Ttef cells, after induction of sexual differentiation for 24 hours. The percentage of sporulation and iodine vapor assays are shown at the bottom of the images. Scale bar, 10 µm.Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (E) RT-qPCRs showing the accumulation of nam1 read-through transcripts in mmi1∆ and rrp6∆ cells. Black arrow and line as in (C). . Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (B, C, E and F).Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (F) ChIPs monitoring the enrichment of H3K9me2 over nam1-byr2 locus and mei4 gene in WT and rrp6∆ cells. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (B, C, E and F).


Figure 4Figure 4 . Mmi1 drives Rrp6 - dependent heterochromatin gene silencing at pericentromeric regions . ( B ) Northern blot showing the level of nam5 / 6 / 7 lncRNA population in mmi1 - ts3 , dcr1 ∆ , clr4 ∆ single mutant cells and in mmi1 - ts3 dcr1 ∆ and mmi1 - ts3 clr4 ∆ double mutant cells , at the permissive ( 25ºC ) and restrictive ( 36ºC ) temperatures . Figure 4 . Mmi1 drives Rrp6 - dependent heterochromatin gene silencing at pericentromeric regions . ( C ) RT - PCRs monitoring the accumulation of nam7 lncRNAs and nam7 - L read - through transcripts , in the same cells and conditions as in ( B ) . Red arrow heads point to the expected PCR products while the other bands correspond to non - specific PCR products . From the scheme and the agarose gels : black arrows , primers used for the three different reverse transcriptions ; black lines and red numbers , expected PCR products of the three different RT - PCRs ; M , DNA ladder Markers ; tub1 , tubulin control . Figure 4 . Mmi1 drives Rrp6 - dependent heterochromatin gene silencing at pericentromeric regions . ( D ) RT - qPCRs showing the levels of nam5 / 6 / 7 lncRNA population in the double mutant rrp6 ∆ dcr1 ∆ cells , relative to the single mutant rrp6 ∆ and dcr1 ∆ cells . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( D , E ) . P value was calculated using a two - tailed Student ' s t - test . Figure 4 . Mmi1 drives Rrp6 - dependent heterochromatin gene silencing at pericentromeric regions . ( E ) RNA - IPs showing that Rrp6 - Myc13 binds to nam5 / 6 / 7 lncRNAs in a Mmi1 - dependent manner . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( D , E ) . P value was calculated using a two - tailed Student ' s t - test .

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

Figure 4Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (B) Northern blot showing the level of nam5/6/7 lncRNA population in mmi1-ts3, dcr1∆, clr4∆ single mutant cells and in mmi1-ts3 dcr1∆ and mmi1-ts3 clr4∆ double mutant cells, at the permissive (25ºC) and restrictive (36ºC) temperatures.Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (C) RT-PCRs monitoring the accumulation of nam7 lncRNAs and nam7-L read-through transcripts, in the same cells and conditions as in (B). Red arrow heads point to the expected PCR products while the other bands correspond to non-specific PCR products. From the scheme and the agarose gels: black arrows, primers used for the three different reverse transcriptions; black lines and red numbers, expected PCR products of the three different RT-PCRs; M, DNA ladder Markers; tub1, tubulin control.Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (D) RT-qPCRs showing the levels of nam5/6/7 lncRNA population in the double mutant rrp6∆ dcr1∆ cells, relative to the single mutant rrp6∆ and dcr1∆ cells. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (D, E). P value was calculated using a two-tailed Student's t-test.Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (E) RNA-IPs showing that Rrp6-Myc13 binds to nam5/6/7 lncRNAs in a Mmi1-dependent manner. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (D, E). P value was calculated using a two-tailed Student's t-test.


Figure 5Figure 5 . Mmi1 - and RNAi - mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle . ( A ) ChIPs assessing the localization of Mmi1 to pericentromeric DNA in WT and clr4 ∆ cells . Figure 5 . Mmi1 - and RNAi - mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle . ( B ) ChIPs showing the localization of Mmi1 ( dashed line ) and H3K9me2 ( black line ) to the pericentromeric nam5 / 6 / 7 DNA regions during the progression of the cell cycle . Cell synchronization was achieved by using cdc25 - ts cells ( see experimental procedures for more details ) . Cell synchronization was monitored by measuring the percentage of cells with a septum ( right part ) . Figure 5 . Mmi1 - and RNAi - mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle . ( C ) RT - qPCRs monitoring the levels of nam5 / 6 / 7 ( left part ) and dg ( middle part ) lncRNA populations during the cell cycle . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( A , B and C ) . Figure 5 . Mmi1 - and RNAi - mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle . ( D ) RT - PCRs monitoring the accumulation of nam7 lncRNAs and nam7 - L read - through transcripts in G2 / M and G1 / S phases from synchronized cells as in ( B ) . Red arrow heads point to the expected PCR products . The other bands are non - specific PCR products . From the scheme : black arrows , primers used for the reverse transcriptions ; black lines and red numbers , regions amplified by PCR .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5Figure 5. Mmi1- and RNAi-mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle. (A) ChIPs assessing the localization of Mmi1 to pericentromeric DNA in WT and clr4∆ cells.Figure 5. Mmi1- and RNAi-mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle. (B) ChIPs showing the localization of Mmi1 (dashed line) and H3K9me2 (black line) to the pericentromeric nam5/6/7 DNA regions during the progression of the cell cycle. Cell synchronization was achieved by using cdc25-ts cells (see experimental procedures for more details). Cell synchronization was monitored by measuring the percentage of cells with a septum (right part).Figure 5. Mmi1- and RNAi-mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle. (C) RT-qPCRs monitoring the levels of nam5/6/7 (left part) and dg (middle part) lncRNA populations during the cell cycle. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (A, B and C).Figure 5. Mmi1- and RNAi-mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle. (D) RT-PCRs monitoring the accumulation of nam7 lncRNAs and nam7-L read-through transcripts in G2/M and G1/S phases from synchronized cells as in (B). Red arrow heads point to the expected PCR products. The other bands are non-specific PCR products. From the scheme: black arrows, primers used for the reverse transcriptions; black lines and red numbers, regions amplified by PCR.


figf1 ( a ) Left , cells were fixed and stained for anti - pTyr - 416 - Src ( green ) and with DAPI ( blue ) . Solid arrows indicate cells with active Src in adhesions ; dashed arrows show cells with active Src in puncta . Scale bars , 20 μm . Right , percentage of cells with active Src localizing to intracellular structures was quantified . Data are presented as mean ± s . d . and significance is P0 . 001 ( n = 3 ) . ( b ) Cells were fixed and stained for anti - FAK ( red ) and anti - pTyr - 416 - Src ( green ) . Merged and higher - magnification images of the outlined areas are also shown . Solid and dashed arrows show Src localization to adhesions or to puncta , respectively . Scale bars , 20 μm . ( c ) Lysates from cells were immunoblotted with anti - FAK , anti - Src , anti - Yes , anti - Fyn , anti - pTyr - 416 - Src and anti - actin antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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 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

figf1(a) Left, cells were fixed and stained for anti-pTyr-416-Src (green) and with DAPI (blue). Solid arrows indicate cells with active Src in adhesions; dashed arrows show cells with active Src in puncta. Scale bars, 20 μm. Right, percentage of cells with active Src localizing to intracellular structures was quantified. Data are presented as mean±s.d. and significance is P0.001 (n=3).(b) Cells were fixed and stained for anti-FAK (red) and anti-pTyr-416-Src (green). Merged and higher-magnification images of the outlined areas are also shown. Solid and dashed arrows show Src localization to adhesions or to puncta, respectively. Scale bars, 20 μm.(c) Lysates from cells were immunoblotted with anti-FAK, anti-Src, anti-Yes, anti-Fyn, anti-pTyr-416-Src and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


figf2 ( a ) Cells were fixed and stained for anti - pTyr - 416 - Src ( red ) and co - stained for anti - LC3B , anti - Atg7 or anti - Atg12 ( all green ) . Scale bars , 20 μm . ( b ) Higher - magnification images of the areas outlined in a . Solid arrows indicate co - localization in focal adhesions and dashed arrows show puncta in the cytoplasm . ( c ) LC3B was immunoprecipitated ( IP ) from FAK + / + and FAK − / − cells and then immunoblotted with anti - Src , anti - pTyr - 416 - Src and anti - LC3B antibodies . ( d ) Cells were transfected with 40 nM scrambled , Atg5 or Atg12 siRNA for 72 h and immunoblotted with anti - Atg5 , anti - Atg12 and anti - actin antibodies ( lower panels ) or fixed and stained for anti - pTyr - 416 - Src ( green ) , anti - paxillin ( red ) and with DAPI ( blue ) . Dashed arrows point to intracellular phospho - Src - containing puncta ; solid arrows point to peripheral phospho - Src . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

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 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

figf2(a) Cells were fixed and stained for anti-pTyr-416-Src (red) and co-stained for anti-LC3B, anti-Atg7 or anti-Atg12 (all green). Scale bars, 20 μm. (b) Higher-magnification images of the areas outlined in a. Solid arrows indicate co-localization in focal adhesions and dashed arrows show puncta in the cytoplasm.(c) LC3B was immunoprecipitated (IP) from FAK+/+ and FAK−/− cells and then immunoblotted with anti-Src, anti-pTyr-416-Src and anti-LC3B antibodies.(d) Cells were transfected with 40 nM scrambled, Atg5 or Atg12 siRNA for 72 h and immunoblotted with anti-Atg5, anti-Atg12 and anti-actin antibodies (lower panels) or fixed and stained for anti-pTyr-416-Src (green), anti-paxillin (red) and with DAPI (blue). Dashed arrows point to intracellular phospho-Src-containing puncta; solid arrows point to peripheral phospho-Src. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


figf3 ( a ) Top , cells were transiently transfected with an RFP - GFP tandem fluorescent - tagged LC3 ( RFP - GFP - LC3 ) . Scale bars , 20 μm . Bottom , the number of yellow puncta and the number of RFP LC3 - positive puncta in the merged images were counted and the total number of puncta per cell was calculated . Data are presented as mean ± s . d . ( n = 3 ) . b ) Cells were treated with 10 μM chloroquine for 24 h and then immunoblotted using anti - pTyr - 416 - Src , anti - Src , anti - LC3B and anti - actin antibodies . Densitometry was carried out to calculate the increase in the amount of LC3B - II relative to actin for each cell type following treatment with chloroquine and is presented as a percentage increase for the immunoblot shown . ( c ) Cells were treated with 200 nM epoxomicin for 4 h and then immunoblotted and probed with anti - pTyr - 416 - Src , anti - Src , anti - p53 and anti - actin antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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 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

figf3(a) Top, cells were transiently transfected with an RFP-GFP tandem fluorescent-tagged LC3 (RFP-GFP-LC3). Scale bars, 20 μm. Bottom, the number of yellow puncta and the number of RFP LC3-positive puncta in the merged images were counted and the total number of puncta per cell was calculated. Data are presented as mean±s.d. (n=3).b) Cells were treated with 10 μM chloroquine for 24 h and then immunoblotted using anti-pTyr-416-Src, anti-Src, anti-LC3B and anti-actin antibodies. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each cell type following treatment with chloroquine and is presented as a percentage increase for the immunoblot shown.(c) Cells were treated with 200 nM epoxomicin for 4 h and then immunoblotted and probed with anti-pTyr-416-Src, anti-Src, anti-p53 and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


figf4 ( a ) Top , FAK − / − cells were treated with 200 nM dasatinib for 24 h and then fixed and stained for anti - Src antibody ( green ) . Solid arrows indicate Src at the cell periphery ; dashed arrows indicate Src in autophagosomes . Scale bars , 20 μm . Bottom , quantification of dasatinib - treated cells with Src in puncta . Data are presented as mean ± s . d . and significance is P0 . 001 ( n = 3 ) . Middle , lysates from cells treated with dasatinib were immunoblotted with anti - Src and anti - pTyr - 416 - Src and lysates from cells treated with dasatinib and chloroquine were immunoblotted with anti - LC3B and anti - actin antibodies . Densitometry was carried out to calculate the increase in the amount of LC3B - II relative to actin for each condition after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown . ( b ) Top , FAK − / − cells were transiently transfected with SrcY527F - GFP or with Src - 251 - GFP ( green ) and stained for anti - LC3B ( red ) . Scale bars , 20 μm . Bottom , quantification of cells with co - localization between Src and LC3B . Data are presented as mean ± s . d . and significance is P0 . 001 ( n = 3 ) . ( c ) Left , FAK − / − cells stably re - expressing wild - type FAK , FAKY397F or FAKY4F - Y9F were fixed and stained for anti - FAK ( red ) and anti - pTyr - 416 - Src ( green ) . Solid arrows indicate co - localization at adhesions and dashed arrows indicate active Src in autophagosomes . Scale bars , 20 μm . Right , lysates from these cells were also immunoblotted with anti - FAK and anti - actin antibodies . ( d ) Left , wild - type FAK , FAKY397F or FAKY4F - Y9F cells were transfected with RFP - GFP - LC3 . Solid arrows indicate co - localization and dotted arrows indicate its absence . Scale bars , 20 μm . ( e ) Lysates were immunoblotted with anti - LC3B and actin antibodies in the absence and presence of chloroquine . Densitometry was carried out to calculate the increase in the amount of LC3B - II relative to actin for each cell type after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown . Uncropped images of blots are shown in Supplementary Fig . S9 .

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 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 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 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

figf4(a) Top, FAK−/− cells were treated with 200 nM dasatinib for 24 h and then fixed and stained for anti-Src antibody (green). Solid arrows indicate Src at the cell periphery; dashed arrows indicate Src in autophagosomes. Scale bars, 20 μm. Bottom, quantification of dasatinib-treated cells with Src in puncta. Data are presented as mean±s.d. and significance is P0.001 (n=3). Middle, lysates from cells treated with dasatinib were immunoblotted with anti-Src and anti-pTyr-416-Src and lysates from cells treated with dasatinib and chloroquine were immunoblotted with anti-LC3B and anti-actin antibodies. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each condition after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown.(b) Top, FAK−/− cells were transiently transfected with SrcY527F-GFP or with Src-251-GFP (green) and stained for anti-LC3B (red). Scale bars, 20 μm. Bottom, quantification of cells with co-localization between Src and LC3B. Data are presented as mean±s.d. and significance is P0.001 (n=3).(c) Left, FAK−/− cells stably re-expressing wild-type FAK, FAKY397F or FAKY4F-Y9F were fixed and stained for anti-FAK (red) and anti-pTyr-416-Src (green). Solid arrows indicate co-localization at adhesions and dashed arrows indicate active Src in autophagosomes. Scale bars, 20 μm. Right, lysates from these cells were also immunoblotted with anti-FAK and anti-actin antibodies.(d) Left, wild-type FAK, FAKY397F or FAKY4F-Y9F cells were transfected with RFP-GFP-LC3. Solid arrows indicate co-localization and dotted arrows indicate its absence. Scale bars, 20 μm.(e) Lysates were immunoblotted with anti-LC3B and actin antibodies in the absence and presence of chloroquine. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each cell type after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown. Uncropped images of blots are shown in Supplementary Fig. S9.


figf5 ( a ) Wild - type FAK cells were suspended in 1 . 4 % methylcellulose solution in growth media and plated on agarose for 3 days . Top and bottom right , cells were recovered and cytospins prepared that were stained for anti - pTyr - 416 - Src ( red ) , LC3B ( green ) and with DAPI ( blue ) or for paxillin ( red ) , LC3B ( green ) and with DAPI ( blue ) . Higher - magnification images of the outlined areas are also shown . Solid arrows indicate localization of pTyr - 416 - Src in LC3B - positive puncta . Scale bars , 20 μm . Bottom left , immunoblotting was carried out on lysates from adherent cells and from cells recovered from methylcellulose using anti - pTyr - 397 FAK , anti - FAK , anti - pTyr - 416 and anti - Src antibodies . ( b ) FAK + / + cells were suspended for 1 h in PBS and cytospins were prepared that were stained for anti - pTyr - 416 - Src ( green ) and with DAPI ( blue ) . A higher - magnification image of the area outlined is also shown . Scale bar , 20 μm . ( c ) LC3B was immunoprecipitated ( IP ) from adherent or suspended FAK + / + and then immunoblotted with anti - Src and anti - LC3B antibodies . Lysates were also immunoblotted with anti - pTyr - 397 - FAK and anti - actin antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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 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

figf5(a) Wild-type FAK cells were suspended in 1.4% methylcellulose solution in growth media and plated on agarose for 3 days. Top and bottom right, cells were recovered and cytospins prepared that were stained for anti-pTyr-416-Src (red), LC3B (green) and with DAPI (blue) or for paxillin (red), LC3B (green) and with DAPI (blue). Higher-magnification images of the outlined areas are also shown. Solid arrows indicate localization of pTyr-416-Src in LC3B-positive puncta. Scale bars, 20 μm. Bottom left, immunoblotting was carried out on lysates from adherent cells and from cells recovered from methylcellulose using anti-pTyr-397 FAK, anti-FAK, anti-pTyr-416 and anti-Src antibodies.(b) FAK+/+ cells were suspended for 1 h in PBS and cytospins were prepared that were stained for anti-pTyr-416-Src (green) and with DAPI (blue). A higher-magnification image of the area outlined is also shown. Scale bar, 20 μm.(c) LC3B was immunoprecipitated (IP) from adherent or suspended FAK+/+ and then immunoblotted with anti-Src and anti-LC3B antibodies. Lysates were also immunoblotted with anti-pTyr-397-FAK and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


figf6 ( a ) Top , FAK − / − cells were sparsely plated and then treated with 3 - MA ( for 48 h ) , chloroquine or dasatinib ( both for 24 h ) . Cells were then left at 37 ° C for 1 week to allow colony formation , fixed and then stained with crystal violet . Bottom , the number of colonies formed was then quantified and normalized against untreated controls . Data are presented as mean ± s . d . and significance is P0 . 001 for 3 - MA , P0 . 01 for chloroquine and P > 0 . 5 for dasatinib ( n = 3 ) . ( b ) Top , wild - type FAK and FAK − / − cells were transiently transfected with two individual Atg5 shRNAs ( 1 and 2 ) , selected in puromycin and then the clonogenic assay was carried out as described above . Bottom , quantification ; data are presented as mean ± s . d . and significance is P > 0 . 1 for Atg5 A and B in wild - type FAK cells and P0 . 01 for Atg5 A and P0 . 05 for Atg5 B in FAK − / − cells ( n = 3 ) . ( c ) Cells were also stained with TUNEL . The positive control was DNase1 - treated cells and the negative control lacked TUNEL reaction mix . Quantification is shown and data are presented as mean ± s . d . and significance is P0 . 005 for 3 - MA and P0 . 001 for Atg5 siRNA ( n = 3 ) . ( d ) FAK − / − cells were untreated or treated with dasatinib , 3 - MA , chloroquine or bafilomycin A ( BFA ) for 24 h , or were transfected with scrambled or Atg5 siRNA for 72 h and then immunoblotted with an anti - PARP antibody . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

figf6(a) Top, FAK−/− cells were sparsely plated and then treated with 3-MA (for 48 h), chloroquine or dasatinib (both for 24 h). Cells were then left at 37 °C for 1 week to allow colony formation, fixed and then stained with crystal violet. Bottom, the number of colonies formed was then quantified and normalized against untreated controls. Data are presented as mean±s.d. and significance is P0.001 for 3-MA, P0.01 for chloroquine and P>0.5 for dasatinib (n=3).(b) Top, wild-type FAK and FAK−/− cells were transiently transfected with two individual Atg5 shRNAs (1 and 2), selected in puromycin and then the clonogenic assay was carried out as described above. Bottom, quantification; data are presented as mean±s.d. and significance is P>0.1 for Atg5 A and B in wild-type FAK cells and P0.01 for Atg5 A and P0.05 for Atg5 B in FAK−/− cells (n=3).(c) Cells were also stained with TUNEL. The positive control was DNase1-treated cells and the negative control lacked TUNEL reaction mix. Quantification is shown and data are presented as mean±s.d. and significance is P0.005 for 3-MA and P0.001 for Atg5 siRNA (n=3).(d) FAK−/− cells were untreated or treated with dasatinib, 3-MA, chloroquine or bafilomycin A (BFA) for 24 h, or were transfected with scrambled or Atg5 siRNA for 72 h and then immunoblotted with an anti-PARP antibody. Uncropped images of blots are shown in Supplementary Fig. S9.


figf7 ( a ) Cells untreated , or treated with chloroquine for 24 h , were fixed and stained for anti - pTyr - 416 - Src ( red ) and anti - c - Cbl ( green ) . Solid arrows indicate co - localization at adhesions and dashed arrows indicate co - localization in autophagosomes . Scale bars , 20 μm . ( b ) c - Cbl was immunoprecipitated ( IP ) from FAK + / + and FAK − / − cells and then immunoblotted with anti - Src , anti - pTyr - 416 - Src and anti - c - Cbl antibodies . ( c , d ) Cells were transfected with scrambled siRNA or with c - Cbl siRNA for 72 h and then immunoblotted with anti - c - Cbl and actin antibodies ( c ) or stained for anti - pTyr - 416 - Src ( red ) and anti - c - Cbl ( green ; d , left ) . Dashed arrows indicate co - localization and solid arrows indicate Src at focal adhesions . Scale bars , 20 μm . Quantification is shown ( d , right ) . Data are presented as mean ± s . d . and significance is P > 0 . 5 for FAK + / + cells and P0 . 001 for FAK − / − cells ( n = 3 ) . ( e ) LC3B was immunoprecipitated from FAK − / − cells expressing either scrambled or c - Cbl siRNA and then immunoblotted with anti - Src and anti - LC3B antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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 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

figf7(a) Cells untreated, or treated with chloroquine for 24 h, were fixed and stained for anti-pTyr-416-Src (red) and anti-c-Cbl (green). Solid arrows indicate co-localization at adhesions and dashed arrows indicate co-localization in autophagosomes. Scale bars, 20 μm.(b) c-Cbl was immunoprecipitated (IP) from FAK+/+ and FAK−/− cells and then immunoblotted with anti-Src, anti-pTyr-416-Src and anti-c-Cbl antibodies.(c,d) Cells were transfected with scrambled siRNA or with c-Cbl siRNA for 72 h and then immunoblotted with anti-c-Cbl and actin antibodies (c) or stained for anti-pTyr-416-Src (red) and anti-c-Cbl (green; d, left). Dashed arrows indicate co-localization and solid arrows indicate Src at focal adhesions. Scale bars, 20 μm. Quantification is shown (d, right). Data are presented as mean±s.d. and significance is P>0.5 for FAK+/+ cells and P0.001 for FAK−/− cells (n=3).(e) LC3B was immunoprecipitated from FAK−/− cells expressing either scrambled or c-Cbl siRNA and then immunoblotted with anti-Src and anti-LC3B antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


figf8 ( a ) c - Cbl siRNA was transfected into FAK − / − cells expressing siRNA resistant , HA - tagged wild - type c - Cbl or c - Cbl mutants with defective E3 ligase activity ( c - CblC381A or c - Cbl - 70Z ) . Left , cells were stained for anti - p - Tyr - 416 - Src ( green ) , anti - HA ( red ) and with DAPI ( blue ) . Solid arrows show co - localization in intracellular puncta . Scale bars , 20 μm . Right , quantification of percentage of cells that contained active Src in intracellular puncta . Data are presented as mean ± s . d . and significance is P > 0 . 5 ( n = 3 ) . ( c ) GST - pulldown assay of HA - tagged wild - type c - Cbl , c - CblAA ( W802A , L805A ) and c - CblAAAA ( W802A , L803A , S804A , L805A ) expressed in HEK293T cells was carried out using GST - LC3 . ( d ) c - Cbl siRNA was transfected into FAK − / − cells expressing siRNA - resistant HA - tagged wild - type c - Cbl or c - CblAAAA . Left , cells were stained for anti - pTyr - 416 - Src ( green ) , anti - HA ( red ) and with DAPI ( blue ) . Solid arrows indicate pTyr - 416 localization to adhesions and dashed arrows indicate localization to intracellular puncta . Scale bars , 20 μm . Right , quantification of percentage of cells that contained active Src in intracellular puncta . Data are presented as mean ± s . d . and significance is P0 . 01 ( n = 3 ) . Uncropped images of blots are shown in Supplementary Fig . S9 .

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 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

figf8(a) c-Cbl siRNA was transfected into FAK−/− cells expressing siRNA resistant, HA-tagged wild-type c-Cbl or c-Cbl mutants with defective E3 ligase activity (c-CblC381A or c-Cbl-70Z). Left, cells were stained for anti-p-Tyr-416-Src (green), anti-HA (red) and with DAPI (blue). Solid arrows show co-localization in intracellular puncta. Scale bars, 20 μm. Right, quantification of percentage of cells that contained active Src in intracellular puncta. Data are presented as mean±s.d. and significance is P>0.5 (n=3).(c) GST-pulldown assay of HA-tagged wild-type c-Cbl, c-CblAA (W802A, L805A) and c-CblAAAA (W802A, L803A, S804A, L805A) expressed in HEK293T cells was carried out using GST-LC3.(d) c-Cbl siRNA was transfected into FAK−/− cells expressing siRNA-resistant HA-tagged wild-type c-Cbl or c-CblAAAA. Left, cells were stained for anti-pTyr-416-Src (green), anti-HA (red) and with DAPI (blue). Solid arrows indicate pTyr-416 localization to adhesions and dashed arrows indicate localization to intracellular puncta. Scale bars, 20 μm. Right, quantification of percentage of cells that contained active Src in intracellular puncta. Data are presented as mean±s.d. and significance is P0.01 (n=3). Uncropped images of blots are shown in Supplementary Fig. S9.


Figure 1A Gene targeting strategy . Left : max genomic locus , targeting construct and max targeted locus . In vitro Flipase ( Flip ) deletion of the selectable markers lacZ and neo results in a max targeted locus with exons 4 and 5 ( coding for bHLHLZip domain ) flanked by two loxP sites ( fl , flox allele ) . Arrows represent PCR primers used for genotyping MaxF9 and MaxF10 . Right : genomic PCR analysis of tail DNA from F1 mice . PCR products of max wt ( 430 bp ) and max flox ( 526 bp ) alleles . M , 1kb ladder . Flow cytometry analyses of B cell populations ( GFP + ) in Bone Marrow ( BM ) and spleen . Single - cell suspensions were prepared from BM or spleen of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against the indicated markers , and analyzed by flow cytometry . For BM analysis , cells were isolated from femora and tibiae . Pro - B cells ( Ly - 6c - CD49b - B220 + IgM - CD43 + ) , pre - B cells ( Ly - 6c - CD49b - B220 + IgM - CD43 - ) , immature B cells ( Ly - 6c - CD49b - B220loIgM + CD43 - ) , and mature B cells ( Ly - 6c - CD49b - B220hiIgM + CD43 - ) To identify KO cells ( GFP + ) all the plots show gated GFP + B lymphocytes . Flow cytometry analyses of B cell populations ( GFP + ) in Bone Marrow ( BM ) Single - cell suspensions were prepared from BM of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against the indicated markers , and analyzed by flow cytometry . For BM analysis , cells were isolated from femora and tibiae . Pro - B cells ( Ly - 6c - CD49b - B220 + IgM - CD43 + ) , pre - B cells ( Ly - 6c - CD49b - B220 + IgM - CD43 - ) Flow cytometry analyses of B cell populations ( GFP + ) in spleen . Single - cell suspensions were prepared from spleen of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against the indicated markers , and analyzed by flow cytometry . spleen follicular B cells ( B220 + CD23 + CD21int ) , marginal zone B cells ( B220 + CD23 - CD21hi ) and immature ( newly formed / transitional stage 1 ) B cells ( B220 + CD23 - CD21lo ) . To identify KO cells ( GFP + ) all the plots show gated GFP + B lymphocytes . F , G , H Absolute numbers of B lymphocytes in Bone Marrow ( F , G ) and spleen ( F , H ) from MaxKO - mb1 ( n = 6 ) , MycKO - mb - 1 ( n = 3 ) , DKO - mb1 ( n = 5 ) and control HET - mb1 ( n = 4 ) mice . Columns represent mean values and error bars standard deviations . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The different B cell populations were identified by flow cytometry Experiment representative of at least three independent experiments . I Cell cycle analysis by flow cytometry of sorted GFP + pre - and pro - B ( B220 + IgM - ) , immature ( B220loIgM + ) and mature ( B220hiIgM + ) bone marrow B cells from MaxKO - mb1 , MycKO - mb1 , DKO - mb1 and HET - mb1 control mice . Mice ( n = 5 ) were injected ( i . p . ) with 200 µg of EdU and analyzed 4 h later . Single - cell suspensions were prepared , stained and sorted . Sorted cells were subsequently stained with propidium iodide for DNA content . Experiment representative of at least three independent experiments . Dotted line is shown for EdU MFI comparison . J Percentage of annexin - V + GFP + cells in the BM of MaxKO - mb1 ( n = 5 ) , MycKO - mb1 ( n = 3 ) , DKO - mb1 ( n = 3 ) and control ( n = 4 ) mice . Pro - B , pre - B , immature and mature B lymphocyte subpopulations were identified by flow cytometry as indicated in E . * p < 0 . 05 , * * * p < 0 . 001 . Experiment 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 O O O 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 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 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 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 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

Figure 1A Gene targeting strategy. Left: max genomic locus, targeting construct and max targeted locus. In vitro Flipase (Flip) deletion of the selectable markers lacZ and neo results in a max targeted locus with exons 4 and 5 (coding for bHLHLZip domain) flanked by two loxP sites (fl, flox allele). Arrows represent PCR primers used for genotyping MaxF9 and MaxF10. Right: genomic PCR analysis of tail DNA from F1 mice. PCR products of max wt (430 bp) and max flox (526 bp) alleles. M, 1kb ladder.Flow cytometry analyses of B cell populations (GFP+) in Bone Marrow (BM) and spleen. Single-cell suspensions were prepared from BM or spleen of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. For BM analysis, cells were isolated from femora and tibiae. Pro-B cells (Ly-6c-CD49b-B220+IgM-CD43+), pre-B cells (Ly-6c-CD49b-B220+IgM-CD43-), immature B cells (Ly-6c-CD49b-B220loIgM+CD43-), and mature B cells (Ly-6c-CD49b-B220hiIgM+CD43-) To identify KO cells (GFP+) all the plots show gated GFP+ B lymphocytes.Flow cytometry analyses of B cell populations (GFP+) in Bone Marrow (BM) Single-cell suspensions were prepared from BM of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. For BM analysis, cells were isolated from femora and tibiae. Pro-B cells (Ly-6c-CD49b-B220+IgM-CD43+), pre-B cells (Ly-6c-CD49b-B220+IgM-CD43-)Flow cytometry analyses of B cell populations (GFP+) in spleen. Single-cell suspensions were prepared from spleen of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. spleen follicular B cells (B220+CD23+CD21int), marginal zone B cells (B220+CD23-CD21hi) and immature (newly formed/transitional stage 1) B cells (B220+CD23-CD21lo). To identify KO cells (GFP+) all the plots show gated GFP+ B lymphocytes.F, G, H Absolute numbers of B lymphocytes in Bone Marrow (F, G) and spleen (F, H) from MaxKO-mb1 (n=6), MycKO-mb-1 (n=3), DKO-mb1 (n=5) and control HET-mb1 (n=4) mice. Columns represent mean values and error bars standard deviations. *p<0.05, **p<0.01, ***p<0.001. The different B cell populations were identified by flow cytometry Experiment representative of at least three independent experiments.I Cell cycle analysis by flow cytometry of sorted GFP+ pre- and pro-B (B220+IgM-), immature (B220loIgM+) and mature (B220hiIgM+) bone marrow B cells from MaxKO-mb1, MycKO-mb1, DKO-mb1 and HET-mb1 control mice. Mice (n=5) were injected (i.p.) with 200 µg of EdU and analyzed 4 h later. Single-cell suspensions were prepared, stained and sorted. Sorted cells were subsequently stained with propidium iodide for DNA content. Experiment representative of at least three independent experiments. Dotted line is shown for EdU MFI comparison.J Percentage of annexin-V+GFP+ cells in the BM of MaxKO-mb1(n=5), MycKO-mb1(n=3), DKO-mb1 (n=3) and control (n=4) mice. Pro-B, pre-B, immature and mature B lymphocyte subpopulations were identified by flow cytometry as indicated in E. *p<0.05, ***p<0.001. Experiment representative of at least three independent experiments.


Figure 2Flow cytometry analysis and absolute numbers of B220loCD138 + ( plasmablasts ) ( A , B ) from HET - cd19 ( n = 6 ) , MaxKO - cd19 ( n = 8 ) , MycKO - cd19 ( n = 4 ) , and DKO - cd19 ( n = 5 ) mice . Spleen B220 + GFP + B lymphocytes were sorted and activated in vitro with LPS plus IL - 4 for 72 h . Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry . * p < 0 . 05 , * * p < 0 . 01 . Columns represent mean values and error bars standard deviations . Experiment representative of at least three independent experiments . Flow cytometry analysis and absolute numbers of B220 + IgG1 + switched B cells ( C , D ) from HET - cd19 ( n = 6 ) , MaxKO - cd19 ( n = 8 ) , MycKO - cd19 ( n = 4 ) , and DKO - cd19 ( n = 5 ) mice . Spleen B220 + GFP + B lymphocytes were sorted and activated in vitro with LPS plus IL - 4 for 72 h . Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry . * p < 0 . 05 , * * p < 0 . 01 . Columns represent mean values and error bars standard deviations . Experiment representative of at least three independent experiments . E Spleen B220 + GFP + B lymphocytes were stained with 7AAD and annexin - V to monitor apoptosis . 7 - AAD - annexin - v - ( alive ) , 7 - AAD - annexin - v + ( early apoptotic ) , 7 - AAD + annexin - v + ( late apoptotic ) , 7 - AAD + annexin - v - ( necrosis ) . F Relative numbers of live , early apoptotic , late apoptotic and necrotic cells from E . G CD69 surface expression in purified B220 + GFP + mature spleen cells . Sorted B cells were activated in vitro with LPS plus IL - 4 for 72 h , stained and analyzed by flow cytometry . The solid grey peak represents the expression levels of CD69 marker in non - activated mature B lymphocytes at day 0 . H Relative numbers of CD69 + B lymphocytes in the cultures from G ( n = 4 ) . Experiment representative of at least three independent experiments . I Analysis of germinal center ( GC ) formation in the spleen of MaxKO - cd19 , Myc - KO - cd19 , DKO - cd19 and heterozygous control mice immunized with TNP - KLH . Representative images of frozen spleen sections stained with IgM ( grey / blue ) , PNA ( GC marker ; red ) , and GFP ( Max - , c - Myc - or c - Myc / Max - deficient B cells ; green ) . Scale bar , 80µm . J Upper , Number of GCs per mouse in spleen sections of immunized MaxKO - cd19 ( n = 3 mice , n = 186 follicles analyzed ) , MycKO - cd19 ( n = 3 mice , n = 178 follicles analyzed ) , DKO - cd19 ( n = 3 mice , n = 191 follicles analyzed ) and heterozygous control mice ( n = 3 mice , n = 203 follicles analyzed ) . Lower , Frequencies of GC ( PNA + ) GFP + ( deleted cells ) or GFP - ( non - deleted cells ) in immunized MaxKO - cd19 ( n = 13 GCs ) , MycKO - cd19 ( n = 21 GCs ) , DKO - cd19 ( n = 27 GCs ) and heterozygous control ( n = 62 GCs ) mice . Analysis was performed at 13 days post - immunization . * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 2Flow cytometry analysis and absolute numbers of B220loCD138+ (plasmablasts) (A, B) from HET-cd19 (n=6), MaxKO-cd19 (n=8), MycKO-cd19 (n=4), and DKO-cd19 (n=5) mice. Spleen B220+GFP+ B lymphocytes were sorted and activated in vitro with LPS plus IL-4 for 72 h. Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry. *p<0.05, **p<0.01. Columns represent mean values and error bars standard deviations. Experiment representative of at least three independent experiments.Flow cytometry analysis and absolute numbers of B220+IgG1+ switched B cells (C, D) from HET-cd19 (n=6), MaxKO-cd19 (n=8), MycKO-cd19 (n=4), and DKO-cd19 (n=5) mice. Spleen B220+GFP+ B lymphocytes were sorted and activated in vitro with LPS plus IL-4 for 72 h. Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry. *p<0.05, **p<0.01. Columns represent mean values and error bars standard deviations. Experiment representative of at least three independent experiments.E Spleen B220+GFP+ B lymphocytes were stained with 7AAD and annexin-V to monitor apoptosis. 7-AAD- annexin-v- (alive), 7-AAD- annexin-v+ (early apoptotic), 7-AAD+ annexin-v+ (late apoptotic), 7-AAD+ annexin-v- (necrosis). F Relative numbers of live, early apoptotic, late apoptotic and necrotic cells from E. G CD69 surface expression in purified B220+GFP+ mature spleen cells. Sorted B cells were activated in vitro with LPS plus IL-4 for 72 h, stained and analyzed by flow cytometry. The solid grey peak represents the expression levels of CD69 marker in non-activated mature B lymphocytes at day 0. H Relative numbers of CD69+ B lymphocytes in the cultures from G (n=4). Experiment representative of at least three independent experiments. I Analysis of germinal center (GC) formation in the spleen of MaxKO-cd19, Myc-KO-cd19, DKO-cd19 and heterozygous control mice immunized with TNP-KLH. Representative images of frozen spleen sections stained with IgM (grey/blue), PNA (GC marker; red), and GFP (Max-, c-Myc- or c-Myc/Max-deficient B cells; green). Scale bar, 80µm.J Upper, Number of GCs per mouse in spleen sections of immunized MaxKO-cd19 (n=3 mice, n=186 follicles analyzed), MycKO-cd19 (n=3 mice, n=178 follicles analyzed), DKO-cd19 (n=3 mice, n=191 follicles analyzed) and heterozygous control mice (n=3 mice, n=203 follicles analyzed). Lower, Frequencies of GC (PNA+) GFP+ (deleted cells) or GFP- (non-deleted cells) in immunized MaxKO-cd19 (n=13 GCs), MycKO-cd19 (n=21 GCs), DKO-cd19 (n=27 GCs) and heterozygous control (n=62 GCs) mice. Analysis was performed at 13 days post-immunization. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


Figure 3A Number of cell divisions in activated mature B lymphocytes . Sorted B220 + GFP + spleen cells from HET - cd19 ( n = 5 ) , MaxKO - cd19 ( n = 5 ) , MycKO - cd19 ( n = 3 ) , and DKO - cd19 ( n = 3 ) mice were stained with a cell tracer , activated in vitro with LPS plus IL - 4 for 72 h and analyzed by flow cytometry . Experiment representative of at least three independent experiments B Cell counts from the cultures C Photographs of B cell cultures Scale bar , 500 µm . 4 × magnification . D Forward scatter ( FSC ) histograms of activated B lymphocytes An arbitrary dashed line is shown for comparison . Cell cycle analysis of sorted B220 + GFP + B lymphocytes activated in vitro . Cells were activated and EdU was added 4 h before harvesting . Cells were stained with propidium iodide to measure DNA content . Experiment representative of at least three independent experiments . An arbitrary dashed line shows the difference of EdU MFI . S phase mean fluorescence intensity ( MFI ) : 7871 HET , 6512 MaxKO , 5860 MycKO , 6420 DKORepresentation of labeling with nucleotide analogues CldU ( red ) and IdU ( green ) and photographs of DNA fibers from sorted B220 + GFP + B lymphocytes activated for 48 h . Scale bar , 10 µm . H Fork rate of DNA fibres from primary B lymphocytes sorted and activated Data is pooled from three replicate experiments . Horizontal bars represent median values . 150 - 250 forks were measured per condition . * * * , p < 0 . 001 ; * * , p < 0 . 01I Western blotting of DNA replication proteins in activated B lymphocytes Sorted B220 + GFP + spleen cells were activated in vitro with LPS plus IL - 4 for 72 h . MEK2 , SMC1 are shown as loading controls . The blots shown are representative of 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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

Figure 3A Number of cell divisions in activated mature B lymphocytes. Sorted B220+GFP+ spleen cells from HET-cd19 (n=5), MaxKO-cd19 (n=5), MycKO-cd19 (n=3), and DKO-cd19 (n=3) mice were stained with a cell tracer, activated in vitro with LPS plus IL-4 for 72 h and analyzed by flow cytometry. Experiment representative of at least three independent experiments B Cell counts from the cultures C Photographs of B cell cultures Scale bar, 500 µm. 4× magnification. D Forward scatter (FSC) histograms of activated B lymphocytes An arbitrary dashed line is shown for comparison.Cell cycle analysis of sorted B220+GFP+ B lymphocytes activated in vitro. Cells were activated and EdU was added 4 h before harvesting. Cells were stained with propidium iodide to measure DNA content. Experiment representative of at least three independent experiments. An arbitrary dashed line shows the difference of EdU MFI. S phase mean fluorescence intensity (MFI): 7871 HET, 6512 MaxKO, 5860 MycKO, 6420 DKORepresentation of labeling with nucleotide analogues CldU (red) and IdU (green) and photographs of DNA fibers from sorted B220+GFP+ B lymphocytes activated for 48 h. Scale bar, 10 µm.H Fork rate of DNA fibres from primary B lymphocytes sorted and activated Data is pooled from three replicate experiments. Horizontal bars represent median values. 150-250 forks were measured per condition. ***, p<0.001; **, p<0.01I Western blotting of DNA replication proteins in activated B lymphocytes Sorted B220+GFP+ spleen cells were activated in vitro with LPS plus IL-4 for 72 h. MEK2, SMC1 are shown as loading controls. The blots shown are representative of three independent experiments.


Figure 4A Bar plot showing the number of DEGs ( adj . p - value < 0 . 01 , FC + 1 . 5 in red , 1 . 5 < FC > - 1 . 5 in white , FC < - 1 . 5 in blue ) . Sorted B220 + GFP + spleen B cells from MaxKO - cd19 , MycKO - cd19 , DKO - cd19 and control HET - cd19 mice were activated with LPS plus IL - 4 for 72 h and analyzed . Two samples for each condition were used . C Hierarchical clustering heat map of DEGs in any comparison ( two samples per condition ) . Dark blue in left panel indicates significance for that contrast column . Colour code : red represents upregulation , white , unchanged , and blue downregulation of expression . Gender : colour code for genetic condition . Pink are female and blue colour male mice . D Venn diagrams of up - ( left ) and down - regulated genes ( right ) for all three conditions ( + / - 1 . 5 - FC and p < 0 . 01 ) . Heatmaps of genes only deregulated in MaxKO - cd19 , MycKO - cd19 , or DKO - cd19 mice are shown .

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 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

Figure 4A Bar plot showing the number of DEGs (adj. p-value <0.01, FC +1.5 in red, 1.5 < FC >-1.5 in white, FC< -1.5 in blue). Sorted B220+GFP+ spleen B cells from MaxKO-cd19, MycKO-cd19, DKO-cd19 and control HET-cd19 mice were activated with LPS plus IL-4 for 72 h and analyzed. Two samples for each condition were used.C Hierarchical clustering heat map of DEGs in any comparison (two samples per condition). Dark blue in left panel indicates significance for that contrast column. Colour code: red represents upregulation, white, unchanged, and blue downregulation of expression. Gender: colour code for genetic condition. Pink are female and blue colour male mice.D Venn diagrams of up- (left) and down-regulated genes (right) for all three conditions (+/-1.5-FC and p<0.01). Heatmaps of genes only deregulated in MaxKO-cd19, MycKO-cd19, or DKO-cd19 mice are shown.


Figure 5B Heatmaps of genes involved in the relevant pathways Colour code : red represents upregulation , white , unchanged , and blue downregulation of expression . C qPCR analysis of up - and down - regulated genes Pooled RNA was prepared from sorted B220 + GFP + spleen B cells of MaxKO - cd19 ( n = 4 ) , MycKO - cd19 ( n = 4 ) , or DKO - cd19 ( n = 4 ) and control HET - cd19 ( n = 4 ) mice activated Genes were randomly selected and qPCR was performed using specific primers for cd72 , med13l , card11 , ssh2 , cdc7 , mrpl12 , rpl39 , and cysc . max and c - myc were included as controls and actin was used for normalization .

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Figure 5B Heatmaps of genes involved in the relevant pathways Colour code: red represents upregulation, white, unchanged, and blue downregulation of expression.C qPCR analysis of up- and down-regulated genes Pooled RNA was prepared from sorted B220+GFP+ spleen B cells of MaxKO-cd19 (n=4), MycKO-cd19 (n=4), or DKO-cd19 (n=4) and control HET-cd19 (n=4) mice activated Genes were randomly selected and qPCR was performed using specific primers for cd72, med13l, card11, ssh2, cdc7, mrpl12, rpl39, and cysc. max and c-myc were included as controls and actin was used for normalization.