( E ) OMA1 interacts with SLP2 . OMA1 - / - FITR293T cells ectopically expressing OMA1 - myc were transfected with pcDNA5 ( Control ) or pcDNA5 - SLP2 - FLAG ( FLAG ) . Isolated mitochondria were solubilized in digitonin and subjected to immunoprecipitation using FLAG - specific antibodies . Native eluates of the precipitate were analyzed by BN - PAGE and immunoblotting using SLP2 - , YME1L - , PARL - , and myc - specific antibodies . In , input ( 8 % ) ; E , eluate ( 100 % ) . Samples were analyzed by SDS - PAGE in parallel ( lower panel ) . ( F ) Quantification of immunoprecipitation efficiencies in ( E ) .

O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) OMA1 interacts with SLP2. OMA1-/-FITR293T cells ectopically expressing OMA1-myc were transfected with pcDNA5 (Control) or pcDNA5-SLP2-FLAG (FLAG). Isolated mitochondria were solubilized in digitonin and subjected to immunoprecipitation using FLAG-specific antibodies. Native eluates of the precipitate were analyzed by BN-PAGE and immunoblotting using SLP2-, YME1L-, PARL-, and myc-specific antibodies. In, input (8%); E, eluate (100%). Samples were analyzed by SDS-PAGE in parallel (lower panel).(F) Quantification of immunoprecipitation efficiencies in (E).


( a ) Western blot analysis showing the replacement of endogenous Beclin 1 with Beclin 1 - EGFP in Becn1 - / - ; Becn1 - EGFP / + mice , as detected by an anti - Beclin 1 antibody .

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

(a) Western blot analysis showing the replacement of endogenous Beclin 1 with Beclin 1-EGFP in Becn1-/-;Becn1-EGFP/+ mice, as detected by an anti-Beclin 1 antibody.


( b ) Coomassie - stained SDS - PAGE showing the Beclin 1 - interacting proteins immuno - isolated from brain and liver of the ' rescued ' mice ( lanes 2 and 4 ) and of control Becn1 + / - littermates ( lanes 1 and 3 ) , using an anti - GFP antibody . Proteins in the gel bands were extracted and identified by mass spectrometry as Vps15 / p150 ( band 1 ) , Vps34 / class III PI ( 3 ) K ( band 3 ) , UVRAG ( band 4 ) , Beclin 1 - EGFP ( band 5 ) , Atg14L ( band 6 , asterisk , gi | 27369860 ) and Rubicon ( band 2 , asterisk , gi | 45708948 ) . UVRAG levels varied with different affinity - purification conditions , suggesting an unstable association of UVRAG with the complex .

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

(b) Coomassie-stained SDS-PAGE showing the Beclin 1-interacting proteins immuno-isolated from brain and liver of the 'rescued' mice (lanes 2 and 4) and of control Becn1+/- littermates (lanes 1 and 3), using an anti-GFP antibody. Proteins in the gel bands were extracted and identified by mass spectrometry as Vps15/p150 (band 1), Vps34/class III PI(3)K (band 3), UVRAG (band 4), Beclin 1-EGFP (band 5), Atg14L (band 6, asterisk, gi|27369860) and Rubicon (band 2, asterisk, gi|45708948). UVRAG levels varied with different affinity-purification conditions, suggesting an unstable association of UVRAG with the complex.


( d ) Western blot analysis of Atg14L , Rubicon , Vps34 and Beclin 1 in gel filtration fractions from wild - type mouse liver extract showed co - elution of these proteins in fractions 38 - 45 . Atg14L was also eluted in later fractions 51 - 56 . The fractions for the peak elution of thyroglobulin ( 670K ) and γ - globulin ( 158K ) are indicated by arrows . Control siRNA - transfected NIH 3T3 cell lysate was loaded as a positive control ( labelled as 1 ) for the migration position of the Atg14L protein on SDS - PAGE ; Atg14L siRNA - transfected NIH 3T3 cell lysate was loaded as a negative control ( labelled as 2 ) .

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

(d) Western blot analysis of Atg14L, Rubicon, Vps34 and Beclin 1 in gel filtration fractions from wild-type mouse liver extract showed co-elution of these proteins in fractions 38-45. Atg14L was also eluted in later fractions 51-56. The fractions for the peak elution of thyroglobulin (670K) and γ-globulin (158K) are indicated by arrows. Control siRNA-transfected NIH 3T3 cell lysate was loaded as a positive control (labelled as 1) for the migration position of the Atg14L protein on SDS-PAGE; Atg14L siRNA-transfected NIH 3T3 cell lysate was loaded as a negative control (labelled as 2).


( e , f ) Co - immunoprecipitation confirmed protein - protein interaction between Atg14L and Rubicon . HEK 293 cells were co - transfected with Atg14L - EGFP and Flag - Rubicon ( e ) or Rubicon - EGFP and Flag - Atg14L ( f ) . Cell lysates were used for immunoprecipitation with an anti - GFP antibody and the resulting immunoprecipitates were blotted with an anti - Flag antibody . Our results show immunoprecipitation of Rubicon by Atg14L ( e ) and vice versa ( f ) . WCL , whole cell lysate ; IP , immunoprecipitated . See Supplementary Information , Fig . S6 for full scans of blots in a , d , e and f .

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

(e, f) Co-immunoprecipitation confirmed protein-protein interaction between Atg14L and Rubicon. HEK 293 cells were co-transfected with Atg14L-EGFP and Flag-Rubicon (e) or Rubicon-EGFP and Flag-Atg14L (f). Cell lysates were used for immunoprecipitation with an anti-GFP antibody and the resulting immunoprecipitates were blotted with an anti-Flag antibody. Our results show immunoprecipitation of Rubicon by Atg14L (e) and vice versa (f). WCL, whole cell lysate; IP, immunoprecipitated. See Supplementary Information, Fig. S6 for full scans of blots in a, d, e and f.


( a ) Beclin 1 or Atg14L siRNA reduced Atg14L levels and increased p62 / SQSTM1 and LC3 II levels under normal and nutrient - starvation conditions in NIH 3T3 cells .

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

(a) Beclin 1 or Atg14L siRNA reduced Atg14L levels and increased p62/SQSTM1 and LC3 II levels under normal and nutrient-starvation conditions in NIH 3T3 cells.


( b ) Compared with control siRNA , Atg14L siRNA decreased long - lived protein degradation in NIH 3T3 cells under normal ( * P = 0 . 007 ) and starvation ( * P = 5 × 10 − 6 ) conditions ( one - tailed Student ' s t - test with equal variances , n = 4 ) . This difference was diminished when the starved cells were treated with 3 - methyladenine ( 3MA , 10 mM ) , a PI ( 3 ) K inhibitor .

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

(b) Compared with control siRNA, Atg14L siRNA decreased long-lived protein degradation in NIH 3T3 cells under normal (*P = 0.007) and starvation (*P = 5 × 10−6) conditions (one-tailed Student's t-test with equal variances, n = 4). This difference was diminished when the starved cells were treated with 3-methyladenine (3MA, 10 mM), a PI(3)K inhibitor.


( c ) Vps34 kinase assay . HEK 293T cells were co - transfected with Myc - Vps34 - Vps15 and Flag - Atg14L or Flag vector , either in the absence or in the presence of Beclin 1 - EGFP . Myc - Vps34 - Vps15 was immunoprecipitated by anti - Myc antibody for the in vitro kinase assay . The resulting radioactive PI ( 3 ) P was separated by thin - layer chromatography ( TLC ) , quantified and normalized against the amount of immunoprecipitated Myc - tagged Vps34 as measured by western blot ( upper panel ) . The quantified results ( lower panel ) show that overexpressing Atg14L significantly upregulated Vps34 kinase activity by 2 . 5 - fold , but only when Beclin 1 was also overexpressed ( * P = 0 . 04 , one - tailed Student ' s t - test with unequal variances , n = 5 ) .

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

(c) Vps34 kinase assay. HEK 293T cells were co-transfected with Myc-Vps34-Vps15 and Flag-Atg14L or Flag vector, either in the absence or in the presence of Beclin 1-EGFP. Myc-Vps34-Vps15 was immunoprecipitated by anti-Myc antibody for the in vitro kinase assay. The resulting radioactive PI(3)P was separated by thin-layer chromatography (TLC), quantified and normalized against the amount of immunoprecipitated Myc-tagged Vps34 as measured by western blot (upper panel). The quantified results (lower panel) show that overexpressing Atg14L significantly upregulated Vps34 kinase activity by 2.5-fold, but only when Beclin 1 was also overexpressed (*P = 0.04, one-tailed Student's t-test with unequal variances, n = 5).


( d ) Colocalization of co - expressed Atg14L - EGFP ( green ) and Beclin 1 - AsRed ( red ) in punctate structures in transiently transfected HeLa cells . Scale bar , 10 μm .

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

(d) Colocalization of co-expressed Atg14L-EGFP (green) and Beclin 1-AsRed (red) in punctate structures in transiently transfected HeLa cells. Scale bar, 10 μm.


( e ) Electron microscopy images show large structures ( asterisks ) that are often enwrapped with double membranes in the HEK 293T cells co - transfected with Atg14L - EGFP and Beclin 1 - AsRed : concentric membrane ' rings ' ( panel 1 ) ; two large structures ( 3 - 5 μm in diameter , panel 2 ) containing material with high electron density ( inset , enwrapping double membranes ) ; numerous autophagosomes ( arrows , panel 3 ) in the cytoplasm ; immuno - electron microscopy image of a Atg14L - Beclin 1 structure ( labelled with anti - GFP antibody and developed by DAB , panel 4 ) enwrapped with concentric membrane ' rings ' . M , mitochondria ; N , nucleus . Scale bar , 500 nm .

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

(e) Electron microscopy images show large structures (asterisks) that are often enwrapped with double membranes in the HEK 293T cells co-transfected with Atg14L-EGFP and Beclin 1-AsRed: concentric membrane 'rings' (panel 1); two large structures (3-5 μm in diameter, panel 2) containing material with high electron density (inset, enwrapping double membranes); numerous autophagosomes (arrows, panel 3) in the cytoplasm; immuno-electron microscopy image of a Atg14L-Beclin 1 structure (labelled with anti-GFP antibody and developed by DAB, panel 4) enwrapped with concentric membrane 'rings'. M, mitochondria; N, nucleus. Scale bar, 500 nm.


( f , g ) EGFP - Atg12 ( f ) or EGFP - Atg5 ( g ) ( green ) was colocalized with the large structures ( arrows ) that were labelled by Atg14L - AsRed ( red ) and Beclin 1 - Myc ( blue ) in transfected HeLa cells . Some of these structures seemed to be ' ring ' - shaped ( yellow arrows and inset ) . Scale bar , 10 μm . See Supplementary Information , Fig . S6 for full scans of blots in a and c .

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

(f, g) EGFP-Atg12 (f) or EGFP-Atg5 (g) (green) was colocalized with the large structures (arrows) that were labelled by Atg14L-AsRed (red) and Beclin 1-Myc (blue) in transfected HeLa cells. Some of these structures seemed to be 'ring'-shaped (yellow arrows and inset). Scale bar, 10 μm. See Supplementary Information, Fig. S6 for full scans of blots in a and c.


( a ) Rubicon siRNA treatment of the NIH 3T3 cells led to decreased levels of p62 and LC3 II under both normal and nutrient - starvation conditions .

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

(a) Rubicon siRNA treatment of the NIH 3T3 cells led to decreased levels of p62 and LC3 II under both normal and nutrient-starvation conditions.


( b ) Overexpression of Rubicon resulted in increased levels of p62 under both normal and nutrient - starved conditions in HEK 293 cells either stably expressing ( upper rows ) or transiently transfected with ( lower rows ) Rubicon - EGFP . The control cells were either stably expressing or transiently transfected with the EGFP - N3 vector .

O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(b) Overexpression of Rubicon resulted in increased levels of p62 under both normal and nutrient-starved conditions in HEK 293 cells either stably expressing (upper rows) or transiently transfected with (lower rows) Rubicon-EGFP. The control cells were either stably expressing or transiently transfected with the EGFP-N3 vector.


( c ) Vps34 kinase activity . HEK 293T cells were co - transfected with Myc - Vps34 - Vps15 and Flag - Rubicon or Flag vector , either in the absence or in the presence of Beclin 1 - EGFP . Myc - Vps34 - Vps15 was immunoprecipitated by an anti - Myc antibody and used for the in vitro kinase assay . The resulting radioactive PI ( 3 ) P was separated by TLC , quantified and normalized against the amount of immunoprecipitated Myc - tagged Vps34 , as measured by western blotting ( upper panel ) . The quantified results ( lower panel ) show that overexpressing Rubicon significantly downregulated the Vps34 kinase activity to 0 . 58 - fold , but only without Beclin 1 overexpression ( * P = 0 . 04 , one - tailed Student ' s t - test with unequal variances , n = 4 ) .

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

(c) Vps34 kinase activity. HEK 293T cells were co-transfected with Myc-Vps34-Vps15 and Flag-Rubicon or Flag vector, either in the absence or in the presence of Beclin 1-EGFP. Myc-Vps34-Vps15 was immunoprecipitated by an anti-Myc antibody and used for the in vitro kinase assay. The resulting radioactive PI(3)P was separated by TLC, quantified and normalized against the amount of immunoprecipitated Myc-tagged Vps34, as measured by western blotting (upper panel). The quantified results (lower panel) show that overexpressing Rubicon significantly downregulated the Vps34 kinase activity to 0.58-fold, but only without Beclin 1 overexpression (*P = 0.04, one-tailed Student's t-test with unequal variances, n = 4).


( d ) Effect of overexpressing Flag - Rubicon on autophagosome acidification , as monitored by mCherry - GFP - LC3 fluorescence . HeLa cells were transiently co - transfected with mCherry - GFP - LC3 and Flag - Rubicon ( or control Flag vector ) . Cells co - expressing mCherry - GFP - LC3 and control Flag vector contained many red - only puncta along with yellow ( indicating the presence of both red and green ) puncta , suggesting the presence of both autolysosomes and nascent autophagosomes ( upper panel ) . In contrast , cells co - expressing mCherry - GFP - LC3 and Flag - Rubicon contained primarily yellow or white puncta , suggesting the presence of only nascent autophagosomes ( lower panel , white arrows ) . Notably , some cells , which were co - transfected with mCherry - GFP - LC3 and Flag - Rubicon but expressed high levels of mCherry - GFP - LC3 and undetectable levels of FLAG - Rubicon , contained many red - only puncta ( lower panel , yellow arrows ) . ( e ) Quantification of the results in d show that overexpressing Flag - Rubicon markedly reduced the percentage of red - only puncta ( mCherry - LC3 ) from 39 % in the control Flag vector - transfected cells to 2 % in the Flag - Rubicon - transfected cells ( * P = 2 × 10 − 26 , one - tailed Student ' s t - test with unequal variances , n = 30 ) , indicating that overexpression of Rubicon blocks autophagosome acidification or maturation . See Supplementary Information , Fig . S6 for full scans of blots in a - c .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(d) Effect of overexpressing Flag-Rubicon on autophagosome acidification, as monitored by mCherry-GFP-LC3 fluorescence. HeLa cells were transiently co-transfected with mCherry-GFP-LC3 and Flag-Rubicon (or control Flag vector). Cells co-expressing mCherry-GFP-LC3 and control Flag vector contained many red-only puncta along with yellow (indicating the presence of both red and green) puncta, suggesting the presence of both autolysosomes and nascent autophagosomes (upper panel). In contrast, cells co-expressing mCherry-GFP-LC3 and Flag-Rubicon contained primarily yellow or white puncta, suggesting the presence of only nascent autophagosomes (lower panel, white arrows). Notably, some cells, which were co-transfected with mCherry-GFP-LC3 and Flag-Rubicon but expressed high levels of mCherry-GFP-LC3 and undetectable levels of FLAG-Rubicon, contained many red-only puncta (lower panel, yellow arrows). (e) Quantification of the results in d show that overexpressing Flag-Rubicon markedly reduced the percentage of red-only puncta (mCherry-LC3) from 39% in the control Flag vector-transfected cells to 2% in the Flag-Rubicon-transfected cells (*P = 2 × 10−26, one-tailed Student's t-test with unequal variances, n = 30), indicating that overexpression of Rubicon blocks autophagosome acidification or maturation. See Supplementary Information, Fig. S6 for full scans of blots in a-c.


( a ) Colocalization of Rubicon - EGFP - associated structures with the late endosome / lysosome marker Lamp1 ( arrows ) in HeLa cells transfected with Rubicon - EGFP . Note that some of the Rubicon - EGFP - associated structures show a ' ring ' shape ( yellow arrows ) . Scale bar , 10 μm .

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

(a) Colocalization of Rubicon-EGFP-associated structures with the late endosome/lysosome marker Lamp1 (arrows) in HeLa cells transfected with Rubicon-EGFP. Note that some of the Rubicon-EGFP-associated structures show a 'ring' shape (yellow arrows). Scale bar, 10 μm.


( b ) Partial colocalization of Rubicon - EGFP - associated structures with the MVB marker LBPA ( arrows ) in HeLa cells transfected with Rubicon - EGFP . Scale bar , 10 μm .

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

(b) Partial colocalization of Rubicon-EGFP-associated structures with the MVB marker LBPA (arrows) in HeLa cells transfected with Rubicon-EGFP. Scale bar, 10 μm.


( c ) Representative ultrastructural images show aberrant expansion of late endosomal / lysosomal structures in HEK 293T cells overexpressing Rubicon - EGFP . These abnormal organelles are large in size , with high ( orange arrows ) or low ( black arrows ) electron density . Some enclose small vesicles ( purple arrows ) and some resemble the MVB ( blue arrows ) . Scale bars , 500 nm .

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

(c) Representative ultrastructural images show aberrant expansion of late endosomal/lysosomal structures in HEK 293T cells overexpressing Rubicon-EGFP. These abnormal organelles are large in size, with high (orange arrows) or low (black arrows) electron density. Some enclose small vesicles (purple arrows) and some resemble the MVB (blue arrows). Scale bars, 500 nm.


( d ) Representative ultrastructural images show late endosome / lysosome - like structures that are labelled with anti - GFP gold particles ( panels 3 , 4 ) in HEK 293T cells transiently transfected with Rubicon - EGFP . These structures are enwrapped by double membranes ( panel 4 inset ) and co - labelled by anti - GFP ( developed by DAB ) and anti - Lamp1 ( gold enhanced ) ( panels 5 - 7 ) antibodies . Note that mitochondria are mostly negative for Rubicon - EGFP ( panel 4 ) . The negative controls are without antibody ( panels 1 - 2 ) . M , mitochondria . Scale bars , 200 nm .

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

(d) Representative ultrastructural images show late endosome/lysosome-like structures that are labelled with anti-GFP gold particles (panels 3, 4) in HEK 293T cells transiently transfected with Rubicon-EGFP. These structures are enwrapped by double membranes (panel 4 inset) and co-labelled by anti-GFP (developed by DAB) and anti-Lamp1 (gold enhanced) (panels 5-7) antibodies. Note that mitochondria are mostly negative for Rubicon-EGFP (panel 4). The negative controls are without antibody (panels 1-2). M, mitochondria. Scale bars, 200 nm.


( b ) Colocalization of the PI ( 3 ) P - enriched lipid domain marker p40 ( phox ) - PX - EGFP ( green ) and Rubicon - AsRed ( red ) on large punctate structures ( arrows ) in the co - transfected HeLa cells ( upper panels ) . Treatment with the PI ( 3 ) K inhibitor wortmannin ( 75 nM ) for 1 h caused disappearance of the PI ( 3 ) P - enriched lipid domains , whereas the Rubicon - AsRed - positive structures were maintained ( lower panels ) . Scale bars , 10 μm .

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

(b) Colocalization of the PI(3)P-enriched lipid domain marker p40 (phox)-PX-EGFP (green) and Rubicon-AsRed (red) on large punctate structures (arrows) in the co-transfected HeLa cells (upper panels). Treatment with the PI(3)K inhibitor wortmannin (75 nM) for 1 h caused disappearance of the PI(3)P-enriched lipid domains, whereas the Rubicon-AsRed-positive structures were maintained (lower panels). Scale bars, 10 μm.


( c ) Subcellular localization of transiently transfected Rubicon - EGFP , RubiconΔRUN - EGFP , RubiconΔC - EGFP or RubiconΔRUNΔC - EGFP in HeLa cells . In contrast to punctate Rubicon - EGFP and RubiconΔRUN - EGFP , RubiconΔC - EGFP and RubiconΔRUNΔC - EGFP were dispersed in the cytoplasm . ΔRUN , RUN domain deletion ; ΔC , cysteine - rich domain deletion . Scale bars , 10 μm .

O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Subcellular localization of transiently transfected Rubicon-EGFP, RubiconΔRUN-EGFP, RubiconΔC-EGFP or RubiconΔRUNΔC-EGFP in HeLa cells. In contrast to punctate Rubicon-EGFP and RubiconΔRUN-EGFP, RubiconΔC-EGFP and RubiconΔRUNΔC-EGFP were dispersed in the cytoplasm. ΔRUN, RUN domain deletion; ΔC, cysteine-rich domain deletion. Scale bars, 10 μm.


( d , e ) Absence of full - length Beclin 1 ( d ) or Beclin 1 - CE mutant ( e ) ( red ) on the Rubicon - EGFP - positive structures ( green ) in HEK 293 cells stably expressing Rubicon - EGFP . These cells were transiently transfected with either Beclin 1 - AsRed ( d ) or Flag - Beclin 1 - CE ( e ; that is , the Flag - tagged Beclin 1 mutant containing both CCD and ECD , which mediate the Beclin 1 - Rubicon interaction as shown in Supplementary Information , Fig . S3h ) . Scale bars , 10 μm .

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

(d, e) Absence of full-length Beclin 1 (d) or Beclin 1-CE mutant (e) (red) on the Rubicon-EGFP-positive structures (green) in HEK 293 cells stably expressing Rubicon-EGFP. These cells were transiently transfected with either Beclin 1-AsRed (d) or Flag-Beclin 1-CE (e; that is, the Flag-tagged Beclin 1 mutant containing both CCD and ECD, which mediate the Beclin 1-Rubicon interaction as shown in Supplementary Information, Fig. S3h). Scale bars, 10 μm.


( f ) The formation of the Rubicon - EGFP - positive structures was not affected by siRNA knockdown of Beclin 1 in HEK 293 cells stably expressing Rubicon - EGFP .

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

(f) The formation of the Rubicon-EGFP-positive structures was not affected by siRNA knockdown of Beclin 1 in HEK 293 cells stably expressing Rubicon-EGFP.


( a ) Beeswarm plot with Factor 1 values for each sample with colors corresponding to three clusters found by 3 - means clustering with low factor values ( LZ ) , intermediate factor values ( IZ ) and high factor values ( HZ )

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

(a) Beeswarm plot with Factor 1 values for each sample with colors corresponding to three clusters found by 3-means clustering with low factor values (LZ), intermediate factor values (IZ) and high factor values (HZ)


( b ) Absolute loadings for the genes with the largest absolute weights in the mRNA data . Plus or minus symbols on the right indicate the sign of the loading . Genes highlighted in orange were previously described as prognostic markers in CLL and associated with IGHV status ( Maloum et al , 2009 ; Morabito et al , 2015 ; Plesingerova et al , 2017 ; Trojani et al , 2012 ; Vasconcelos et al , 2005 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Absolute loadings for the genes with the largest absolute weights in the mRNA data. Plus or minus symbols on the right indicate the sign of the loading. Genes highlighted in orange were previously described as prognostic markers in CLL and associated with IGHV status (Maloum et al, 2009; Morabito et al, 2015; Plesingerova et al, 2017; Trojani et al, 2012; Vasconcelos et al, 2005)


( c ) Heatmap of gene expression values for genes with the largest weights as in

(c) Heatmap of gene expression values for genes with the largest weights as in b


( d ) Absolute loadings of the drugs with the largest weights , annotated by target

(d) Absolute loadings of the drugs with the largest weights, annotated by target category


( e ) Drug response curves for two of the drugs , stratified by the clusters as in

(e) Drug response curves for two of the drugs, stratified by the clusters as in a


B . Quantification of PLAs between BRCA2 and DPP9 in control HeLa WT cells treated with non - targeting siRNA ( siNT ) or silenced with the indicated oligos . 300 ng / mL MMC was added for 24 h . Each dot represents the number of PLA events in a single cell , from two to seven biological replicates ( siNT - MMC ( n = 6 ) , siNT + MMC ( n = 7 ) , siDPP9 - MMC ( n = 4 ) , siDPP9 + MMC ( n = 5 ) , siBRCA2 - MMC ( n = 2 ) , siBRCA2 + MMC ( n = 3 ) , siFLNA - MMC ( n = 5 ) , siFLNA + MMC ( n = 6 ) , NgtCntrl - MMC ( n = 3 ) , NgtCntrl + MMC ( n = 6 ) ) . The number of foci are shown based on their cellular localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * = p ≤ 0 . 05 , * * * * = p ≤ 0 . 0001 ) . C . Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa WT cells . Exposure of cells to MMC triggers more PLA events ( white ) . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _

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

B. Quantification of PLAs between BRCA2 and DPP9 in control HeLa WT cells treated with non-targeting siRNA (siNT) or silenced with the indicated oligos. 300 ng / mL MMC was added for 24 h. Each dot represents the number of PLA events in a single cell, from two to seven biological replicates (siNT-MMC (n = 6), siNT+MMC (n = 7), siDPP9-MMC (n = 4), siDPP9+MMC (n = 5), siBRCA2-MMC (n = 2), siBRCA2+MMC (n = 3), siFLNA-MMC (n = 5), siFLNA+MMC (n = 6), NgtCntrl-MMC (n = 3), NgtCntrl+MMC (n = 6)). The number of foci are shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (* = p ≤ 0.05, **** = p ≤ 0.0001). C. Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa WT cells. Exposure of cells to MMC triggers more PLA events (white). Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_2889071


D . Quantification of PLAs between BRCA2 and DPP9 in asynchronous HeLa WT cells or in cells in S - Phase . Each dot represents the number of PLA events in a single cell , from three biological replicates . Cells were synchronised with a double thymidine block and released for 3 h before addition of NCS ( 250 ng / mL for 30 min ) . Cells were sampled prior to NCS treatment , after NCS treatment and with 1 . 5 h and 3 h of recovery after NCS treatment . The number of foci is shown based on their cellular localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * = p ≤ 0 . 05 , * * = p ≤ 0 . 01 , * * * * = p ≤ 0 . 0001 ) . E . Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa cells . Exposure of cells to NCS triggers more PLA events ( white ) , which increase in time . Cells in S - Phase show a greater fold - increase in the number of PLA events in comparison to asynchronous cells . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Antibodies as in ( C ) .

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

D. Quantification of PLAs between BRCA2 and DPP9 in asynchronous HeLa WT cells or in cells in S-Phase. Each dot represents the number of PLA events in a single cell, from three biological replicates. Cells were synchronised with a double thymidine block and released for 3 h before addition of NCS (250 ng / mL for 30 min). Cells were sampled prior to NCS treatment, after NCS treatment and with 1.5 h and 3 h of recovery after NCS treatment. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (* = p ≤ 0.05, ** = p ≤ 0.01, **** = p ≤ 0.0001). E. Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa cells. Exposure of cells to NCS triggers more PLA events (white), which increase in time. Cells in S-Phase show a greater fold-increase in the number of PLA events in comparison to asynchronous cells. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Antibodies as in (C).


F . Co - immunoprecipitation assays showing binding of BRCA2 and DPP9 - SWT . HEK293 DPP9 KO + DPP9WT cells , were treated with 1µg / mL Dox ( 24 h ) to induce the expression of DPP9 - FLAG . DNA damage was induced with 300 nM MMC treatment for 24h . Control cells do not express DPP9 ( - Dox ) . Bound proteins were eluted with a FLAG peptide and analysed by Western Blotting ( anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _ 731947 , anti - FLAG RRID : AB _ 262044 ) .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

F. Co-immunoprecipitation assays showing binding of BRCA2 and DPP9-SWT. HEK293 DPP9 KO + DPP9WT cells, were treated with 1µg/mL Dox (24 h) to induce the expression of DPP9-FLAG. DNA damage was induced with 300 nM MMC treatment for 24h. Control cells do not express DPP9 (- Dox). Bound proteins were eluted with a FLAG peptide and analysed by Western Blotting (anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_731947, anti-FLAG RRID:AB_262044).


B . Pull - down assay showing direct binding of purified recombinant DPP9 to a BRCA2 N - terminal1 - 40 fragment immobilized on HA - beads . The DPP9 inhibitors 1G244 or SLRFLYEG compete with BRCA21 - 40HA for interaction with DPP9 . Representative data of three technical replicates is shown . Anti - HA : RRID : AB _ 2565334 , anti - DPP9 : RRID : AB _ 2889071 .

O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

B. Pull-down assay showing direct binding of purified recombinant DPP9 to a BRCA2 N-terminal1-40 fragment immobilized on HA-beads. The DPP9 inhibitors 1G244 or SLRFLYEG compete with BRCA21-40HA for interaction with DPP9. Representative data of three technical replicates is shown. Anti-HA: RRID:AB_2565334, anti-DPP9: RRID:AB_2889071.


C . Quantification of PLAs showing fewer MMC - induced DPP9 - BRCA2 PLA events in HeLa cells treated with 10µM 1G244 . Each dot represents the number of PLA events in a single cell , from three biological replicates . The number of foci is shown based on their cellular localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * = p ≤ 0 . 001 , * * * * = p ≤ 0 . 0001 ) . D . Representative images of DPP9 - BRCA2 PLA in the presence of 1G244 - a competitive inhibitor of DPP9 . Control cells were mock treated with DMSO . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _

O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O

C. Quantification of PLAs showing fewer MMC-induced DPP9-BRCA2 PLA events in HeLa cells treated with 10µM 1G244. Each dot represents the number of PLA events in a single cell, from three biological replicates. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (*** = p ≤ 0.001, **** = p ≤ 0.0001). D. Representative images of DPP9-BRCA2 PLA in the presence of 1G244 - a competitive inhibitor of DPP9. Control cells were mock treated with DMSO. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_2889071


F . Pull - down assay showing a competition between DPP9 and PALB2 for binding to the BRCA2 N - terminus . While each protein can bind to the BRCA2 N - terminal peptide , in the presence of DPP9 , the PALB2 - BRCA2 binding is negatively affected . Representative data of three technical replicates is shown . Anti - PALB2 : RRID : AB _ 890607 , anti - HA : RRID : AB _ 2565334 , anti - DPP9 : RRID : AB _ 2889071 .

O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

F. Pull-down assay showing a competition between DPP9 and PALB2 for binding to the BRCA2 N-terminus. While each protein can bind to the BRCA2 N-terminal peptide, in the presence of DPP9, the PALB2-BRCA2 binding is negatively affected. Representative data of three technical replicates is shown. Anti-PALB2: RRID:AB_890607, anti-HA: RRID:AB_2565334, anti-DPP9: RRID:AB_2889071.


A - B . Representative Western Blots and accompanying graph from more than three biological replicates show that MMC ( 300 nM , 24 h ) induces a rapid turnover of endogenous BRCA2 in HeLa WT cells , which is less pronounced in HeLa WT cells treated with the proteasome inhibitor MG132 ( 100µM ) , in HeLa DPP9 KD cells and in cells transiently silenced for DPP9 ( siDPP9 ) . RAD51 stability is not altered in HeLa DPP9 KD cells . Vinculin is a loading control for BRCA2 , Tubulin is a loading control for RAD51 . Shown images originate from one representative cycloheximide ( CHX ) chase assay . The ratios of BRCA2 to Vinculin and RAD51 to Tubulin are defined as 100 % at time 0 h . Mean ± SEM , data were analysed by a paired two - tailed t - test . ( * = p ≤ 0 . 03 ) . Anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _ 731947 , anti - Vinculin : RRID : AB _ 477629 , anti - Tubulin : RRID : AB _ 628412 , anti - RAD51 : RRID : AB _ 1142428 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

A-B. Representative Western Blots and accompanying graph from more than three biological replicates show that MMC (300 nM, 24 h) induces a rapid turnover of endogenous BRCA2 in HeLa WT cells, which is less pronounced in HeLa WT cells treated with the proteasome inhibitor MG132 (100µM), in HeLa DPP9 KD cells and in cells transiently silenced for DPP9 (siDPP9). RAD51 stability is not altered in HeLa DPP9 KD cells. Vinculin is a loading control for BRCA2, Tubulin is a loading control for RAD51. Shown images originate from one representative cycloheximide (CHX) chase assay. The ratios of BRCA2 to Vinculin and RAD51 to Tubulin are defined as 100% at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test. (*= p ≤ 0.03). Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_731947, anti-Vinculin: RRID:AB_477629, anti-Tubulin: RRID:AB_628412, anti-RAD51: RRID:AB_1142428.


C - D . Representative Western Blots and graph summarizing results of more than three biological replicates . CHX chase assays show that the MMC - induced degradation of BRCA2 is less pronounced in HEK293 DPP9 KO cells as well as in HEK293 DPP9 KO + DPP9 - SS729A cells overexpressing an inactive DPP9 - S mutant . Expression of DPP9 was induced ( + Dox , 1µg / ml ) simultaneously with MMC ( 300 nM ) , 24 h . Cells that were overexpressing the active variant ( DPP9 - SWT ) show similar levels of MMC - induced BRCA2 degradation . RAD51 stability is not altered by MMC and is similar in all cell lines . Vinculin is a loading control for BRCA2 , Tubulin is a loading control for RAD51 . Shown images originate from one representative cycloheximide ( CHX ) chase assay . The ratios of BRCA2 to Vinculin are defined as 100 % at time 0 h . Mean ± SEM , data were analysed by a paired two - tailed t - test ( * = p ≤ 0 . 03 , * * = p ≤ 0 . 002 ) . Antibodies as described in ( A - B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-D. Representative Western Blots and graph summarizing results of more than three biological replicates. CHX chase assays show that the MMC-induced degradation of BRCA2 is less pronounced in HEK293 DPP9 KO cells as well as in HEK293 DPP9 KO + DPP9-SS729A cells overexpressing an inactive DPP9-S mutant. Expression of DPP9 was induced (+ Dox, 1µg/ml) simultaneously with MMC (300 nM), 24 h. Cells that were overexpressing the active variant (DPP9-SWT) show similar levels of MMC-induced BRCA2 degradation. RAD51 stability is not altered by MMC and is similar in all cell lines. Vinculin is a loading control for BRCA2, Tubulin is a loading control for RAD51. Shown images originate from one representative cycloheximide (CHX) chase assay. The ratios of BRCA2 to Vinculin are defined as 100% at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test (*= p ≤ 0.03, **= p ≤ 0.002). Antibodies as described in (A-B).


F - G . Representative Western Blots and graphs summarizing CHX assays from three biological replicates showing that the BRCA2 ∆ MP3 - 1000 truncation mutant is degraded at a higher rate compared to the untruncated BRCA21 - 1000 . Cells were co - transfected with a GFP expressing plasmid , as transfection and loading control . Control cells were transfected with GFP only ( U . T ) . Image originates from one representative experiment . BRCA2 - FLAG signals are related to the transfection and loading control GFP . BRCA2 levels in relation to GFP were defined as 100 % at time 0 h . Mean ± SEM , data were analysed by a paired two - tailed t - test ( * = p ≤ 0 . 03 ) . Anti - FLAG : RRID : AB _ 262044 , anti - GFP : RRID : AB _ 641123 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-G. Representative Western Blots and graphs summarizing CHX assays from three biological replicates showing that the BRCA2∆MP3-1000 truncation mutant is degraded at a higher rate compared to the untruncated BRCA21-1000. Cells were co-transfected with a GFP expressing plasmid, as transfection and loading control. Control cells were transfected with GFP only (U.T). Image originates from one representative experiment. BRCA2-FLAG signals are related to the transfection and loading control GFP. BRCA2 levels in relation to GFP were defined as 100 % at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test (*= p ≤ 0.03). Anti-FLAG: RRID:AB_262044, anti-GFP: RRID:AB_641123.


A . Representative immunofluorescence images of γH2AX signals ( white , RRID : AB _ 309864 ) in gki MEF DPP9S729A , showing more γH2AX in gki MEF DPP9S729A cells following removal of Neocarzinostatin ( NCS ) , quantification in ( B ) . MEF DPP9S729A and control MEF cells were treated with 250 ng / mL NCS for 30 min and allowed to recover for the indicated time - points . γH2AX signals at time 0 , reflect 30 min of NCS and no recovery time . Nuclei are shown in blue ( DAPI ) . Scale bar 10 µm . B . Quantification of γH2AX in gki MEF DPP9S729A and control MEF cells as described in ( A ) . Signals from more than 1700 cells were quantified per condition per experiment . Mean ± SEM from six biological replicates , each in technical duplicates . Data were analysed by an unpaired two - way ANOVA with Sidak ' s multiple comparison test ( * = p ≤ 0 . 05 , * * = p ≤ 0 . 01 ) .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative immunofluorescence images of γH2AX signals (white, RRID:AB_309864) in gki MEF DPP9S729A, showing more γH2AX in gki MEF DPP9S729A cells following removal of Neocarzinostatin (NCS), quantification in (B). MEF DPP9S729A and control MEF cells were treated with 250 ng/mL NCS for 30 min and allowed to recover for the indicated time-points. γH2AX signals at time 0, reflect 30 min of NCS and no recovery time. Nuclei are shown in blue (DAPI). Scale bar 10 µm. B. Quantification of γH2AX in gki MEF DPP9S729A and control MEF cells as described in (A). Signals from more than 1700 cells were quantified per condition per experiment. Mean ± SEM from six biological replicates, each in technical duplicates. Data were analysed by an unpaired two-way ANOVA with Sidak's multiple comparison test (* = p ≤ 0.05, ** = p ≤ 0.01).


C . Representative PLA images of BRCA2 - PALB2 PLA experiments showing more PLA events ( white ) in HeLa cells silenced for DPP9 ( siDPP9 ) with respect to cells treated with non - targeting siRNA ( siNT ) . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Anti - PALB2 : RRID : AB _ 890607 , anti - BRCA2 : RRID : AB _ 2259370 .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O

C. Representative PLA images of BRCA2-PALB2 PLA experiments showing more PLA events (white) in HeLa cells silenced for DPP9 (siDPP9) with respect to cells treated with non-targeting siRNA (siNT). Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-PALB2: RRID:AB_890607, anti-BRCA2: RRID:AB_2259370.


D . Quantification of PLAs experiments showing more BRCA2 - PALB2 PLA events in cells silenced for DPP9 ( siDPP9 ) in comparison to non - targeting controls ( siNT ) . Each dot represents the number of PLA events in a single cell , from four biological replicates . The technical control samples ( NgtCntrl ) omitted the BRCA2 antibody . The number of foci is shown based on their cellular localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * * = p ≤ 0 . 0001 ) .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Quantification of PLAs experiments showing more BRCA2-PALB2 PLA events in cells silenced for DPP9 (siDPP9) in comparison to non-targeting controls (siNT). Each dot represents the number of PLA events in a single cell, from four biological replicates. The technical control samples (NgtCntrl) omitted the BRCA2 antibody. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).


E . Representative PLA images of γH2Ax - BRCA2 PLA experiment showing a reduction in the number of PLA events ( white ) between γH2Ax and BRCA2 in HeLa DPP9 KD cells . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Anti - γH2Ax : RRID : AB _ 2118009 , anti - BRCA2 : RRID : AB _ 2259370 F . Quantification of PLAs showing fewer MMC - induced γH2Ax - BRCA2 PLA events in HeLa DPP9 KD cells , in comparison to HeLa WT cells . Each dot represents the number of PLA events in a single cell , from three biological replicates . The technical control samples ( NgtCntrl ) omitted the γH2Ax antibody . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * * = p ≤ 0 . 0001 ) .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Representative PLA images of γH2Ax-BRCA2 PLA experiment showing a reduction in the number of PLA events (white) between γH2Ax and BRCA2 in HeLa DPP9 KD cells. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-γH2Ax: RRID:AB_2118009, anti-BRCA2: RRID:AB_2259370 F. Quantification of PLAs showing fewer MMC-induced γH2Ax-BRCA2 PLA events in HeLa DPP9 KD cells, in comparison to HeLa WT cells. Each dot represents the number of PLA events in a single cell, from three biological replicates. The technical control samples (NgtCntrl) omitted the γH2Ax antibody. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).


A . Representative immunofluorescence images showing that re - expression of DPP9 - SWT leads to an increase in the number of RAD51 foci formed following exposure to MMC in HEK293 DPP9 KO + DPP9 - SWT cells . Expression of DPP9 was induced ( + Dox , 1µg / ml ) simultaneously with MMC ( 300 nM ) , 24 h . RAD51 foci are shown in white , nuclei ( DAPI ) are shown in blue . Scale bar 10 µm . Anti - RAD51 : RRID : AB _ 1142428 . B . Graph showing the number of RAD51 foci following induction of DPP9 - SWT expression , compared to uninduced HEK293 DPP9 KO + DPP9WT cells ( - Dox ) . Induction of HEK293 DPP9 KO + DPP9S729A for expression of DPP9 - SS729A did not result in more RAD51 foci . Each dot represents the number of RAD51 foci in a single cell , from three biological replicates . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * * = p ≤ 0 . 0001 ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative immunofluorescence images showing that re-expression of DPP9-SWT leads to an increase in the number of RAD51 foci formed following exposure to MMC in HEK293 DPP9 KO + DPP9-SWT cells. Expression of DPP9 was induced (+ Dox, 1µg/ml) simultaneously with MMC (300 nM), 24 h. RAD51 foci are shown in white, nuclei (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428. B. Graph showing the number of RAD51 foci following induction of DPP9-SWT expression, compared to uninduced HEK293 DPP9 KO + DPP9WT cells (-Dox). Induction of HEK293 DPP9 KO + DPP9S729A for expression of DPP9-SS729A did not result in more RAD51 foci. Each dot represents the number of RAD51 foci in a single cell, from three biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).


Representative images ( C , and summarizing graph ( D showing the number of RAD51 foci per nucleus in HeLa WT cells ( C , D ) Where stated , cells were treated with control siRNA ( siNT ) or silenced for BRCA2 , and transiently transfected with the BRCA21 ‑ 3418 or BRCA23 - 3418 constructs . Both BRCA2 constructs can rescue the RAD51 foci formation phenotype to the control levels in HeLa WT cells ( C , D ) . Each dot represents the number of RAD51 foci in a single cell , from three ( D ) biological replicates . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * * = p ≤ 0 . 0001 ) . RAD51 foci are shown in white , nuclei ( DAPI ) are shown in blue . Scale bar 10 µm . Anti - RAD51 : RRID : AB _ 1142428 ; ; anti - BRCA2 : RRID : AB _ 2259370 .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O

Representative images (C, and summarizing graph (D showing the number of RAD51 foci per nucleus in HeLa WT cells (C, D) Where stated, cells were treated with control siRNA (siNT) or silenced for BRCA2, and transiently transfected with the BRCA21‑3418 or BRCA23-3418 constructs. Both BRCA2 constructs can rescue the RAD51 foci formation phenotype to the control levels in HeLa WT cells (C, D). Each dot represents the number of RAD51 foci in a single cell, from three (D) biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001). RAD51 foci are shown in white, nuclei (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428; ; anti-BRCA2: RRID:AB_2259370.


Representative images , E ) and summarizing graph F ) showing the number of RAD51 foci per nucleus in HeLa DPP9 KD cells ( E , F ) . In HeLa DPP9 KD cells significantly more RAD51 foci were present in cells transfected with BRCA2 ∆ MP3 - 3418 compared to BRCA2 silenced cells and cells expressing the untruncated BRCA21 - 3418 ( E , F ) . Each dot represents the number of RAD51 foci in a single cell , from four ( F ) biological replicates . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * * = p ≤ 0 . 0001 ) . RAD51 foci are shown in white , nuclei ( DAPI ) are shown in blue . Scale bar 10 µm . Anti - RAD51 : RRID : AB _ 1142428 ; anti - BRCA2 : RRID : AB _ 2259370 .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O

Representative images , E) and summarizing graph F) showing the number of RAD51 foci per nucleus in HeLa DPP9 KD cells (E, F). In HeLa DPP9 KD cells significantly more RAD51 foci were present in cells transfected with BRCA2∆MP3-3418 compared to BRCA2 silenced cells and cells expressing the untruncated BRCA21-3418 (E, F). Each dot represents the number of RAD51 foci in a single cell, from four (F) biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001). RAD51 foci are shown in white, nuclei (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428; anti-BRCA2: RRID:AB_2259370.


B , C CDKL51 - 352 autophosphorylation is shown . WT CDKL5 uses ATPγS , KD is inactive , F89A uses benzyl - ATPγS with reduced efficiency , F89A / C152A mutant has rescued activity . Quantification of Thio - P signal is normalized for CDKL5 levels ( HA ) and KD background is subtracted . Fisher ' s LSD : n = 4 replicates . bn = benzyl

O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

B, C CDKL51-352 autophosphorylation is shown. WT CDKL5 uses ATPγS, KD is inactive, F89A uses benzyl-ATPγS with reduced efficiency, F89A/ C152A mutant has rescued activity. Quantification of Thio-P signal is normalized for CDKL5 levels (HA) and KD background is subtracted. Fisher's LSD: n = 4 replicates. bn = benzyl


E - H In vitro kinase assays showing efficient MAP1S phosphorylation by CDKL5 . 50 ng ( 40 nM ) AS - CDKL5 phosphorylates 150 ng ( 50 nM ) MAP1S very rapidly ( E , F ) Quantification of phosphorylated MAP1S is normalized to maximum intensity . n = 2

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

E - H In vitro kinase assays showing efficient MAP1S phosphorylation by CDKL5. 50 ng (40 nM) AS-CDKL5 phosphorylates 150 ng (50 nM) MAP1S very rapidly (E, F) Quantification of phosphorylated MAP1S is normalized to maximum intensity. n = 2 replicates


E - H In vitro kinase assays showing efficient MAP1S phosphorylation by CDKL5 In 30 minutes of incubation , 150 ng MAP1S is phosphorylated by small amounts of CDKL5 ( G , H ) . Quantification of phosphorylated MAP1S is normalized to maximum intensity . n = 2

O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O

E - H In vitro kinase assays showing efficient MAP1S phosphorylation by CDKL5 In 30 minutes of incubation, 150 ng MAP1S is phosphorylated by small amounts of CDKL5 (G, H). Quantification of phosphorylated MAP1S is normalized to maximum intensity. n = 2 replicates


I - L Western blots of in vitro kinase assays showing loss of phosphorylation in phosphomutants ARHGEF2 S122A ( I Substrate levels are shown by coommassie staining

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

I - L Western blots of in vitro kinase assays showing loss of phosphorylation in phosphomutants ARHGEF2 S122A (I Substrate levels are shown by coommassie staining underneath


I - L Western blots of in vitro kinase assays showing loss of phosphorylation in phosphomutant EB2 S222A ( J Substrate levels are shown by coommassie staining

I - L Western blots of in vitro kinase assays showing loss of phosphorylation in phosphomutant EB2 S222A (J Substrate levels are shown by coommassie staining underneath


I - L Western blots of in vitro kinase assays showing loss of phosphorylation in phosphomutant MAP1S S786 / 812A ( K ) . Substrate levels are shown by coommassie staining underneath . MAP1S phosphorylation is quantified in ( L ) . Dunnett ' s multiple comparison : n = 3

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

I - L Western blots of in vitro kinase assays showing loss of phosphorylation in phosphomutant MAP1S S786/812A (K). Substrate levels are shown by coommassie staining underneath. MAP1S phosphorylation is quantified in (L). Dunnett's multiple comparison: n = 3 replicates


A , B In vivo substrate phosphorylations are assessed by Western blotting of 5 WT ( CDKL5 + / Y ) and 5 CDKL5 KO ( CDKL5 - / Y ) mouse cortical lysates using phosphospecific antibodies . EB2 pS222 and MAP1S pS812 are dramatically reduced in KOs . Quantification of substrate phosphorylation is normalized for total protein level . Student ' s t - test : n = 5 animals per

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B In vivo substrate phosphorylations are assessed by Western blotting of 5 WT (CDKL5 +/Y) and 5 CDKL5 KO (CDKL5 -/Y) mouse cortical lysates using phosphospecific antibodies. EB2 pS222 and MAP1S pS812 are dramatically reduced in KOs. Quantification of substrate phosphorylation is normalized for total protein level. Student's t-test: n = 5 animals per genotype


C , D Developmental expression of CDKL5 and levels of its substrate phosphorylations in P4 - P50 cortex . CDKL5 - dependent substrate phosphorylation is quantified by subtracting KO from WT levels for each age . n = 3 animals per age per genotype

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

C, D Developmental expression of CDKL5 and levels of its substrate phosphorylations in P4 - P50 cortex. CDKL5-dependent substrate phosphorylation is quantified by subtracting KO from WT levels for each age. n = 3 animals per age per genotype


E , F Expression of CDKL5 and levels of substrate phosphorylation in P8 Nex - Cre conditional KO cortex . CDKL5 protein expression is almost completely lost . Quantification of substrate phosphorylation is normalized for total protein level . Student ' s t - test : n = 3 animals per genotype

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

E, F Expression of CDKL5 and levels of substrate phosphorylation in P8 Nex-Cre conditional KO cortex. CDKL5 protein expression is almost completely lost. Quantification of substrate phosphorylation is normalized for total protein level. Student's t-test: n = 3 animals per genotype


G CDKL5 WT and CDKL5 KO neurons in culture are co - stained with neuronal dendrite marker MAP2 , EB2 and EB2 pS222 . EB2 pS222 is lost in CDKL5 KO dendrites . Scale bar is 20 μm

O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O

G CDKL5 WT and CDKL5 KO neurons in culture are co-stained with neuronal dendrite marker MAP2, EB2 and EB2 pS222. EB2 pS222 is lost in CDKL5 KO dendrites. Scale bar is 20 μm


A - C Protein lysates were obtained from cortical neuronal cultures treated with 50 mM KCl applied directly to culture media for indicated durations . 100 µm AP5 was used to block NMDAR activity during KCl treatment . Quantification of phosphorylated EB2 ( B ) and total CDKL5 ( C ) are normalized to 0 minutes time point . EB2 phosphorylation is significantly decreased after 10 minutes of KCl treatment compared to 0 minutes . CDKL5 protein levels are slightly reduced after 40 minutes compared to 0 minutes . Dunnett ' s multiple comparison : n = 3

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

A - C Protein lysates were obtained from cortical neuronal cultures treated with 50 mM KCl applied directly to culture media for indicated durations. 100 µm AP5 was used to block NMDAR activity during KCl treatment. Quantification of phosphorylated EB2 (B) and total CDKL5 (C) are normalized to 0 minutes time point. EB2 phosphorylation is significantly decreased after 10 minutes of KCl treatment compared to 0 minutes. CDKL5 protein levels are slightly reduced after 40 minutes compared to 0 minutes. Dunnett's multiple comparison: n = 3 replicates


D , E Protein lysates were obtained from cortical neuronal cultures treated with 50 μM NMDA applied directly to culture media for indicated durations . Quantification of phosphorylated EB2 and total CDKL5 is normalized to 0 minutes time point . EB2 S222 phosphorylation is significantly reduced after 10 minutes compared to 0 minutes . Dunnett ' s multiple comparison : n = 3 replicates

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

D, E Protein lysates were obtained from cortical neuronal cultures treated with 50 μM NMDA applied directly to culture media for indicated durations. Quantification of phosphorylated EB2 and total CDKL5 is normalized to 0 minutes time point. EB2 S222 phosphorylation is significantly reduced after 10 minutes compared to 0 minutes. Dunnett's multiple comparison: n = 3 replicates


A Representative kymographs of EB3 - tdTomato in cortical neuron dendrites at DIV14

A Representative kymographs of EB3-tdTomato in cortical neuron dendrites at DIV14


B - D In CDKL5 KOs ( CDKL5 - / Y ) , comet distance ( B ) and lifetime ( C ) is increased when compared to WT ( CDKL5 + / Y ) . Comet velocity ( D ) is unchanged . Student ' s t - test : n = 16 / 615 and 15 / 381 neurons / comets in WT and KO , respectively

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

B - D In CDKL5 KOs (CDKL5 -/Y), comet distance (B) and lifetime (C) is increased when compared to WT (CDKL5 +/Y). Comet velocity (D) is unchanged. Student's t-test: n = 16/615 and 15/381 neurons/comets in WT and KO, respectively


E Representative kymographs of CDKL5 WT and KO neurons transfected with control shRNA and MAP1S shRNA

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


F Comet lifetime is higher in control shRNA expressing KOs than WTs . MAP1S shRNA expression reduces comet lifetime in KO neurons . EB2 shRNA - mediated knockdown does not alter microtubule dynamics in WT or KO . Tukey ' s multiple comparison : n = 12 - 15 neurons , 388 - 741 comets per condition

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

F Comet lifetime is higher in control shRNA expressing KOs than WTs. MAP1S shRNA expression reduces comet lifetime in KO neurons. EB2 shRNA-mediated knockdown does not alter microtubule dynamics in WT or KO. Tukey's multiple comparison: n = 12-15 neurons, 388-741 comets per condition


A , B Representative stills ( A ) and kymographs ( B ) of TrkB - RFP overexpressed in dendrites of WT and CDKL5 KO DIV14 cortical neurons . Scale bar is 5 μm . C , D No significant change is observed in the ratio of dynamic vs stationary vesicles ( C ) or anterograde vs retrograde runs ( D ) . S = stationary , D = dynamic , A = anterograde , R = retrograde . Vesicles with a net distance > 5 μm were considered dynamic . E , F Both anterograde and retrograde runs show a trend towards reduced velocity in CDKL5 KO neurons ( E ) . Anterograde run lengths are significantly reduced in KO compared to WT neurons ( F )

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

A, B Representative stills (A) and kymographs (B) of TrkB-RFP overexpressed in dendrites of WT and CDKL5 KO DIV14 cortical neurons. Scale bar is 5 μm. C, D No significant change is observed in the ratio of dynamic vs stationary vesicles (C) or anterograde vs retrograde runs (D). S = stationary, D = dynamic, A = anterograde, R = retrograde. Vesicles with a net distance > 5 μm were considered dynamic. E, F Both anterograde and retrograde runs show a trend towards reduced velocity in CDKL5 KO neurons (E). Anterograde run lengths are significantly reduced in KO compared to WT neurons (F)


B COS - 7 cells expressing HA - MAP1S LC , Flag - CDKL5 WT / kinase dead ( KD ) is shown with endogenous α - tubulin staining . MAP1S does not localize to microtubules when phosphorylated by CDKL5 . Scale bar is 20 μm

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

B COS-7 cells expressing HA-MAP1S LC, Flag-CDKL5 WT / kinase dead (KD) is shown with endogenous α-tubulin staining. MAP1S does not localize to microtubules when phosphorylated by CDKL5. Scale bar is 20 μm


C , D The percentage of cells where α - tubulin and MAP1S LC are colocalised ( C ) and the quantification of MAP1S LC and α - tubulin colocalisation by Pearson correlation coefficient ( D ) . Tukey ' s multiple comparison : n = 22 - 24 cells per condition

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

C, D The percentage of cells where α-tubulin and MAP1S LC are colocalised (C) and the quantification of MAP1S LC and α-tubulin colocalisation by Pearson correlation coefficient (D). Tukey's multiple comparison: n = 22-24 cells per condition


E , F Taxol - stabilised microtubule co - sedimentation assay shows direct binding of MAP1S LC to microtubules is reduced upon prior phosphorylation by CDKL5 . Quantification of soluble MAP1S in the supernatant ( s / n ) fraction is normalized to the MT - bound pellet fraction . 3 : 1 MT : MAP1S LC ratio used here . Tukey ' s multiple comparison : n = 3 technical repeats

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

E, F Taxol-stabilised microtubule co-sedimentation assay shows direct binding of MAP1S LC to microtubules is reduced upon prior phosphorylation by CDKL5. Quantification of soluble MAP1S in the supernatant (s/n) fraction is normalized to the MT-bound pellet fraction. 3:1 MT:MAP1S LC ratio used here. Tukey's multiple comparison: n = 3 technical repeats


G HA - MAP1S LC is overexpressed and stained with anti - HA antibody in cultured cortical neurons ( red ) . Numerous loops at the dendrites ' tips are observed ( arrowheads ) . Scale bar is 5 μm

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

G HA-MAP1S LC is overexpressed and stained with anti-HA antibody in cultured cortical neurons (red). Numerous loops at the dendrites' tips are observed (arrowheads). Scale bar is 5 μm


A , B Western blots for total EB2 , pS223 and tubulin using protein lysates obtained from neurons derived from three patients with CDD and their related control . 1 = p . D135 _ F154del , male ; 2 = p . R59X , male ; 3 = p . R59X , female . Quantification of EB2 phosphorylation is normalized for total protein level . Student ' s t - test : n = 3 patients / controls . * p < 0 . 05 , error bars are SEM

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

A, B Western blots for total EB2, pS223 and tubulin using protein lysates obtained from neurons derived from three patients with CDD and their related control. 1 = p.D135_F154del, male; 2 = p.R59X, male; 3 = p.R59X, female. Quantification of EB2 phosphorylation is normalized for total protein level. Student's t-test: n = 3 patients/controls. * p < 0.05, error bars are SEM


C Representative images of human iPSC - derived neurons co - stained with the neuronal dendrite marker MAP2 , EB2 and EB2 pS223 . EB2 phosphorylation is present in dendrites of control neurons but not in CDD neurons . Enlarged images of the boxed areas are shown on the right panel . Scale bar is 20 μm

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

C Representative images of human iPSC-derived neurons co-stained with the neuronal dendrite marker MAP2, EB2 and EB2 pS223. EB2 phosphorylation is present in dendrites of control neurons but not in CDD neurons. Enlarged images of the boxed areas are shown on the right panel. Scale bar is 20 μm


A . Quantitative analysis of developmental expression of Lrig1 mRNA in rat hippocampus by real - time RT - PCR . The results are shown as mean SEM of n = 3 independent assays . The levels of Lrig1 mRNA were normalized using the expression of the housekeeping gene Tbp ( TATA binding protein ) . The insert shows the expression of Lrig1 in embryonic E17 . 5 rat hippocampus examined by RT - PCR . Control sample without reverse transcriptase ( - RT ) is also shown .

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

A. Quantitative analysis of developmental expression of Lrig1 mRNA in rat hippocampus by real-time RT-PCR. The results are shown as mean SEM of n=3 independent assays. The levels of Lrig1 mRNA were normalized using the expression of the housekeeping gene Tbp (TATA binding protein). The insert shows the expression of Lrig1 in embryonic E17.5 rat hippocampus examined by RT-PCR. Control sample without reverse transcriptase (-RT) is also shown.


B . Localization of Lrig1 ( red ) in coronal sections from P15 ratbrain by immunofluorescence . Anti - Lrig1ECD antibodies label dentate gyrus cells , CA1 - CA3 hippocampalneurons and pyramidal corticalneurons ( * ) . Scale bar , 400 m .

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

B. Localization of Lrig1 (red) in coronal sections from P15 ratbrain by immunofluorescence. Anti-Lrig1ECD antibodies label dentate gyrus cells, CA1-CA3 hippocampalneurons and pyramidal corticalneurons (*). Scale bar, 400 m.


C - E . Confocal image of cortical ( C ) and hippocampal ( D - E ) pyramidal neurons stained with rabbit anti - Lrig1ECD antibody . Arrows indicate Lrig1 staining in proximal segments of apical dendrites of CA1 - CA3hippocampal and pyramidal cortical neurons ( layer V ) . Scale bars , 20 m .

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

C-E. Confocal image of cortical (C) and hippocampal (D-E) pyramidal neurons stained with rabbit anti-Lrig1ECD antibody. Arrows indicate Lrig1 staining in proximal segments of apical dendrites of CA1-CA3hippocampal and pyramidal cortical neurons (layer V). Scale bars, 20 m.


F . Immunofluorescence staining of Lrig1 ( red ) with anti - Lrig1ECD antibody and the neuronal marker ßIII tubulin ( green ) in dissociated hippocampal cells cultured for 7 DIV . Yellow indicates neuronal expression of Lrig1 . Scale bar , 15 m .

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

F. Immunofluorescence staining of Lrig1 (red) with anti-Lrig1ECD antibody and the neuronal marker ßIII tubulin (green) in dissociated hippocampal cells cultured for 7 DIV. Yellow indicates neuronal expression of Lrig1. Scale bar, 15 m.


G . Immunofluorescence staining of Lrig1 ( red ) with anti - Lrig1ECD antibody and the astrocytic marker S100 ( green ) in dissociated hippocampal cells cultured for 7 DIV . Scale bar , 20 m .

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

G. Immunofluorescence staining of Lrig1 (red) with anti-Lrig1ECD antibody and the astrocytic marker S100 (green) in dissociated hippocampal cells cultured for 7 DIV. Scale bar, 20 m.


H . Localization of Lrig1 ( red ) with anti - Lrig1ECD antibody and the somatodendritic marker MAP - 2 ( blue ) by immunocytochemistry in dissociated rat hippocampalneurons after 12 DIV . Scale bar , 20 m .

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

H. Localization of Lrig1 (red) with anti-Lrig1ECD antibody and the somatodendritic marker MAP-2 (blue) by immunocytochemistry in dissociated rat hippocampalneurons after 12 DIV. Scale bar, 20 m.


A . Representative images of mouse hippocampal neurons transfected with either GFP - expressing control or Lrig1 shRNA vector at 9 DIV and maintained for 3 additional days in vitro ( 9 + 3 DIV ) . Scale bar , 15 m . Boxed area represents a higher - magnification image showing the profuse proximal dendritic arborization of Lrig1 - shRNA transfected neurons . B . Sholl analysis of the dendritic arbor from hippocampal neurons transfected with either control or Lrig1 shRNA - GFP vector at 9 DIV and maintained for 3 additional days in vitro ( 9 + 3 DIV ) . Data are shown as mean SEM of n = 3 independent experiments . * p < 0 . 05 and * * p < 0 . 01 by two - way ANOVA followed by Bonferroni multiple comparisons test . C - G . Quantification of primary ( C ) , and secondary ( D ) dendrites as well as total dendritic branching ( E ) , terminal dendritic points ( F ) , and total dendritic length ( G ) of hippocampal neurons transfected with either control or Lrig1 shRNA - GFP vector . The results are shown as mean SEM of n = 3 independent experiments . * p < 0 . 05 by Students t test .

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

A. Representative images of mouse hippocampal neurons transfected with either GFP-expressing control or Lrig1 shRNA vector at 9 DIV and maintained for 3 additional days in vitro (9+3 DIV). Scale bar, 15 m. Boxed area represents a higher-magnification image showing the profuse proximal dendritic arborization of Lrig1-shRNA transfected neurons.B. Sholl analysis of the dendritic arbor from hippocampal neurons transfected with either control or Lrig1 shRNA-GFP vector at 9 DIV and maintained for 3 additional days in vitro (9+3 DIV). Data are shown as mean SEM of n=3 independent experiments. *p<0.05 and **p<0.01 by two-way ANOVA followed by Bonferroni multiple comparisons test.C-G. Quantification of primary (C), and secondary (D) dendrites as well as total dendritic branching (E), terminal dendritic points (F), and total dendritic length (G) of hippocampal neurons transfected with either control or Lrig1 shRNA-GFP vector. The results are shown as mean SEM of n=3 independent experiments. *p<0.05 by Students t test.


H . Knockdown efficiency was analyzed by real - time RT - PCR in MN1 cells transfected with control or Lrig1 shRNA vectors . Transfected cells were enriched by puromycin treatment in order to increase the population of cells expressing control or Lrig1 shRNA constructs . Data are shown as individual values of a representative assay measured in triplicates . n = 2 independent experiments were performed .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

H. Knockdown efficiency was analyzed by real-time RT-PCR in MN1 cells transfected with control or Lrig1 shRNA vectors. Transfected cells were enriched by puromycin treatment in order to increase the population of cells expressing control or Lrig1 shRNA constructs. Data are shown as individual values of a representative assay measured in triplicates. n=2 independent experiments were performed.


I . Representative images of MAP - 2 immunostained hippocampalneurons obtained from wild - type and Lrig1 - deficient mice cultured for 7 days in vitro ( 7 DIV ) . Scale bar , 15 m . J . Sholl analysis of the dendritic arbor from MAP - 2 stained hippocampalneurons ( 7 DIV ) isolated from wild type and Lrig1 - deficient mice . Data are shown as mean SEM of n = 3 independent experiments . * p < 0 . 05 by two - way ANOVA followed by Bonferroni multiple comparisons test . K - M . Quantification of the number of primary dendrites ( K ) , secondary dendrites ( L ) and total dendritic branching ( M ) from MAP - 2 stained hippocampalneurons ( 7 DIV ) isolated from wild - type and Lrig1 - deficient mice . The results are shown as mean SEM of n = 3 independent experiments . * p < 0 . 05 by Students t test .

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

I. Representative images of MAP-2 immunostained hippocampalneurons obtained from wild-type and Lrig1-deficient mice cultured for 7 days in vitro (7 DIV). Scale bar, 15 m.J. Sholl analysis of the dendritic arbor from MAP-2 stained hippocampalneurons (7 DIV) isolated from wild type and Lrig1-deficient mice. Data are shown as mean SEM of n=3 independent experiments. *p<0.05 by two-way ANOVA followed by Bonferroni multiple comparisons test.K-M. Quantification of the number of primary dendrites (K), secondary dendrites (L) and total dendritic branching (M) from MAP-2 stained hippocampalneurons (7 DIV) isolated from wild-type and Lrig1-deficient mice. The results are shown as mean SEM of n=3 independent experiments. *p<0.05 by Students t test.


A . Representative images and drawings of Golgi - stainedhippocampalCA1pyramidal neurons from 4 weeks - old wild type and Lrig1 - null littermate mice . Scale bar , 15 m . B . Quantification of the number of primary dendrites and branching of apical and basal dendritic arbors of hippocampalCA1pyramidal neurons from 4 - 5 weeks - old control ( wild type / heterozygous ) and Lrig1 - null littermate mice . The results are shown as mean SEM of independent determinations performed in n = 4 mice of each genotype . * p < 0 . 05 , * * p < 0 . 001 by Students t test . NS , not significant . C . Cummulative dendrite crossings of concentric circles of increasing radius ( 10 μm ring interval ) centering the reference point at the cell body . These values were obtained by Sholl analysis and represent the summatory of the dendritic crossings registered within the first 60 m closest to the soma . The results are shown as mean SEM of independent determinations performed in n = 4 mice of each genotype . * p < 0 . 05 by Students t test . D . Sholl analysis of apical and basal dendritic arbors of hippocampalCA1pyramidal neurons from 4 - 5 weeks - old control ( wild type / heterozygous ) and Lrig1 - null littermate mice . The results are shown as mean SEM . * p < 0 . 05 , * * p < 0 . 001 by two - way ANOVA followed by Bonferroni multiple comparisons test . Quantifications showed in B - D were performed in n = 60 neurons from 4 wild type / heterozygous mice and 4 Lrig1 - null littermate mice ( n = 4 ) .

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

A. Representative images and drawings of Golgi-stainedhippocampalCA1pyramidal neurons from 4 weeks-old wild type and Lrig1-null littermate mice. Scale bar, 15 m.B. Quantification of the number of primary dendrites and branching of apical and basal dendritic arbors of hippocampalCA1pyramidal neurons from 4-5 weeks-old control (wild type/heterozygous) and Lrig1-null littermate mice. The results are shown as mean SEM of independent determinations performed in n=4 mice of each genotype. *p<0.05, **p<0.001 by Students t test. NS, not significant.C. Cummulative dendrite crossings of concentric circles of increasing radius (10 μm ring interval) centering the reference point at the cell body. These values were obtained by Sholl analysis and represent the summatory of the dendritic crossings registered within the first 60 m closest to the soma. The results are shown as mean SEM of independent determinations performed in n=4 mice of each genotype. *p<0.05 by Students t test.D. Sholl analysis of apical and basal dendritic arbors of hippocampalCA1pyramidal neurons from 4-5 weeks-old control (wild type/heterozygous) and Lrig1-null littermate mice. The results are shown as mean SEM. *p<0.05, **p<0.001 by two-way ANOVA followed by Bonferroni multiple comparisons test.Quantifications showed in B-D were performed in n=60 neurons from 4 wild type/heterozygous mice and 4 Lrig1-null littermate mice (n=4).


A . Schematic diagram of the social interaction device indicating the social and the empty chambers . B , C . Mice were simultaneously exposed to an empty container and a caged unfamiliar juvenile mouse ( social enclosure , stranger 1 ) . Lrig1 - mutant mice exhibit social interaction defects as determined by the time spent interacting ( sniffing ) with the stranger enclosure ( B ) and the percentage of total interaction time with stranger in the three - chamber social interaction test ( C ) . Dashed line in ( C ) represents chance - level performance ( i . e . 50 % ) when mice equally explore the social enclosure and the empty container . Data represent means ± SEM of independent determinations performed in n = 8 - 9 mice of each genotype , and the statistical significance between wt and knockout mice are * p < 0 . 05 by Students t test .

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

A. Schematic diagram of the social interaction device indicating the social and the empty chambers.B, C. Mice were simultaneously exposed to an empty container and a caged unfamiliar juvenile mouse (social enclosure, stranger 1). Lrig1-mutant mice exhibit social interaction defects as determined by the time spent interacting (sniffing) with the stranger enclosure (B) and the percentage of total interaction time with stranger in the three-chamber social interaction test (C). Dashed line in (C) represents chance-level performance (i.e. 50%) when mice equally explore the social enclosure and the empty container. Data represent means ± SEM of independent determinations performed in n=8-9 mice of each genotype, and the statistical significance between wt and knockout mice are *p<0.05 by Students t test.


D . Schematic diagram of the social novelty test . In the test for social novelty , a second stranger ( stranger 2 ) mouse was introduced in the empty container . E . In the preference for social novelty task , wt mice showed a preference for social novelty , while mutant mice showed no significant preference for novel target ( stranger 2 ) . Mutants also spent significantly less time interacting with the novel mice compared to controls . Data represent means ± SEM of independent determinations performed in n = 8 - 9 mice of each genotype , and the statistical significance are as follow : * p < 0 . 05 and * * p < 0 . 005 by ANOVA followed by Student - Neuman Keuls multiple comparison test .

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

D. Schematic diagram of the social novelty test. In the test for social novelty, a second stranger (stranger 2) mouse was introduced in the empty container.E. In the preference for social novelty task, wt mice showed a preference for social novelty, while mutant mice showed no significant preference for novel target (stranger 2). Mutants also spent significantly less time interacting with the novel mice compared to controls. Data represent means ± SEM of independent determinations performed in n=8-9 mice of each genotype, and the statistical significance are as follow: *p<0.05 and **p<0.005 by ANOVA followed by Student-Neuman Keuls multiple comparison test.


A . Schematic representation of Lrig1 mutants are shown on the left . Expression levels of these mutants were analysed in transfected cell extracts by immunoblotting with anti - Flag antibodies .

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

A. Schematic representation of Lrig1 mutants are shown on the left. Expression levels of these mutants were analysed in transfected cell extracts by immunoblotting with anti-Flag antibodies.


B . Representative images of rathippocampal neurons transfected at DIV8 with empty vector , full length ( FL ) Flag - tagged Lrig1 , or Lrig1 mutant lacking the LRR domain ( LRR ) in combination with an enhanced green fluorescent protein ( GFP ) expression vector . After transfection at 9 DIV , neurons were cultured in the absence or in the presence of BDNF ( 30 ng / ml ) for 48 h . Then , hippocampal cultures at 11 DIV were fixed and stained with anti - Flag antibodies to control Lrig1 expression . Scale bar , 15 m .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

B. Representative images of rathippocampal neurons transfected at DIV8 with empty vector, full length (FL) Flag-tagged Lrig1, or Lrig1 mutant lacking the LRR domain (LRR) in combination with an enhanced green fluorescent protein (GFP) expression vector. After transfection at 9 DIV, neurons were cultured in the absence or in the presence of BDNF (30 ng/ml) for 48 h. Then, hippocampal cultures at 11 DIV were fixed and stained with anti-Flag antibodies to control Lrig1 expression. Scale bar, 15 m.


C , D . Quantification of the effects of Flag - Lrig1 constructs on BDNF - induced primary ( C ) and secondary ( D ) dendrite formation of hippocampal neurons treated as indicated in A . The results are shown as mean SEM of 3 independent experiments . * p < 0 . 05 , * * p < 0 . 01 by one - way ANOVA followed by Tukey ´ s multiple comparison test . NS , not significant .

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

C, D. Quantification of the effects of Flag-Lrig1 constructs on BDNF-induced primary (C) and secondary (D) dendrite formation of hippocampal neurons treated as indicated in A. The results are shown as mean SEM of 3 independent experiments. *p<0.05, **p<0.01 by one-way ANOVA followed by Tukey´s multiple comparison test. NS, not significant.


E . Representative confocal images of dendritic shafts containing spines from hippocampal neurons transfected at 15 DIV with either control vector or Flag - Lrig1 construct together with GFP . After transfection , neurons were cultured in the absence or in the presence of BDNF ( 30 ng / ml ) for 48 h ( 15 + 3 DIV ) . Scale bar , 5 m . F . Quantification of the effect of Lrig1 overexpression on neurotrophin - induced spine density . The results are shown as mean SEM of n = 3 independent experiments . * p < 0 . 05 by one - way ANOVA followed by Student - Neuman Keuls multiple comparison test .

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Representative confocal images of dendritic shafts containing spines from hippocampal neurons transfected at 15 DIV with either control vector or Flag-Lrig1 construct together with GFP. After transfection, neurons were cultured in the absence or in the presence of BDNF (30 ng/ml) for 48 h (15+3 DIV). Scale bar, 5 m.F. Quantification of the effect of Lrig1 overexpression on neurotrophin-induced spine density. The results are shown as mean SEM of n=3 independent experiments. *p<0.05 by one-way ANOVA followed by Student-Neuman Keuls multiple comparison test.


A . Coexpression of Lrig1 ( red ) and TrkB ( green ) in primary rat hippocampal neurons . Boxed area represents a higher - magnification image showing a high colocalization between TrkB and Lrig1 in a pyramidal neuron . Yellow indicates regions of colocalization . Scale bar , 30 m . Data represent n = 3 independent experiments .

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

A. Coexpression of Lrig1 (red) and TrkB (green) in primary rat hippocampal neurons. Boxed area represents a higher-magnification image showing a high colocalization between TrkB and Lrig1 in a pyramidal neuron. Yellow indicates regions of colocalization. Scale bar, 30 m. Data represent n=3 independent experiments.


B . Quantitative analysis of Lrig1 mRNA expression by real - time RT - PCR . Rat hippocampal cultures ( 10 DIV ) were treated with BDNF ( 50 ng / ml ) during the indicated times . The levels were normalized using the expression of the housekeeping gene Tbp . The results are shown as mean SD of n = 3 independent experiments . * p < 0 . 05 vs . control group by one - way ANOVA followed by Dunnetts test .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Quantitative analysis of Lrig1 mRNA expression by real-time RT-PCR. Rat hippocampal cultures (10 DIV) were treated with BDNF (50 ng/ml) during the indicated times. The levels were normalized using the expression of the housekeeping gene Tbp. The results are shown as mean SD of n=3 independent experiments. *p<0.05 vs. control group by one-way ANOVA followed by Dunnetts test.