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-GENEPROD O O B-SUBCELLULAR O B-CELL_LINE B-GENEPROD O O O O O O O O O B-CELL_LINE B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD 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-GENEPROD O O B-EXP_ASSAY O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY

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 B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY 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 B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-ORGANISM B-TISSUE O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY 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 B-EXP_ASSAY O B-GENEPROD O O O O O B-EXP_ASSAY O O O O B-TISSUE O B-GENEPROD O O O B-EXP_ASSAY 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 B-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O B-TISSUE O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY 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-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-SUBCELLULAR 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 B-CELL_TYPE I-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD 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 B-CELL_TYPE I-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O B-EXP_ASSAY 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

O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE B-SUBCELLULAR O O

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-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE 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 B-EXP_ASSAY O B-GENEPROD O O O B-CELL_TYPE O O O O O B-GENEPROD 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-GENEPROD O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-CELL_TYPE 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 B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O B-CELL_TYPE 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 B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY O B-SUBCELLULAR O B-EXP_ASSAY O B-GENEPROD 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-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY 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 B-SMALL_MOLECULE O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O B-GENEPROD 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 B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-SUBCELLULAR 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 B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O O O O B-CELL_TYPE O O O B-ORGANISM O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-ORGANISM 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 B-EXP_ASSAY O B-ORGANISM O O O B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O O B-SUBCELLULAR O O B-CELL_TYPE O O O B-DISEASE B-CELL_TYPE O O B-EXP_ASSAY 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-GENEPROD O O B-ORGANISM B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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-GENEPROD O O O O O O O O B-ORGANISM O B-EXP_ASSAY O O O B-GENEPROD O O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE 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 B-EXP_ASSAY O O B-TISSUE O O O O B-TISSUE O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O B-TISSUE O B-TISSUE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 B-EXP_ASSAY O O B-GENEPROD O O O O O O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O O O O B-TISSUE O O O O O O O O B-CELL_TYPE O O B-GENEPROD 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 B-EXP_ASSAY O O B-GENEPROD O O O O O O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O O O O O B-TISSUE 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-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O B-ORGANISM B-CELL_TYPE 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 B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O B-GENEPROD O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O 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-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-CELL_TYPE O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-GENEPROD O O O B-ORGANISM 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 B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY 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 B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD 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 B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O 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-GENEPROD O O O O B-GENEPROD O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE 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-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-TISSUE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O 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.


C , D . Coimmunoprecipitation between Flag - Lrig1 and HA - TrkB ( C ) or between Flag - Lrig3 and HA - TrkB ( D ) overexpressed in HEK293 cells . Cell extracts were analysed by immunoprecipitation with anti - Flag antibodies followed by immunoblot ( IB ) with antibodies against HA . Reprobing of the same blots with anti - Flag antibodies is shown below . The bottom panel shows HA expression in total lysates . Data represent n = 3 independent experiments .

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

C, D. Coimmunoprecipitation between Flag-Lrig1 and HA-TrkB (C) or between Flag-Lrig3 and HA-TrkB (D) overexpressed in HEK293 cells. Cell extracts were analysed by immunoprecipitation with anti-Flag antibodies followed by immunoblot (IB) with antibodies against HA. Reprobing of the same blots with anti-Flag antibodies is shown below. The bottom panel shows HA expression in total lysates. Data represent n=3 independent experiments.


E . Coimmunoprecipitation between HA - TrkB and Flag - Lrig1 or between HA - TrkB and HA - Lrig2 exogenously expressed in HEK293 cells . Cell extracts were analysed by IP with anti - pan - Trk antibodies followed by IB with antibodies against Flag or Lrig2 . Reprobing of the same blots with anti - TrkB antibodies is shown below . The bottom panels show Flag and Lrig2 expression in total lysates . Data represent n = 2 independent assays .

O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O

E. Coimmunoprecipitation between HA-TrkB and Flag-Lrig1 or between HA-TrkB and HA-Lrig2 exogenously expressed in HEK293 cells. Cell extracts were analysed by IP with anti-pan-Trk antibodies followed by IB with antibodies against Flag or Lrig2. Reprobing of the same blots with anti-TrkB antibodies is shown below. The bottom panels show Flag and Lrig2 expression in total lysates. Data represent n=2 independent assays.


F . In vivo interaction between Lrig1 and TrkB . Coimmunoprecipitation between Lrig1 and TrkB receptor endogenously expressed from P15 rat hippocampal tissue extracts . Samples were equally divided in two parts and then the analysis was done by immunoprecipitation with control ( anti - HA epitope tag antibody ) or anti - Lrig1 antibodies , followed by immunoblotting with anti - TrkB antibody . Reprobing of the same blot with anti - Lrig1 antibody is also shown . Expression of TrkB in one aliquot of the starting material ( indicated as lysate ) is included . Arrow indicates the band of TrkB coimmunoprecipitated with anti - Lrig1 antibody . Similar results were obtained in n = 3 independent assays .

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F. In vivo interaction between Lrig1 and TrkB. Coimmunoprecipitation between Lrig1 and TrkB receptor endogenously expressed from P15 rat hippocampal tissue extracts. Samples were equally divided in two parts and then the analysis was done by immunoprecipitation with control (anti-HA epitope tag antibody) or anti-Lrig1 antibodies, followed by immunoblotting with anti-TrkB antibody. Reprobing of the same blot with anti-Lrig1 antibody is also shown. Expression of TrkB in one aliquot of the starting material (indicated as lysate) is included. Arrow indicates the band of TrkB coimmunoprecipitated with anti-Lrig1 antibody. Similar results were obtained in n=3 independent assays.


G . Ligand - dependent activation of TrkB ( p - TrkB ) was evaluated by transient transfection of HA - TrkB plasmid with either a control or a Flag - Lrig1 vector into HEK cells . After 36 h cells were serum - starved and stimulated with or without BDNF ( 30 ng / ml ) for 15 min . The level of TrkB activation ( p - TrkB ) was evaluated in total cell lysates by immunoblotting ( IB ) with a specific antibody that recognizes TrkB phosphorylated in tyrosine 705 ( pY705 ) . Reprobing of the same blot with anti - HA and anti - Flag antibodies is shown . Fold of p - TrkB change relative to total TrkB is indicated . Similar results were obtained in n = 3 independent assays .

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G. Ligand-dependent activation of TrkB (p-TrkB) was evaluated by transient transfection of HA-TrkB plasmid with either a control or a Flag- Lrig1 vector into HEK cells. After 36 h cells were serum-starved and stimulated with or without BDNF (30 ng/ml) for 15 min. The level of TrkB activation (p-TrkB) was evaluated in total cell lysates by immunoblotting (IB) with a specific antibody that recognizes TrkB phosphorylated in tyrosine 705 (pY705). Reprobing of the same blot with anti-HA and anti-Flag antibodies is shown. Fold of p-TrkB change relative to total TrkB is indicated. Similar results were obtained in n=3 independent assays.


H . TrkB ubiquitination was evaluated by transient transfection of HA - TrkB plasmid with either a control or a Flag - Lrig1 vector into MN1 cells . After 36 h , cells were serum - starved , pre - treated with the cell - permeable proteasome inhibitor MG - 132 ( 20 M ) and stimulated with BDNF for 15 min . Total lysates were immunoprecipitated with anti - HA antibodies followed by immunoblot ( IB ) with antibodies against ubiquitin . Reprobing of the same blot with anti - HA antibodies is also shown . TrkB activation ( p - TrkB ) was evaluated in cell lysates . Reprobing of the same blot with anti - TrkB and anti - Flag antibodies is also shown . Fold of p - TrkB ( p - Y705 ) change relative to total TrkB is indicated . Data represent n = 3 independent assays .

O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O

H. TrkB ubiquitination was evaluated by transient transfection of HA-TrkB plasmid with either a control or a Flag-Lrig1 vector into MN1 cells. After 36 h, cells were serum-starved, pre-treated with the cell-permeable proteasome inhibitor MG-132 (20 M) and stimulated with BDNF for 15 min. Total lysates were immunoprecipitated with anti-HA antibodies followed by immunoblot (IB) with antibodies against ubiquitin. Reprobing of the same blot with anti-HA antibodies is also shown. TrkB activation (p-TrkB) was evaluated in cell lysates. Reprobing of the same blot with anti-TrkB and anti-Flag antibodies is also shown. Fold of p-TrkB (p-Y705) change relative to total TrkB is indicated. Data represent n=3 independent assays.


A . Immunoblot showing TrkB activation in hippocampal neurons cultured from Lrig1 heterozygous ( + / - ) and Lrig1 knockout ( - / - ) mice littermates treated in the absence or in the presence of BDNF ( 30 ng / ml ) for 30 minutes . Reprobing of the same blot with anti - ßIII - Tubulin is shown as loading control . B . Fold of TrkB activation relative to unstimulated control group ( phospho - TrkB at Tyr705 ) in hippocampal neurons cultured from Lrig1 ( + / + ; + / - ) and Lrig1 ( - / - ) mice treated in the absence or in the presence of BDNF ( 30 ng / ml ) for 30 minutes . Results are presented as mean SEM of n = 4 independent experiments . ( * p < 0 . 05 by Students t test ) .

O O B-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O O O O O O 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. Immunoblot showing TrkB activation in hippocampal neurons cultured from Lrig1 heterozygous (+/-) and Lrig1 knockout (-/-) mice littermates treated in the absence or in the presence of BDNF (30 ng/ml) for 30 minutes. Reprobing of the same blot with anti- ßIII-Tubulin is shown as loading control.B. Fold of TrkB activation relative to unstimulated control group (phospho-TrkB at Tyr705) in hippocampal neurons cultured from Lrig1 (+/+; +/-) and Lrig1 (-/-) mice treated in the absence or in the presence of BDNF (30 ng/ml) for 30 minutes. Results are presented as mean SEM of n=4 independent experiments. (*p<0.05 by Students t test).


C . Immunoblot showing MAPK activation in hippocampal neurons cultured from Lrig1 wild - type ( + / + ) and Lrig1 - deficient ( - / - ) mice littermates treated in the absence or in the presence of BDNF ( 30 ng / ml ) for the indicated times . Reprobing of the same blot with anti - ßIII - Tubulin is shown as loading control . Fold of MAPK activation relative to tubulin is indicated . D . Fold of MAPK activation ( P - MAPK ) relative to untreated control group in hippocampal neurons cultured from Lrig1 ( + / + ; + / - ) and Lrig1 ( - / - ) mice treated in the absence or in the presence of BDNF ( 30 ng / ml ) for 30 minutes . Results are presented as mean SEM of n = 4 independent experiments . ( * p < 0 . 05 by Students t test ) .

O O B-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O O O O O O 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. Immunoblot showing MAPK activation in hippocampal neurons cultured from Lrig1 wild-type (+/+) and Lrig1-deficient (-/-) mice littermates treated in the absence or in the presence of BDNF (30 ng/ml) for the indicated times. Reprobing of the same blot with anti- ßIII-Tubulin is shown as loading control. Fold of MAPK activation relative to tubulin is indicated.D. Fold of MAPK activation (P-MAPK) relative to untreated control group in hippocampal neurons cultured from Lrig1 (+/+; +/-) and Lrig1 (-/-) mice treated in the absence or in the presence of BDNF (30 ng/ml) for 30 minutes. Results are presented as mean SEM of n=4 independent experiments. (*p<0.05 by Students t test).


E . Representative images of mousehippocampal neurons transfected at 4 DIV with either GFP - expressing control vector or Lrig1shRNA - GFP plasmid . After transfection , neurons were cultured in the absence or in the presence of BDNF ( 25 ng / ml ) for 48 h ( 7 DIV ) . Arrows indicate branching points along the principal dendrite . Scale bar , 10 m .

O O O O O B-ORGANISM B-CELL_TYPE O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O

E. Representative images of mousehippocampal neurons transfected at 4 DIV with either GFP-expressing control vector or Lrig1shRNA-GFP plasmid. After transfection, neurons were cultured in the absence or in the presence of BDNF (25 ng/ml) for 48 h (7 DIV). Arrows indicate branching points along the principal dendrite. Scale bar, 10 m.


F - I . Quantification of primary dendrites ( F ) , secondary dendrites ( G ) , total branching ( H ) and branchig of the principal dendrite ( I ) of hippocampal neurons treated as indicated in E . The results are shown as individual values and the means of a representative assay . A total of 30 neurons were analyzed per experimental condition . Similar results were obtained in n = 3 independent experiments .

O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O 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-I. Quantification of primary dendrites (F), secondary dendrites (G), total branching (H) and branchig of the principal dendrite (I) of hippocampal neurons treated as indicated in E. The results are shown as individual values and the means of a representative assay. A total of 30 neurons were analyzed per experimental condition. Similar results were obtained in n=3 independent experiments.


J . Lrig1 + / + and Lrig1 - / - hippocampal lysates from postnatal day 11 ( P11 ) mice were immunoblotted against phospho - TrkB ( p - TrkB ) at Tyr705 and total TrkB . Quantification ( mean SD ) of the p - TrkB / TrkB ratio between Lrig1 + / + ( n = 4 ) and Lrig1 - / - ( n = 3 ) hippocampi is also shown .

O O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O B-ORGANISM O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-TISSUE O O O O

J. Lrig1+/+ and Lrig1-/- hippocampal lysates from postnatal day 11 (P11) mice were immunoblotted against phospho-TrkB (p-TrkB) at Tyr705 and total TrkB. Quantification (mean SD) of the p-TrkB/TrkB ratio between Lrig1+/+ (n=4) and Lrig1-/- (n=3) hippocampi is also shown.


B , C , Analysis of GFP transfer after transplantation of Nrl : : GFP donor photoreceptors into wildtype C57BL / 6J ( B ) and Nrl - / - ( C ) recipient retinas after 14 ( wt , n = 9 ; Nrl - / - , n = 7 ) and 21 ( wt , n = 6 ; Nrl - / - , n = 4 ) and 90 ( wt , n = 5 ; Nrl - / - , n = 4 ) days . Tissue is stained for GFP and nuclei ( Hoechst ) . In this and all following figures , donor cells ( D ) are located above , and acceptor cells with GFP ( A ) are located below the outer limiting membrane ( white dashed line ) , which delimits the apical border of the outer nuclear layer ( ONL ) . In wildtype recipients , donor cells are typically located above the outer segment region ( between the grey dashed lines ) . In Nrl - / - recipients , donor cells are located in the inner segment ( IS ) region . Plotted is the ratio of the numbers of GFP + photoreceptors in the ONL ( acceptor photoreceptors ) and GFP + donor photoreceptors in the subretinal space ( SRS ) over time .

O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O B-TISSUE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O B-TISSUE O O B-CELL_TYPE O O B-GENEPROD O O B-CELL_TYPE O O B-TISSUE I-TISSUE O B-TISSUE O O O O

B, C, Analysis of GFP transfer after transplantation of Nrl::GFP donor photoreceptors into wildtype C57BL/6J (B) and Nrl-/- (C) recipient retinas after 14 (wt, n=9; Nrl-/-, n=7) and 21 (wt, n=6; Nrl-/-, n=4) and 90 (wt, n=5; Nrl-/-, n=4) days. Tissue is stained for GFP and nuclei (Hoechst). In this and all following figures, donor cells (D) are located above, and acceptor cells with GFP (A) are located below the outer limiting membrane (white dashed line), which delimits the apical border of the outer nuclear layer (ONL). In wildtype recipients, donor cells are typically located above the outer segment region (between the grey dashed lines). In Nrl-/- recipients, donor cells are located in the inner segment (IS) region. Plotted is the ratio of the numbers of GFP+ photoreceptors in the ONL (acceptor photoreceptors) and GFP+ donor photoreceptors in the subretinal space (SRS) over time.


F , Co - transplanted P3 - 4 Crx : : Cre and P3 - 5 ROSAmT / mG donor photoreceptors into C57BL / 6J recipient retinas show Cre reporter gene induction in donor cells located in the SRS 14 days post - transplantation .

O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-TISSUE O B-GENEPROD O O O O O O O O O B-TISSUE O O O O O O

F, Co-transplanted P3-4 Crx::Cre and P3-5 ROSAmT/mG donor photoreceptors into C57BL/6J recipient retinas show Cre reporter gene induction in donor cells located in the SRS 14 days post-transplantation.


A - C , Combined FISH and IHF to detect GFP ( A , B ) and GNAT1 ( C ) 21 days post - transplantation of Nrl : : GFP photoreceptors into C57BL / 6J ( A ) and Nrl - / - ( B , C ) recipients .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-GENEPROD B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O O

A-C, Combined FISH and IHF to detect GFP (A, B) and GNAT1 (C) 21 days post-transplantation of Nrl::GFP photoreceptors into C57BL/6J (A) and Nrl-/- (B, C) recipients.


F - K , FISH and IHF for GFP and GFP transcripts in C57BL / 6J recipients transplanted with lentiviral - infected donor cells expressing cytoplasmic GFP ( Lenti - GFP ) ( F , I ) and nuclear - localized GFP ( Lenti - nls - GFP ) ( G , J ) . H , MT index for cytosolic ( Lenti - GFP , n = 5 ) and nuclear localized ( Lenti - nls - GFP , n = 3 ) . K , Quantification of transcripts in donor and acceptor photoreceptors . ( Lenti - GFP , n = 6 images from 3 transplanted animals ; Lenti - nls - GFP , n = 8 images from 4 transplanted animals ) .

O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O B-SUBCELLULAR B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY B-GENEPROD O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O B-ORGANISM O B-GENEPROD O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_TYPE O O B-ORGANISM O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O

F-K, FISH and IHF for GFP and GFP transcripts in C57BL/6J recipients transplanted with lentiviral-infected donor cells expressing cytoplasmic GFP (Lenti -GFP) (F, I) and nuclear-localized GFP (Lenti-nls-GFP) (G, J). H, MT index for cytosolic (Lenti-GFP, n=5) and nuclear localized (Lenti-nls-GFP, n=3). K, Quantification of transcripts in donor and acceptor photoreceptors. (Lenti-GFP, n=6 images from 3 transplanted animals; Lenti-nls-GFP, n=8 images from 4 transplanted animals).


B - D ' ' , Higher magnification of regions in A ( indicated by yellow dashed boxes ) showing colocalization of ATP5B and Mito - DsRed - labelled mitochondria in the outer segments ( B ) , soma ( C ) , and the axon ( D ) of acceptor photoreceptors .

O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O B-CELL_TYPE O

B-D'', Higher magnification of regions in A (indicated by yellow dashed boxes) showing colocalization of ATP5B and Mito-DsRed-labelled mitochondria in the outer segments (B), soma (C), and the axon (D) of acceptor photoreceptors.


F , G , C57BL / 6J cells with mitochondria labelled with MitoTrackerTM - Green ( MTG ) or MitoTrackerTM - Red ( MTR ) exchange mitochondria after 3 DIV ( 0 DIV , n = 6 ; 3 DIV , n = 6 ) . White dashed line delimitates both cell somas and the protrusion connecting them ( F ) . No exchange is observed when the two photoreceptor populations are separated by a transwell ( n = 3 ) , nor in co - cultures of live MTG - labelled and lysed MTR - labelled cells ( n = 3 ) ( G ) .

O O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

F, G, C57BL/6J cells with mitochondria labelled with MitoTrackerTM-Green (MTG) or MitoTrackerTM-Red (MTR) exchange mitochondria after 3 DIV (0 DIV, n=6; 3 DIV, n=6). White dashed line delimitates both cell somas and the protrusion connecting them (F). No exchange is observed when the two photoreceptor populations are separated by a transwell (n=3), nor in co-cultures of live MTG-labelled and lysed MTR-labelled cells (n=3) (G).


A , Maximum intensity projection of a confocal micrograph of a cryostat section from a Nrl - / - recipient retina 21 days after transplantation with Nrl : : GFP donor photoreceptors , showing protrusions ( pink arrows ) connecting donor and acceptor photoreceptors .

O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-TISSUE O O O O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O

A, Maximum intensity projection of a confocal micrograph of a cryostat section from a Nrl-/- recipient retina 21 days after transplantation with Nrl::GFP donor photoreceptors, showing protrusions (pink arrows) connecting donor and acceptor photoreceptors.


D , E , Left : 3D reconstruction of two - photon ( D ) and light sheet microscopy images of intact cleared ROSAmT / mG eye showing that GFP + - donor and - acceptor photoreceptors are in the same area . D , Right : A high magnification image showing GFP + - donor and - acceptor photoreceptors connected by protrusions ( pink arrows ) . E , Right : In detail , donor and acceptor photoreceptors also appear connected through cell protrusions ( pink arrow ) .

O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-TISSUE O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O

D, E, Left: 3D reconstruction of two-photon (D) and light sheet microscopy images of intact cleared ROSAmT/mG eye showing that GFP+-donor and -acceptor photoreceptors are in the same area. D, Right: A high magnification image showing GFP+-donor and -acceptor photoreceptors connected by protrusions (pink arrows). E, Right: In detail, donor and acceptor photoreceptors also appear connected through cell protrusions (pink arrow).


A , Confocal image shows a Lenti - GFP infected donor photoreceptor ( yellow arrow ) connected to an acceptor photoreceptor . The donor protrusion ( pink arrow ) co - localizes with α - tubulin but not the outer segment marker GNAT1 .

O O B-EXP_ASSAY O O O B-ORGANISM O B-GENEPROD O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O

A, Confocal image shows a Lenti-GFP infected donor photoreceptor (yellow arrow) connected to an acceptor photoreceptor. The donor protrusion (pink arrow) co-localizes with α-tubulin but not the outer segment marker GNAT1.


B , Nrl : : GFP donor photoreceptors transplanted in a Prphr2 - / - recipient retina exhibit polarized Prphr2 expression ( yellow arrow ) on the apical side of the donor photoreceptor . Basal protrusions ( pink arrows ) indicate donor protrusions that contact the host retina .

O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O B-GENEPROD O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-TISSUE O

B, Nrl::GFP donor photoreceptors transplanted in a Prphr2-/- recipient retina exhibit polarized Prphr2 expression (yellow arrow) on the apical side of the donor photoreceptor. Basal protrusions (pink arrows) indicate donor protrusions that contact the host retina.


C , D , Expression of β - tubulin ( C ) , SV2 ( D ) and GFP in the protrusions connecting Lenti - GFP - infected C57BL / 6J donor photoreceptors with acceptor photoreceptors . Pink arrows point to the protrusions connecting donor and acceptor cells .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O O O O O O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O O O O

C, D, Expression of β-tubulin (C), SV2 (D) and GFP in the protrusions connecting Lenti-GFP-infected C57BL/6J donor photoreceptors with acceptor photoreceptors. Pink arrows point to the protrusions connecting donor and acceptor cells.


A , B , Examples of live Nrl : : GFP and C57BL / 6J retinal cultures showing actin + ( SirActin ) and GFP + ( A ) and tubulin + ( ViaFluor ) and MitoTracker Green + ( MTG ) protrusions ( B ) connecting photoreceptors . DIC , differential interference contrast microscopy .

O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-TISSUE O O B-SUBCELLULAR O O B-GENEPROD O O B-GENEPROD O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

A, B, Examples of live Nrl::GFP and C57BL/6J retinal cultures showing actin+ (SirActin) and GFP+ (A) and tubulin+ (ViaFluor) and MitoTracker Green+ (MTG) protrusions (B) connecting photoreceptors. DIC, differential interference contrast microscopy.


E , Time lapse imaging showing movement of MitoTracker Green + puncta in photoreceptors connected by a protrusion . White arrows point MTG + puncta inside of the protrusion during the time lapse imaging .

O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

E, Time lapse imaging showing movement of MitoTracker Green+ puncta in photoreceptors connected by a protrusion. White arrows point MTG+ puncta inside of the protrusion during the time lapse imaging.


C , Confocal microscopy image of cryostat sections of C57BL / 6J recipient retinas 21 days after transplant of C57BL / J6 photoreceptors infected with empty vector ( n = 5 ) , RHOA overexpression ( RHOA OE ) ( n = 7 ) or RAC1 dominant negative ( RAC1 DN ) ( n = 7 ) lentiviruses . Quantification of MT in retinas transplanted with control , RHOA and RAC1 DN lentiviruses .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-TISSUE O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O B-TISSUE O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O

C, Confocal microscopy image of cryostat sections of C57BL/6J recipient retinas 21 days after transplant of C57BL/J6 photoreceptors infected with empty vector (n=5), RHOA overexpression (RHOA OE) (n=7) or RAC1 dominant negative (RAC1 DN) (n=7) lentiviruses. Quantification of MT in retinas transplanted with control, RHOA and RAC1 DN lentiviruses.


D , GFP staining of whole - mounted retinas after transplantation with control , RHOA and RAC1 DN lentivirus infected donor cells ( n = 4 per group ) . Quantification shows a significant reduction in the average protrusion length per cell and in the total protrusion length per cell after RHOA OE and RAC1 DN infection compared to the empty vector control .

O O B-GENEPROD B-EXP_ASSAY O O O O B-TISSUE O O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O

D, GFP staining of whole-mounted retinas after transplantation with control, RHOA and RAC1 DN lentivirus infected donor cells (n=4 per group). Quantification shows a significant reduction in the average protrusion length per cell and in the total protrusion length per cell after RHOA OE and RAC1 DN infection compared to the empty vector control.


A Genome - wide nucleosome maps were generated from the frequency distribution of the read midpoint positions of pair - end sequenced MNase - resistant fragments in the size range 138 - 161 bp ( spanning the nucleosome footprint ) . Figure shows a section of a nucleosome map taken from chromosome 12 derived from pluripotent iPSC ( pl - iPSC ) to show examples of nucleosomes selected as highly positioned , marked * .

O O O O B-SUBCELLULAR O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O

A Genome-wide nucleosome maps were generated from the frequency distribution of the read midpoint positions of pair-end sequenced MNase-resistant fragments in the size range 138-161 bp (spanning the nucleosome footprint). Figure shows a section of a nucleosome map taken from chromosome 12 derived from pluripotent iPSC (pl-iPSC) to show examples of nucleosomes selected as highly positioned, marked *.


B The distribution of positioned nucleosomes ( derived 138 - 161 bp size class ) and sub - nucleosome fragments ( derived 112 - 137 bp size class ) for pl - iPSC and iPSC - derived NPC ( NPC ) . Actual calculated numbers are presented above each column .

O O B-EXP_ASSAY O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O

B The distribution of positioned nucleosomes (derived 138-161 bp size class) and sub-nucleosome fragments (derived 112-137 bp size class) for pl-iPSC and iPSC-derived NPC (NPC). Actual calculated numbers are presented above each column.


D Average frequency distribution of nucleosome positions relative to each other . Data was aligned to each mapped nucleosome in the genome and plotted for a 600 bp window for pl - iPSC , NPC and the chronic myelogenous leukaemia ( Cml ) derived cell line , K562 . In all cases , a prominent single nucleosome is presented with only minor flanking peaks , indicating that the majority of nucleosomes do not occur as evenly spaced arrays in human cells .

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D Average frequency distribution of nucleosome positions relative to each other. Data was aligned to each mapped nucleosome in the genome and plotted for a 600 bp window for pl-iPSC, NPC and the chronic myelogenous leukaemia (Cml) derived cell line, K562. In all cases, a prominent single nucleosome is presented with only minor flanking peaks, indicating that the majority of nucleosomes do not occur as evenly spaced arrays in human cells.


E Distribution of nucleosome array sizes for pl - iPSC , NPC and K562 , calculated as the number of nucleosomes within a distance of 150 - 200 bp of each other . Bars show the number of nucleosomes arrayed as singletons ( 1 ) , pairs ( 2 ) , triplets ( 3 ) and greater than 3 ( > 3 ) , displayed as log10 values and the percentage distribution within a cell line is shown above the column .

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E Distribution of nucleosome array sizes for pl-iPSC, NPC and K562, calculated as the number of nucleosomes within a distance of 150-200 bp of each other. Bars show the number of nucleosomes arrayed as singletons (1), pairs (2), triplets (3) and greater than 3 (>3), displayed as log10 values and the percentage distribution within a cell line is shown above the column.


A Bar chart showing the number of nucleosomes that map to selected chromatin states [ 20 ] . Active ( State 1 ) ; Insulator ( State 8 ) ; Repressed , Rep , ( State 12 ) and heterochromatin , Hetero ( State 13 ) , and to open chromatin ( based on ATAC - seq ) . Shown are the observed number of nucleosomes per chromatin state ( black ) and the expected number calculated assuming a random distribution across the genome ( grey ) .

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A Bar chart showing the number of nucleosomes that map to selected chromatin states [20]. Active (State 1); Insulator (State 8); Repressed, Rep, (State 12) and heterochromatin, Hetero (State 13), and to open chromatin (based on ATAC-seq). Shown are the observed number of nucleosomes per chromatin state (black) and the expected number calculated assuming a random distribution across the genome (grey).


A , B Nucleosome maps at and surrounding , the TSS region of the NANOG and GRIA1 genes in pl - iPSC ( upper , black ) and in NPC cells ( lower , grey ) . The TSS is indicated by a dashed line at chr 12 : 7 , 941 , 991 for NANOG and chr5 : 152 , 870 , 105 for GRIA1 . * indicates the position of a highly positioned nucleosome associated with the TSS of the active gene .

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A, B Nucleosome maps at and surrounding, the TSS region of the NANOG and GRIA1 genes in pl-iPSC (upper, black) and in NPC cells (lower, grey). The TSS is indicated by a dashed line at chr 12: 7,941,991 for NANOG and chr5: 152,870,105 for GRIA1. * indicates the position of a highly positioned nucleosome associated with the TSS of the active gene.


C Global frequency distribution of nucleosome distributions at TSS of protein - coding genes . Alignment of nucleosome maps centred on TSS using the non - redundant list of 66 , 047 mapped human protein - coding gene TSS . The features corresponding to the nucleosome free region ( NFR ) and - 1 to + 2 nucleosomes are marked .

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C Global frequency distribution of nucleosome distributions at TSS of protein-coding genes. Alignment of nucleosome maps centred on TSS using the non-redundant list of 66,047 mapped human protein-coding gene TSS. The features corresponding to the nucleosome free region (NFR) and -1 to +2 nucleosomes are marked.


D Genome - wide positioning of nucleosomes within ± 300 bp of a TSS of an active gene . The number of nucleosomes observed are plotted for both pl - iPSC and NPC cell types for gene sets selected either as uniquely expressed in pl - iPSC ( n = 3 , 833 ) or NPC ( n = 2 , 082 ) . If positioned nucleosomes are a strong predictor of gene activity , high numbers of nucleosomes would be expected for pluripotent - specific genes in pl - iPSC , but not NPC stages , and for NPC specific genes in NPC , but not pl - iPSC stages . No correlation between gene activity and positioned nucleosomes was observed .

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D Genome-wide positioning of nucleosomes within ± 300 bp of a TSS of an active gene. The number of nucleosomes observed are plotted for both pl-iPSC and NPC cell types for gene sets selected either as uniquely expressed in pl-iPSC (n=3,833) or NPC (n=2,082). If positioned nucleosomes are a strong predictor of gene activity, high numbers of nucleosomes would be expected for pluripotent-specific genes in pl-iPSC, but not NPC stages, and for NPC specific genes in NPC, but not pl-iPSC stages. No correlation between gene activity and positioned nucleosomes was observed.


A The average distribution of nucleosomes centred on the RE1 site ( n = 871 ) , spanning 2 Kb of flanking genomic sequence .

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A The average distribution of nucleosomes centred on the RE1 site (n=871), spanning 2 Kb of flanking genomic sequence.


B , C Aligned pair - end sequence read data were stratified into two size classes , ( B ) 122 - 137 bp and ( C ) 162 - 188 bp for pl - iPSC and NPC . Plots show the average distribution of nucleosomes centred at and surrounding the RE1 site ( n = 871 ) . Peaks correspond to the NRSF / REST complex , and nucleosomes in B and C respectively .

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B, C Aligned pair-end sequence read data were stratified into two size classes, (B) 122-137 bp and (C) 162-188 bp for pl-iPSC and NPC. Plots show the average distribution of nucleosomes centred at and surrounding the RE1 site (n=871). Peaks correspond to the NRSF/REST complex, and nucleosomes in B and C respectively.


D Cluster analysis of nucleosome positioning centred on the RE1 site ± 300 bp . RE1 sites were clustered based on the 122 - 137 bp mid - point sequence read values ( NRSF / REST protein complex ) from pl - iPSC , shown as a frequency distribution in upper left panel . The frequency distribution data for the same sites was plotted using 122 - 137 bp data from NPC ( upper right panel ) and 162 - 188 bp data ( nucleosomes ; bottom panels ) for the strongest ( solid line ) and weakest ( dotted line ) clusters .

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D Cluster analysis of nucleosome positioning centred on the RE1 site ± 300 bp. RE1 sites were clustered based on the 122-137 bp mid-point sequence read values (NRSF/REST protein complex) from pl-iPSC, shown as a frequency distribution in upper left panel. The frequency distribution data for the same sites was plotted using 122-137 bp data from NPC (upper right panel) and 162-188 bp data (nucleosomes; bottom panels) for the strongest (solid line) and weakest (dotted line) clusters.


A Average frequency distribution for sequence read mid - point data in the size range of 122 - 137 bp centred on the CTCF binding site ( n = 9516 ) . This shows a distinctive peak corresponding to the CTCF protein complex , which is present in pl - iPSC , but reduced in NPC . B Average frequency distribution for sequence read mid - point data centred on the CTCF binding site in the size range of 162 - 188 bp , corresponding to larger nucleosome footprints , for pl - iPSC and NPC cells . This shows that the pattern of nucleosome positioning is independent of the amount CTCF protein complex . Although positioned nucleosomes are retained flanking CTCF sites , their positions are shifted closer to the CTCF site and their spacing is altered . C Average frequency distribution for sequence read mid - point data centred on the CTCF site for pl - iPSC ( 162 - 188 bp ) and K562 ( total MNase - seq data ) cells . Positioned nucleosomes are retained flanking CTCF sites in K562 cells , but with shifted positions and altered spacing .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-CELL_TYPE O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-EXP_ASSAY B-SUBCELLULAR O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O B-CELL_LINE O O B-GENEPROD O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O B-GENEPROD O O B-CELL_LINE O O O O O B-EXP_ASSAY O O O O

A Average frequency distribution for sequence read mid-point data in the size range of 122-137 bp centred on the CTCF binding site (n=9516). This shows a distinctive peak corresponding to the CTCF protein complex, which is present in pl-iPSC, but reduced in NPC. B Average frequency distribution for sequence read mid-point data centred on the CTCF binding site in the size range of 162-188 bp, corresponding to larger nucleosome footprints, for pl-iPSC and NPC cells. This shows that the pattern of nucleosome positioning is independent of the amount CTCF protein complex. Although positioned nucleosomes are retained flanking CTCF sites, their positions are shifted closer to the CTCF site and their spacing is altered. C Average frequency distribution for sequence read mid-point data centred on the CTCF site for pl-iPSC (162-188 bp) and K562 (total MNase-seq data) cells. Positioned nucleosomes are retained flanking CTCF sites in K562 cells, but with shifted positions and altered spacing.


D Average frequency distribution for sequence read mid - point data in the size range of 122 - 137 bp for pl - iPSC and NPC cells centred on the 2 , 522 CTCF binding sites identified from NPC specific ChIP - seq data . A distinctive peak corresponding to the CTCF protein complex is present in the NPC state , but reduced to background levels in pl - iPSC . E Average frequency distribution for sequence read mid - point data centred on the CTCF binding sites in the size range of 162 - 188 bp , corresponding to larger nucleosome footprints , for pl - iPSC and NPC centred on the 2 , 522 CTCF binding sites identified from NPC specific ChIP - seq data . Nucleosome arrays are present in both cell states , independent of the CTCF protein complex . Nucleosomes in the CTCF sites in pl - iPSC tend to position closer to the CTCF site than in NPC . The data derived from the unique NPC CTCF sites has greater noise due to the smaller number of sites analysed .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O B-GENEPROD O O O O B-CELL_TYPE O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

D Average frequency distribution for sequence read mid-point data in the size range of 122-137 bp for pl-iPSC and NPC cells centred on the 2,522 CTCF binding sites identified from NPC specific ChIP-seq data. A distinctive peak corresponding to the CTCF protein complex is present in the NPC state, but reduced to background levels in pl-iPSC. E Average frequency distribution for sequence read mid-point data centred on the CTCF binding sites in the size range of 162-188 bp, corresponding to larger nucleosome footprints, for pl-iPSC and NPC centred on the 2,522 CTCF binding sites identified from NPC specific ChIP-seq data. Nucleosome arrays are present in both cell states, independent of the CTCF protein complex. Nucleosomes in the CTCF sites in pl-iPSC tend to position closer to the CTCF site than in NPC. The data derived from the unique NPC CTCF sites has greater noise due to the smaller number of sites analysed.


( a ) HACE1 mRNA expression is markedly increased in ischaemic and dilated cardiomyopathy ( ICM and DCM ) heart failure ( HF ) patient heart tissue compared with non - failing hearts ( NF ) . Expression levels were normalized to HPRT and GAPDH mRNAs . n = 15 per group . Control , non - failing heart .

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(a) HACE1 mRNA expression is markedly increased in ischaemic and dilated cardiomyopathy (ICM and DCM) heart failure (HF) patient heart tissue compared with non-failing hearts (NF). Expression levels were normalized to HPRT and GAPDH mRNAs. n=15 per group. Control, non-failing heart.


( b ) Elevated Hace1 mRNA expression in sTAC mousehearts compared with sham controls , expression levels were normalized to Hprt and Gapdh mRNAs . n = 6 - 9 per group , P = 0 . 004 ( unpaired two - tailed Student ' s t - tests ) .

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(b) Elevated Hace1 mRNA expression in sTAC mousehearts compared with sham controls, expression levels were normalized to Hprt and Gapdh mRNAs. n=6-9 per group, P=0.004 (unpaired two-tailed Student's t-tests).


( c - e ) Hace1 − / − and WT mice were subjected to sTAC or sham surgery and analysed 4 days after the operation . Echocardiography measurement of ( c ) LV fractional shortening ( FS = ( LVEDD − LVESD ) / LVEDD × 100 % ) ; ( d ) LV end - diastolic dimension ( LVEDD ) ( mm ) and ( e ) LV end - systolic dimension ( LVESD ) ( mm ) are shown . n = 6 - 8 for each group .

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(c-e) Hace1−/− and WT mice were subjected to sTAC or sham surgery and analysed 4 days after the operation. Echocardiography measurement of (c) LV fractional shortening (FS=(LVEDD−LVESD)/LVEDD × 100%); (d) LV end-diastolic dimension (LVEDD) (mm) and (e) LV end-systolic dimension (LVESD) (mm) are shown. n=6-8 for each group.


( f ) Mortality rate of WT and Hace1 − / − mice after sTAC is shown by Kaplan - Meier plots as the survival proportion of each cohort of mice . Survival incidence in mice over the indicated follow - up period is shown as the ratio of the number of mice survived / total mice analysed for each group . This represents 60 % ( 12 / 20 ) for Hace1 − / − sTAC mice , 84 % ( 16 / 19 ) for Hace1 + / + sTAC mice and 100 % ( 12 / 12 ) for both Hace1 − / − and Hace1 + / + sham mice on day 4 ; P value as indicated ( log - rank χ2 - test ) .

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(f) Mortality rate of WT and Hace1−/− mice after sTAC is shown by Kaplan-Meier plots as the survival proportion of each cohort of mice. Survival incidence in mice over the indicated follow-up period is shown as the ratio of the number of mice survived/total mice analysed for each group. This represents 60% (12/20) for Hace1−/− sTAC mice, 84% (16/19) for Hace1+/+ sTAC mice and 100% (12/12) for both Hace1−/− and Hace1+/+ sham mice on day 4; P value as indicated (log-rank χ2-test).


( g ) Heart weight / body weight ratios ( HW / BW ) and ( h ) lung weight / body weight ratio ( LW / BW ) of WT and Hace1 − / − mice 4 days after sTAC or sham surgery . n = 15 - 21 for each group .

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(g) Heart weight/body weight ratios (HW/BW) and (h) lung weight/body weight ratio (LW/BW) of WT and Hace1−/− mice 4 days after sTAC or sham surgery. n=15-21 for each group.


( i ) Representative haematoxylin and eosin staining transverse heart cross - sections showing myocyte cross - sectional area ( scale bars , 20 μm ) .

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(i) Representative haematoxylin and eosin staining transverse heart cross-sections showing myocyte cross-sectional area (scale bars, 20 μm).


( j ) Real - time RT - PCR analyses reveal a significant increase in the expression of the atrial natriuretic peptide gene ( Nppa ) , a clinical biomarker for heart hypertrophy , in Hace1 − / − sTAC heart ; expression levels were normalized to Hprt and Gapdh mRNAs . n = 6 - 9 per group . In all panels , error bars represent s . e . m . , one - way analysis of variance were used to calculate P - values .

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(j) Real-time RT-PCR analyses reveal a significant increase in the expression of the atrial natriuretic peptide gene (Nppa), a clinical biomarker for heart hypertrophy, in Hace1−/− sTAC heart; expression levels were normalized to Hprt and Gapdh mRNAs. n=6-9 per group. In all panels, error bars represent s.e.m., one-way analysis of variance were used to calculate P-values.


Representative immunohistochemistry staining for ( a ) ubiquitinated protein ( brown ) ; ( b ) p62 ( brown ) and ( c ) GFP - LC3 ( brown ) in paraffin sections of heart revealing increased accumulation of p62 , ubiquitinated protein and LC3 in Hace1 − / − sTAChearts . Hearts from three mice in each group were analysed ( scale bars , 10 μm ) .

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Representative immunohistochemistry staining for (a) ubiquitinated protein (brown); (b) p62 (brown) and (c) GFP-LC3 (brown) in paraffin sections of heart revealing increased accumulation of p62, ubiquitinated protein and LC3 in Hace1−/− sTAChearts. Hearts from three mice in each group were analysed (scale bars, 10 μm).


Representative immunoblot and quantification for ( d , e ) Ub - conjugated proteins , ( d , f ) p62 and ( g , h ) LC3 in heart extracts ( n = 3 per group ) , Gapdh were used as a loading control .

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Representative immunoblot and quantification for (d,e) Ub-conjugated proteins, (d,f) p62 and (g,h) LC3 in heart extracts (n=3 per group), Gapdh were used as a loading control.


( i ) Increased Ub proteasome activity in Hace1 − / − sTAC hearts as measured by 20S proteasome activity assay ( n = 4 - 6 per group ) . In all panels , error bars represent s . e . m . , P value between KO and WT sTAC as indicated ( one - way analysis of variance ) .

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(i) Increased Ub proteasome activity in Hace1−/− sTAC hearts as measured by 20S proteasome activity assay (n=4-6 per group). In all panels, error bars represent s.e.m., P value between KO and WT sTAC as indicated (one-way analysis of variance).


( a ) Representative confocal microscopy images showing HACE1 ( green ) localizing to the Ub + protein aggregates ( red ) induced by puromycin treatment in HA - HACE1 MEF . Arrows indicate co - localized puncta . Scale bar , 10 μm .

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(a) Representative confocal microscopy images showing HACE1 (green) localizing to the Ub+ protein aggregates (red) induced by puromycin treatment in HA-HACE1 MEF. Arrows indicate co-localized puncta. Scale bar, 10 μm.


( b ) Representative confocal micrographs of Hace1 − / − and Hace1 + / + NCM treated with puromycin for 4 h ( puromycin ) , puromycin followed by 8 h recover ( puromycin + recover ) . Vehicle ( PBS ) - treated cells were used as control . Hace1 deficiency in cardiomyocytes ( Hace1 − / − NCM ) blocks ubiquitinated protein aggregate degradation . Scale bars , 10 μm .

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(b) Representative confocal micrographs of Hace1−/− and Hace1+/+ NCM treated with puromycin for 4 h (puromycin), puromycin followed by 8 h recover (puromycin+recover). Vehicle (PBS)-treated cells were used as control. Hace1 deficiency in cardiomyocytes (Hace1−/− NCM) blocks ubiquitinated protein aggregate degradation. Scale bars, 10 μm.