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

Figure 2A. 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.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.


Figure 3A . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. 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).


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

Figure 4A. 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.


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

Figure 5A. 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.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.


Figure 6A . 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 . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. 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.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.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.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.


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

Figure 7A. 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).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.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.


Figure 1 ( A ) Heteroplasmy levels by PCR - RFLP at position 3243 in 82 hiPSCs clones at passage 2 , derived from three MELAS patients ( MitoA , MitoB , MitoC ) . The black dots represent the mutation load for a specific hiPSC clone ; n is the total number of individual hiPSC clones analyzed per patient . The red stars represent the heteroplasmy levels for the parental fibroblasts . ( A ) Heteroplasmy levels by PCR - RFLP at position 3243 in 82 hiPSCs clones at passage 2 , derived from three MELAS patients ( MitoA , MitoB , MitoC ) . The black dots represent the mutation load for a specific hiPSC clone ; n is the total number of individual hiPSC clones analyzed per patient . The red stars represent the heteroplasmy levels for the parental fibroblasts .

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

Figure 1(A) Heteroplasmy levels by PCR-RFLP at position 3243 in 82 hiPSCs clones at passage 2, derived from three MELAS patients (MitoA, MitoB, MitoC). The black dots represent the mutation load for a specific hiPSC clone; n is the total number of individual hiPSC clones analyzed per patient. The red stars represent the heteroplasmy levels for the parental fibroblasts.(A) Heteroplasmy levels by PCR-RFLP at position 3243 in 82 hiPSCs clones at passage 2, derived from three MELAS patients (MitoA, MitoB, MitoC). The black dots represent the mutation load for a specific hiPSC clone; n is the total number of individual hiPSC clones analyzed per patient. The red stars represent the heteroplasmy levels for the parental fibroblasts.


Figure 2 ( A ) mtDNA NGS of MitoA fibroblasts ( MitoA FB at passage 5 ) and MitoA - derived hiPSCs ( passages 7 - 9 ) ; n = 13 . ( B ) mtDNA NGS of MitoB fibroblasts ( MitoB FB at passage 3 ) and MitoB - derived hiPSCs ( passages 5 - 11 ) ; n = 12 . ( C ) mtDNA NGS of MitoC fibroblasts ( MitoC FB at passage 7 ) and MitoC - derived hiPSCs ( passages 5 - 11 ) ; n = 11 . GPMs are represented by black diamonds at the specific mtDNA positions ; small black diamonds denote mutation load between 1 and 10 % , medium black diamonds 10 to 40 % , and large black diamonds > 40 % . Red diamonds : point mutation A3243G ( MELAS causing disease mutation ) . Numbers beside diamonds indicate the specific mutation load at that position .

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Figure 2(A) mtDNA NGS of MitoA fibroblasts (MitoA FB at passage 5) and MitoA-derived hiPSCs (passages 7-9); n=13.(B) mtDNA NGS of MitoB fibroblasts (MitoB FB at passage 3) and MitoB-derived hiPSCs (passages 5-11); n=12.(C) mtDNA NGS of MitoC fibroblasts (MitoC FB at passage 7) and MitoC-derived hiPSCs (passages 5-11); n=11.GPMs are represented by black diamonds at the specific mtDNA positions; small black diamonds denote mutation load between 1 and 10%, medium black diamonds 10 to 40%, and large black diamonds > 40%. Red diamonds: point mutation A3243G (MELAS causing disease mutation). Numbers beside diamonds indicate the specific mutation load at that position.


Figure 3 ( B ) Distribution of GPMs with mutation load lower or greater than 10 % , in control and MELAS groups . The majority of GPMs are present at levels lower than 10 % in either group . ( C ) Location of GPMs greater than 10 % , in mtDNA .

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Figure 3(B) Distribution of GPMs with mutation load lower or greater than 10%, in control and MELAS groups. The majority of GPMs are present at levels lower than 10% in either group.(C) Location of GPMs greater than 10%, in mtDNA.


Figure 4 ( B ) mtDNA NGS showing the position and mutation load of GPMs in seven isogenic MitoA - derived hiPSCs clones and their differentiated counterparts . The mtDNA genotype is stable during in vitro cardiac differentiation and lactate enrichment . MitoA hiPSCs 57 and 65 ( wild - type ) are labelled in black , MitoA hiPSCs 61 and 69 ( m . 3243A > G mutant ) are labelled in red and MitoA hiPSCs 58 , 59 and 217 ( with amplified GPMs in nuclear reprogramming ) are labelled in blue . ( C ) Quantification of the percentage of mature hiPSCs - derived cardiomyocytes in direct differentiation of hiPSC clones . Representative histograms showing the percentage of the cells expressing mature cardiac markers : myosin heavy chain ( MF20 ) , troponin T ( cTnT ) and troponin I ( cTnI ) by flow cytometry , in MitoA - 57 - derived cardiomyocytes ( wild - type ) and MitoA - 69 - derived cardiomyocytes ( 83 % m . 3243A > G ) . For complete quantification of cardiac markers in all hiPSCs - derived cardiomyocytes generated . ( D ) Immunofluorescence images of cardiac markers ( cTnT and MF20 ) showing similar sarcomere structures in hiPSC - derived cardiomyocytes wild - type ( MitoA - 57 ) and mutant ( MitoA - 69 ) . ( E ) Oxygen consumption rate ( OCR ) in hiPSCs . OCR reported as a % of that in MitoA - 57 hiPSC , the control line . MitoA - 57 and MitoA - 65 hiPSCs wild - type ( in black ) , MitoA - 61 and MitoA - 69 m . 3243A > G mutant ( in red ) , MitoA - 58 , MitoA - 59 and MitoA - 217 hiPSCs amplified GPMs ( in blue ) . Error bars are mean ± SD and OCR data are representative 3 biological replicates in all samples . ( F ) Oxygen consumption rate in hiPSCs - derived cardiomyocytes at day 19 of cardiac differentiation . OCR reported as % of that in MitoA - 57 - derived cardiomyocytes , the control line . MitoA - 57 and MitoA - 65 derived cardiomyocytes wild - type ( in black ) , MitoA - 61 and MitoA - 69 derived cardiomyocytes mutants m . 3243A > G ( in red ) , MitoA - 58 , MitoA - 59 and MitoA - 217 derived cardiomyocytes with amplified GPMs ( in blue ) . Error bars are mean ± SD and OCR data are representative of two independent in vitro cardiac differentiation experiments , with at least 3 biological replicates in each sample , and significance established using Student ' s t - test .

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

Figure 4(B) mtDNA NGS showing the position and mutation load of GPMs in seven isogenic MitoA-derived hiPSCs clones and their differentiated counterparts. The mtDNA genotype is stable during in vitro cardiac differentiation and lactate enrichment. MitoA hiPSCs 57 and 65 (wild-type) are labelled in black, MitoA hiPSCs 61 and 69 (m.3243A>G mutant) are labelled in red and MitoA hiPSCs 58, 59 and 217 (with amplified GPMs in nuclear reprogramming) are labelled in blue.(C) Quantification of the percentage of mature hiPSCs-derived cardiomyocytes in direct differentiation of hiPSC clones. Representative histograms showing the percentage of the cells expressing mature cardiac markers: myosin heavy chain (MF20), troponin T (cTnT) and troponin I (cTnI) by flow cytometry, in MitoA-57-derived cardiomyocytes (wild-type) and MitoA-69-derived cardiomyocytes (83% m.3243A>G). For complete quantification of cardiac markers in all hiPSCs-derived cardiomyocytes generated.(D) Immunofluorescence images of cardiac markers (cTnT and MF20) showing similar sarcomere structures in hiPSC-derived cardiomyocytes wild-type (MitoA-57) and mutant (MitoA-69).(E) Oxygen consumption rate (OCR) in hiPSCs. OCR reported as a % of that in MitoA-57 hiPSC, the control line. MitoA-57 and MitoA-65 hiPSCs wild-type (in black), MitoA-61 and MitoA-69 m.3243A>G mutant (in red), MitoA-58, MitoA-59 and MitoA-217 hiPSCs amplified GPMs (in blue). Error bars are mean ± SD and OCR data are representative 3 biological replicates in all samples.(F) Oxygen consumption rate in hiPSCs-derived cardiomyocytes at day 19 of cardiac differentiation. OCR reported as % of that in MitoA-57-derived cardiomyocytes, the control line. MitoA-57 and MitoA-65 derived cardiomyocytes wild-type (in black), MitoA-61 and MitoA-69 derived cardiomyocytes mutants m.3243A>G (in red), MitoA-58, MitoA-59 and MitoA-217 derived cardiomyocytes with amplified GPMs (in blue). Error bars are mean ± SD and OCR data are representative of two independent in vitro cardiac differentiation experiments, with at least 3 biological replicates in each sample, and significance established using Student's t-test.


Figure 1a - b , Representative images of cilia ( a ) and dot plots ( b ) showing primary cilia length variation at different time points after serum starvation ( SS ) . Markers in ( a ) show ARL13B ( cilia ) and PCNT ( Basal body ) . The graph shows individual length of cilia from 3 biological replicates ( n > 150 cilia per sample and experiment ) . Scale bar : 1µm . Data shown in include mean ± s . d . are calculated by two - tailed unpaired student t - test . d , Representative images show cilia segmentation based on acetylated tubulin ( Ac tub ) , polyglutamylated tubulin ( Glu tub ) , ARL13B and PCNT . The cartoon on the left indicates proximal segments ( PS , Glu tub rich - region ) and distal segments ( DS ) . DS were determined by calculating the difference between PS and total cilia length based on ARL13B staining as depicted by the dashed lines on the images . Scale bar : 1 . 5µm . e , Quantification of d . Comparative analysis of average cilia length based on Ac tubulin and ARL13B ( cilia ) staining shown for specified cilia length groups ( 2 < , 2 - 3 , 3 - 4 and > 4µm ) . Three biological replicates , n = 400 cilia per sample and repetition . The numbers above the cartoons show the ratio of the calculated Ac tub / whole cilia length . Data shown include mean ± s . d . are calculated by two - tailed unpaired student t - test .

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Figure 1a-b, Representative images of cilia (a) and dot plots (b) showing primary cilia length variation at different time points after serum starvation (SS). Markers in (a) show ARL13B (cilia) and PCNT (Basal body). The graph shows individual length of cilia from 3 biological replicates (n>150 cilia per sample and experiment). Scale bar: 1µm. Data shown in include mean ±s.d. are calculated by two-tailed unpaired student t-test.d, Representative images show cilia segmentation based on acetylated tubulin (Ac tub), polyglutamylated tubulin (Glu tub), ARL13B and PCNT. The cartoon on the left indicates proximal segments (PS, Glu tub rich-region) and distal segments (DS). DS were determined by calculating the difference between PS and total cilia length based on ARL13B staining as depicted by the dashed lines on the images. Scale bar: 1.5µm. e, Quantification of d. Comparative analysis of average cilia length based on Ac tubulin and ARL13B (cilia) staining shown for specified cilia length groups (2<, 2-3, 3-4 and >4µm). Three biological replicates, n=400 cilia per sample and repetition. The numbers above the cartoons show the ratio of the calculated Ac tub/whole cilia length. Data shown include mean ±s.d. are calculated by two-tailed unpaired student t-test.


Figure 2a , Representative images of 48h serum starved RPE1 cells treated with solvent control ( DMSO ) , cytochalasin D ( CytoD ) and / or nocodazole ( Noco ) for 3h before immunostaining with the indicated antibodies . Scale bar : 3µm . b , Quantification of ( a ) showing average length of proximal segment ( PS , Glu tub ) and whole cilia ( ARL13B ) . The numbers above the cartoons show the ratio of the calculated distal segment ( DS ) / whole cilia length . Three biological replicates , n = 100 cilia per sample and repetition . Data shown include mean ± s . d . ; P values are calculated by two - tailed unpaired student t - test . RPE1 cells were treated with the indicated siRNAs before serum starvation for 48h . Representative immunostained images with indicated antibodies Scale bar : 2µm . e - f , Cells were treated with the indicated siRNAs before serum starvation for 48h and treatment with DMSO or Noco for 3h before immunostaining with the indicated antibodies . Representative images ( e ) and quantification of cilia length based on ARL13B ( f ) are shown . Scale bar : 2µm . Three biological replicates , n = 100 cilia per sample and repetition . Data shown include mean ± s . d . ; P values are calculated by two - tailed unpaired student t - test .

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

Figure 2a, Representative images of 48h serum starved RPE1 cells treated with solvent control (DMSO), cytochalasin D (CytoD) and/or nocodazole (Noco) for 3h before immunostaining with the indicated antibodies. Scale bar: 3µm. b, Quantification of (a) showing average length of proximal segment (PS, Glu tub) and whole cilia (ARL13B). The numbers above the cartoons show the ratio of the calculated distal segment (DS)/whole cilia length. Three biological replicates, n=100 cilia per sample and repetition. Data shown include mean ±s.d.; P values are calculated by two-tailed unpaired student t-test.RPE1 cells were treated with the indicated siRNAs before serum starvation for 48h. Representative immunostained images with indicated antibodies Scale bar: 2µm.e-f, Cells were treated with the indicated siRNAs before serum starvation for 48h and treatment with DMSO or Noco for 3h before immunostaining with the indicated antibodies. Representative images (e) and quantification of cilia length based on ARL13B (f) are shown. Scale bar: 2µm. Three biological replicates, n=100 cilia per sample and repetition. Data shown include mean ±s.d.; P values are calculated by two-tailed unpaired student t-test.


Figure 3a , Images show the localisation of endogeneous SEPT2 and SEPT7 in RPE1 ARLB13B - mRuby2 cells with basal body staining . The insets to the right show enlargements of the cilium . Scale bar : 4µm ( large ) and 2µm ( small ) . b , STED microscopy images showing ciliated RPE1 cells expressing EGFP - SEPT2 or SEPT7 - EGFP and stained for ARL13B . Scale bar : 1µm . d , Representative images showing co - localisation of MKS3 or CEP290 in ciliated RPE1 ARLB13B - mRuby2 and GFP - SEPT2 cells . The white arrows point to the specific MKS3 or CEP290 signals . Scale bar : 1µm . e , Line scan measurements show the normalised signal intensities along the cilium for the indicated proteins as specified in ( d ) ( dashed lines ) . f , Effect of nocodazole ( Noco ) treatment ( 3h ) on the localisation of endogenous SEPT2 and SEPT7 at cilia in RPE1 cells . The insets show magnifications of the cilium . Scale bar : 5µm ( large ) and 2 . 5µm ( small ) . g , Relative signal intensity of endogenous SEPT2 or SEPT7 along the cilium from ( f ) . SEPT2 or SEPT7 signals at cilia were normalized to the total cilia length . Three biological replicates , n > 90 cilia per sample and repetition . Data show in ( g ) includes mean ± s . d . ; P values are calculated by two - tailed unpaired student t - test . See source data .

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Figure 3a, Images show the localisation of endogeneous SEPT2 and SEPT7 in RPE1 ARLB13B-mRuby2 cells with basal body staining. The insets to the right show enlargements of the cilium. Scale bar: 4µm (large) and 2µm (small). b, STED microscopy images showing ciliated RPE1 cells expressing EGFP-SEPT2 or SEPT7-EGFP and stained for ARL13B. Scale bar: 1µm.d, Representative images showing co-localisation of MKS3 or CEP290 in ciliated RPE1 ARLB13B-mRuby2 and GFP-SEPT2 cells. The white arrows point to the specific MKS3 or CEP290 signals. Scale bar: 1µm. e, Line scan measurements show the normalised signal intensities along the cilium for the indicated proteins as specified in (d) (dashed lines).f, Effect of nocodazole (Noco) treatment (3h) on the localisation of endogenous SEPT2 and SEPT7 at cilia in RPE1 cells. The insets show magnifications of the cilium. Scale bar: 5µm (large) and 2.5µm (small). g, Relative signal intensity of endogenous SEPT2 or SEPT7 along the cilium from (f). SEPT2 or SEPT7 signals at cilia were normalized to the total cilia length. Three biological replicates, n>90 cilia per sample and repetition. Data show in (g) includes mean ±s.d.; P values are calculated by two-tailed unpaired student t-test. See source data.


Figure 4g , Electron microscopy of RPE1 WT and SEPT2 - KO cilia . Black arrows point to the axoneme and basal body ( BB ) . Scale bar : 400nm . h , Quantification of ( g ) . Only cilia detected from bottom ( BB ) to tip in serial sections were quantified . N > 15 cilia from two independent experiments . Data include mean ± s . d . ; P values are calculated by unpaired Wilcoxon - Mann - Whitney Rank Sum Testi - j , RPE1 WT and SEPT2 - KO cells after 48h SS were treated with solvent control ( DMSO ) , cytochalasin D ( CytoD ) and / or nocodazole ( Noco ) for 3h before immunostaining with the indicated antibodies . Representative images ( i ) and quantifications of PS ( Glu tub ) and whole cilia ( ARL13B ) length ( j ) are shown . The numbers above the cartoons in ( j ) show the ratio of the calculated DS / whole cilia length . Scale bar : 3µm . Three biological replicates , n = 100 cilia per sample and repetition . Data show include mean ± s . d . ; P values are calculated by two - tailed unpaired student t -

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

Figure 4g, Electron microscopy of RPE1 WT and SEPT2-KO cilia. Black arrows point to the axoneme and basal body (BB). Scale bar: 400nm. h, Quantification of (g). Only cilia detected from bottom (BB) to tip in serial sections were quantified. N>15 cilia from two independent experiments. Data include mean ±s.d.; P values are calculated by unpaired Wilcoxon-Mann-Whitney Rank Sum Testi-j, RPE1 WT and SEPT2-KO cells after 48h SS were treated with solvent control (DMSO), cytochalasin D (CytoD) and/or nocodazole (Noco) for 3h before immunostaining with the indicated antibodies. Representative images (i) and quantifications of PS (Glu tub) and whole cilia (ARL13B) length (j) are shown. The numbers above the cartoons in (j) show the ratio of the calculated DS/whole cilia length. Scale bar: 3µm. Three biological replicates, n=100 cilia per sample and repetition. Data show include mean ±s.d.; P values are calculated by two-tailed unpaired student t-test


Figure 5quantification of the relative signal intensities for the indicated proteins ( b ) . IFT88 and KIF17 signal intensities were normalized to the whole cilia length . KIF7 signal intensity was quantified at ciliary tip or base Three biological replicates , n > 80 cilia per sample and repetition . Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test . c - d , KIF7 localisation at the tip and base of cilia ( indicated by arrows ) in RPE1 cells after 48h SS treated for 3h with solvent control ( DMSO ) or nocodazole ( Noco ) and quantification of the relative signal intensities for the ciliary base and tip of KIF7 ( d ) . Scale bar : 2µm . KIF7 signal intensity was quantified at the ciliary tip or base as indicated in ( c ) ( white arrows ) . Three biological replicates , at least 80 cilia per sample and repetition . Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test . e - f , RPE1 WT and SEPT2 - KO cells were treated with control or KIF7 siRNA after 48h SS and immunostained with the indicated antibodies . Representative images ( e ) and quantifications of proximal segment ( Glu tub ) and whole cilia ( ARL13B ) length ( f ) are shown . The numbers above the cartoons in ( f ) show the ratio of the calculated distal segments / whole cilia length . Three biological replicates , n = 100 cilia per sample and repetition . Scale bar : 2µm . Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test .

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Figure 5quantification of the relative signal intensities for the indicated proteins (b). IFT88 and KIF17 signal intensities were normalized to the whole cilia length. KIF7 signal intensity was quantified at ciliary tip or base Three biological replicates, n>80 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.c-d, KIF7 localisation at the tip and base of cilia (indicated by arrows) in RPE1 cells after 48h SS treated for 3h with solvent control (DMSO) or nocodazole (Noco) and quantification of the relative signal intensities for the ciliary base and tip of KIF7 (d). Scale bar: 2µm. KIF7 signal intensity was quantified at the ciliary tip or base as indicated in (c) (white arrows). Three biological replicates, at least 80 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.e-f, RPE1 WT and SEPT2-KO cells were treated with control or KIF7 siRNA after 48h SS and immunostained with the indicated antibodies. Representative images (e) and quantifications of proximal segment (Glu tub) and whole cilia (ARL13B) length (f) are shown. The numbers above the cartoons in (f) show the ratio of the calculated distal segments /whole cilia length. Three biological replicates, n=100 cilia per sample and repetition. Scale bar: 2µm. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


Figure 6d , FRAP experiment of SSTR3 - GFP in RPE1 WT and SEPT2 KO cells . Cells were starved for 48h before the beginning of inspection and imaged every 30sec for 10mins after bleaching . Scale bar : 5µm . e , Percentage of normalised SSTR3 - GFP signal recovery at whole cilia after photobleching from ( d ) . n = 20 cilia are quantified from three biological replicates Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test . f - g , Influence of MKS3 depletion on cilia length of RPE WT and SEPT2 - KO cells . Cells are treated with the indicated siRNA and serum starved ( SS ) for 48h before immunostaining with the indicated antibodies . Representative images ( f ) and quantifications of proximal segment ( Glu tub ) and whole cilia ( ARL13B ) length ( g ) are shown . The numbers above the cartoons in ( g ) show the ratio of the calculated distal segments / whole cilia length . Scale bar : 2µm . Three 3 biological replicates , n = 100 cilia per sample and repetition . Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test . h - i , A functional TZ is required for KIF7 cilia tip localisation in RPE1 cells . KIF7 signal intensities are measured in ciliated RPE1 cells treated with control or MKS3 siRNA and 48h SS . Representative images ( h ) and quantification of KIF7 signal intensity at the base and tip of cilia ( h ) are shown . Scale bar : 2µm . Three biological replicates , at least 60 cilia per sample and repetition . Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test .

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

Figure 6d, FRAP experiment of SSTR3-GFP in RPE1 WT and SEPT2 KO cells. Cells were starved for 48h before the beginning of inspection and imaged every 30sec for 10mins after bleaching. Scale bar: 5µm. e, Percentage of normalised SSTR3-GFP signal recovery at whole cilia after photobleching from (d). n=20 cilia are quantified from three biological replicates Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.f-g, Influence of MKS3 depletion on cilia length of RPE WT and SEPT2-KO cells. Cells are treated with the indicated siRNA and serum starved (SS) for 48h before immunostaining with the indicated antibodies. Representative images (f) and quantifications of proximal segment (Glu tub) and whole cilia (ARL13B) length (g) are shown. The numbers above the cartoons in (g) show the ratio of the calculated distal segments/whole cilia length. Scale bar: 2µm. Three 3 biological replicates, n=100 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.h-i, A functional TZ is required for KIF7 cilia tip localisation in RPE1 cells. KIF7 signal intensities are measured in ciliated RPE1 cells treated with control or MKS3 siRNA and 48h SS. Representative images (h) and quantification of KIF7 signal intensity at the base and tip of cilia (h) are shown. Scale bar: 2µm. Three biological replicates, at least 60 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


Figure 7a , Time - lapse images of RPE1 WT and SEPT2 - KO cells stably expressing ARL13B - GFP and γ - tubulin - mRuby2 . Cells are starved for 32h before the beginning of inspection and imaged every 15min for 9 . 5h . Scale bar : 5µm . b , Quantification of ( a ) showing changes in cilia length during time - lapse imaging . Three biological replicates , 10 cilia per sample and repetition . c , Time - lapse images show ectocytosis events observed in RPE1 SEPT2 - KO cells . Experiments are done as in ( a ) with exception that cells are imaged every 15min . Scale bar : 2 . 5µm . g - h , RPE1 SEPT2 - KO cells expressing ARL13B - mRuby2 and Neongreen - EFHC1 after 48h SS and immunostained with Glu - tub antibody . Representative images ( g ) and quantifications of proximal segment ( Glu tub ) , EFHC1 and whole cilia ( ARL13B ) length ( h ) are shown . Three biological replicates , n = 100 cilia per sample and repetition . Scale bar : 2µm .

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

Figure 7a, Time-lapse images of RPE1 WT and SEPT2-KO cells stably expressing ARL13B-GFP and γ-tubulin-mRuby2. Cells are starved for 32h before the beginning of inspection and imaged every 15min for 9.5h. Scale bar: 5µm. b, Quantification of (a) showing changes in cilia length during time-lapse imaging. Three biological replicates, 10 cilia per sample and repetition. c, Time-lapse images show ectocytosis events observed in RPE1 SEPT2-KO cells. Experiments are done as in (a) with exception that cells are imaged every 15min. Scale bar: 2.5µm.g-h, RPE1 SEPT2-KO cells expressing ARL13B-mRuby2 and Neongreen-EFHC1 after 48h SS and immunostained with Glu-tub antibody. Representative images (g) and quantifications of proximal segment (Glu tub), EFHC1 and whole cilia (ARL13B) length (h) are shown. Three biological replicates, n=100 cilia per sample and repetition. Scale bar: 2µm.


Figure 1B HeLa cells were infected with x - light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , and then samples were labelled with antibody for SEPT7 . The scale bar represents 5 μm . Graph represents mean % ± SEM of Shigella responding to IPTG outside ( - ) or inside ( + ) SEPT7 cages from 4 independent experiments . Student ' s t - test , * * * = p < 0 . 001 . C HeLa cells were infected with x - light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , and then samples were labelled with antibody for p62 . The scale bar represents 5 μm . Graph represents mean % ± SEM of Shigella responding to IPTG without ( - ) or with ( + ) p62 from at least 4 independent experiments . Student ' s t - test , * * * = p < 0 . 001 . D Control ( CTRL ) or isopropanol - treated x - light Shigella GFP were induced with IPTG for 30 min , then labelled with SYTOX Orange for 10 min . The scale bar represents 5 μm . E HeLa cells were infected for 1 h 30 min , treated with ethanol ( CTRL ) or erythromycin ( EM ) for 2 h prior to fixation , and then labelled with antibody for SEPT7 . Graph represents mean % ± SEM of Shigella in SEPT7 cages from 3 independent experiments . Student ' s t - test , * * * = p < 0 . 001 .

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Figure 1B HeLa cells were infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for SEPT7. The scale bar represents 5 μm. Graph represents mean % ± SEM of Shigella responding to IPTG outside (-) or inside (+) SEPT7 cages from 4 independent experiments. Student's t-test, *** = p<0.001.C HeLa cells were infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for p62. The scale bar represents 5 μm. Graph represents mean % ± SEM of Shigella responding to IPTG without (-) or with (+) p62 from at least 4 independent experiments. Student's t-test, *** = p<0.001.D Control (CTRL) or isopropanol-treated x-light Shigella GFP were induced with IPTG for 30 min, then labelled with SYTOX Orange for 10 min. The scale bar represents 5 μm.E HeLa cells were infected for 1 h 30 min, treated with ethanol (CTRL) or erythromycin (EM) for 2 h prior to fixation, and then labelled with antibody for SEPT7. Graph represents mean % ± SEM of Shigella in SEPT7 cages from 3 independent experiments. Student's t-test, *** = p<0.001.


Figure 2B HeLa cells stably expressing SEPT6 - GFP were infected with S . flexneri AfaI for 4 h , harvested , and then tested with Co - IP . Empty vector and / or uninfected HeLa cells were used in parallel as control . GFP - trap magnetic agarose beads were used to isolate SEPT6 - GFP from cells , and extracts were immunoblotted for SEPT6 , GFP , SEPT7 , or p62 . The lysates were immunoblotted for GAPDH as control for cellular protein levels .

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Figure 2B HeLa cells stably expressing SEPT6-GFP were infected with S. flexneri AfaI for 4 h, harvested, and then tested with Co-IP. Empty vector and/or uninfected HeLa cells were used in parallel as control. GFP-trap magnetic agarose beads were used to isolate SEPT6-GFP from cells, and extracts were immunoblotted for SEPT6, GFP, SEPT7, or p62. The lysates were immunoblotted for GAPDH as control for cellular protein levels.


Figure 3A HeLa cells were labelled with MitoTracker Red CMXRos , treated with cytochalasin D for 30 min , then fixed and labelled for endogenous SEPT7 for confocal microscopy . The scale bar represents 0 . 5 μm . See also Fig EV3A . B HeLa cells were infected with S . flexneri for 4 h 40 min , labelled with MitoTracker Red CMXRos , fixed and labelled with antibody for endogenous SEPT7 for quantitative confocal microscopy . The scale bar represents 1 μm . C HeLa cells stably expressing SEPT6 - GFP were transfected with Mito - BFP for 24 h , infected with Shigella - mCherry for 4 h 40 min , and processed for CLEM . SEPT6 is shown in green , mitochondria in red , and Shigella - mCherry in blue . Septin cages identified by fluorescent light microscopy ( FLM ) were processed for TEM . The right - most image is enlarged from the boxed region in the TEM image , and shows the bacterium ( Sf ) surrounded by a double membrane of autophagy ( Au ) and the mitochondrial membrane ( Mt ) . The scale bar represents 1 μm . See also Fig EV3B . D HeLa cells stably expressing SEPT6 - GFP were transfected with Mito - BFP for 24 h , then infected with Shigella - mCherry for 2 h for live confocal microscopy . Time frame sequence shows a septin cage assembling around Shigella . Mitochondria ( circle ) support sites of septin ring assembly around the bacterium . Each frame was acquired every 2 min . The scale bar represents 1 μm . See also Movie EV1 . E HeLa cells were treated with control ( CTRL ) , Drp1 , or Mfn1 siRNA for 72 h . Whole cell lysates were immunoblotted for Drp1 or Mfn1 to show the efficiency of depletion . GAPDH was used as loading control . siRNA - treated cells were infected with S . flexneri for 4 h 40 min , then fixed for microscopy and stained for endogenous SEPT7 for quantitative confocal microscopy . Graph represents the mean % ± SEM of Shigella inside SEPT7 cages from at least 4 independent experiments per treatment . Student ' s t - test , * * = p < 0 . 01 ; * * * = p < 0 . 001 .

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Figure 3A HeLa cells were labelled with MitoTracker Red CMXRos, treated with cytochalasin D for 30 min, then fixed and labelled for endogenous SEPT7 for confocal microscopy. The scale bar represents 0.5 μm. See also Fig EV3A.B HeLa cells were infected with S. flexneri for 4 h 40 min, labelled with MitoTracker Red CMXRos, fixed and labelled with antibody for endogenous SEPT7 for quantitative confocal microscopy. The scale bar represents 1 μm.C HeLa cells stably expressing SEPT6-GFP were transfected with Mito-BFP for 24 h, infected with Shigella-mCherry for 4 h 40 min, and processed for CLEM. SEPT6 is shown in green, mitochondria in red, and Shigella-mCherry in blue. Septin cages identified by fluorescent light microscopy (FLM) were processed for TEM. The right-most image is enlarged from the boxed region in the TEM image, and shows the bacterium (Sf) surrounded by a double membrane of autophagy (Au) and the mitochondrial membrane (Mt). The scale bar represents 1 μm. See also Fig EV3B.D HeLa cells stably expressing SEPT6-GFP were transfected with Mito-BFP for 24 h, then infected with Shigella-mCherry for 2 h for live confocal microscopy. Time frame sequence shows a septin cage assembling around Shigella. Mitochondria (circle) support sites of septin ring assembly around the bacterium. Each frame was acquired every 2 min. The scale bar represents 1 μm. See also Movie EV1.E HeLa cells were treated with control (CTRL), Drp1, or Mfn1 siRNA for 72 h. Whole cell lysates were immunoblotted for Drp1 or Mfn1 to show the efficiency of depletion. GAPDH was used as loading control. siRNA-treated cells were infected with S. flexneri for 4 h 40 min, then fixed for microscopy and stained for endogenous SEPT7 for quantitative confocal microscopy. Graph represents the mean % ± SEM of Shigella inside SEPT7 cages from at least 4 independent experiments per treatment. Student's t-test, ** = p <0.01; *** = p<0.001.


Figure 4A HeLa cells expressing SEPT6 - GFP were stained with MitoTracker Red CMXRos , and fixed for confocal microscopy . Inset image highlights location of septins at sites of mitochondrial fission . The scale bar represents 5 μm . B HeLa cells were fixed and labelled with antibody against SEPT7 and secondary antibody coupled to HRP , and processed for TEM . The right image is enlarged from the boxed region in the TEM image , and shows a SEPT7 ( S7 ) filament ( labelled in black ) intersecting with the mitochondrial ( Mt ) fission site . The scale bar represents 100 nm . See also Fig EV4 . C HeLa cells expressing SEPT6 - GFP were stained with MitoTracker Red CMXRos for live SOFI - based super resolution microscopy . Each image was reconstructed from 100 raw TIRF frames using a custom made SOFI - based algorithm . Arrows highlight SEPT6 association with location of mitochondrial fission . The scale bar represents 1 µm . See also Movie EV3 . D HeLa cells expressing SEPT6 - GFP were labelled with MitoTracker Red CMXRos , fixed for widefield immunofluorescence microscopy , and labelled with antibodies to Drp1 . Arrows highlight one example of Drp1 and SEPT6 association with location of mitochondrial fission . The scale bar represents 1 μm . E HeLa cells were transfected with Mito - BFP and ActA - SEPT7 - mCherry , fixed and labelled with antibody to Drp1 for widefield immunofluorescence microscopy . The scale bar represents 5 μm . Inset images highlight Drp1 and SEPT7 associated with sites of mitochondrial fission . Pearson ' s correlation coefficient from 3 independent experiments ( mean ± SEM ) shows that mitochondria in ActA - SEPT7 transfected cells recruit significantly more Drp1 than control cells ( Mito - BFP transfected alone ) . Student ' s t - test , * = p < 0 . 05 .

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Figure 4A HeLa cells expressing SEPT6-GFP were stained with MitoTracker Red CMXRos, and fixed for confocal microscopy. Inset image highlights location of septins at sites of mitochondrial fission. The scale bar represents 5 μm.B HeLa cells were fixed and labelled with antibody against SEPT7 and secondary antibody coupled to HRP, and processed for TEM. The right image is enlarged from the boxed region in the TEM image, and shows a SEPT7 (S7) filament (labelled in black) intersecting with the mitochondrial (Mt) fission site. The scale bar represents 100 nm. See also Fig EV4.C HeLa cells expressing SEPT6-GFP were stained with MitoTracker Red CMXRos for live SOFI-based super resolution microscopy. Each image was reconstructed from 100 raw TIRF frames using a custom made SOFI-based algorithm. Arrows highlight SEPT6 association with location of mitochondrial fission. The scale bar represents 1 µm. See also Movie EV3.D HeLa cells expressing SEPT6-GFP were labelled with MitoTracker Red CMXRos, fixed for widefield immunofluorescence microscopy, and labelled with antibodies to Drp1. Arrows highlight one example of Drp1 and SEPT6 association with location of mitochondrial fission. The scale bar represents 1 μm.E HeLa cells were transfected with Mito-BFP and ActA-SEPT7-mCherry, fixed and labelled with antibody to Drp1 for widefield immunofluorescence microscopy. The scale bar represents 5 μm. Inset images highlight Drp1 and SEPT7 associated with sites of mitochondrial fission. Pearson's correlation coefficient from 3 independent experiments (mean ± SEM) shows that mitochondria in ActA-SEPT7 transfected cells recruit significantly more Drp1 than control cells (Mito-BFP transfected alone). Student's t-test, * = p<0.05.


Figure 5A HeLa cells were treated with control ( CTRL ) , SEPT7 , or Drp1 siRNA for 72 h . Whole cell lysate of siRNA - treated cells were immunoblotted for GAPDH , SEPT7 , or Drp1 to show the efficiency of siRNA depletion . GAPDH was used as loading control . siRNA - treated cells were labelled with MitoTracker Red CMXRos , and fixed for confocal microscopy . The scale bar represents 5 μm . B HeLa cells were treated with control ( CTRL ) , SEPT2 , SEPT7 , SEPT9 , Drp1 , or SEPT7 + Drp1 siRNA for 72 h , labelled with MitoTracker Red CMXRos , and fixed for quantitative confocal microscopy . Graph represents the distribution in length ( μm ; whiskers from min to max ) of mitochondria in siRNA - treated cells from 3 independent experiments ( analysis of at least 100 measurements per biological replicate ) . Student ' s t - test , * * * = p < 0 . 001 . C HeLa cells stably expressing SEPT6 - GFP were harvested , and then tested with Co - IP . HeLa cells expressing GFP were used in parallel as control . GFP - trap magnetic agarose beads were used to isolate SEPT6 - GFP from cells , and extracts were immunoblotted for Drp1 , SEPT2 , SEPT7 , or GFP . The lysates were immunoblotted for GAPDH as control for cellular protein levels . D HeLa cells were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , and whole cell lysates were immunoblotted for SEPT7 , Drp1 , phosphorylated Drp1 ( P - Drp1 ) , or Mfn1 . GAPDH was used as loading control . Graph represents the mean % ± SEM of the relative amount of Drp1 , P - Drp1 , Mfn1 , or SEPT7 proteins quantified by densitometry from 4 independent experiments . Student ' s t - test , ns = non - significant ; * * = p < 0 . 01 , * * * = p < 0 . 001 . E HeLa cells were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , labelled with MitoTracker Red CMXRos , then fixed and labelled for endogenous P - Drp1 for quantitative confocal microscopy . Representative images shown here . The scale bar represents 1 μm . The recruitment of P - Drp1 to mitochondria was significantly decreased as compared to control cells as measured by Pearson ' s correlation coefficient from at least 5 independent experiments ( mean ± SEM , analysis of at least 250 cells per biological replicate ) . Student ' s t - test , * = p < 0 . 05 . F HeLa cells were treated with control ( CTRL ) or SEPT7 siRNA for 72 h , labelled with MitoTracker Red CMXRos , then fixed and labelled for endogenous Mfn1 for quantitative confocal microscopy . Representative images shown here . The scale bar represents 1 μm . The recruitment of Mfn1 to mitochondria was significantly increased as compared to control cells as measured by Pearson ' s correlation coefficient from at least 5 independent experiments ( mean ± SEM , analysis of at least 250 cells per biological replicate ) . Student ' s t - test , * = p < 0 . 05 .

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

Figure 5A HeLa cells were treated with control (CTRL), SEPT7, or Drp1 siRNA for 72 h. Whole cell lysate of siRNA-treated cells were immunoblotted for GAPDH, SEPT7, or Drp1 to show the efficiency of siRNA depletion. GAPDH was used as loading control. siRNA-treated cells were labelled with MitoTracker Red CMXRos, and fixed for confocal microscopy. The scale bar represents 5 μm.B HeLa cells were treated with control (CTRL), SEPT2, SEPT7, SEPT9, Drp1, or SEPT7+ Drp1 siRNA for 72 h, labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Graph represents the distribution in length (μm; whiskers from min to max) of mitochondria in siRNA-treated cells from 3 independent experiments (analysis of at least 100 measurements per biological replicate). Student's t-test, *** = p<0.001.C HeLa cells stably expressing SEPT6-GFP were harvested, and then tested with Co-IP. HeLa cells expressing GFP were used in parallel as control. GFP-trap magnetic agarose beads were used to isolate SEPT6-GFP from cells, and extracts were immunoblotted for Drp1, SEPT2, SEPT7, or GFP. The lysates were immunoblotted for GAPDH as control for cellular protein levels.D HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, and whole cell lysates were immunoblotted for SEPT7, Drp1, phosphorylated Drp1 (P-Drp1), or Mfn1. GAPDH was used as loading control. Graph represents the mean % ± SEM of the relative amount of Drp1, P-Drp1, Mfn1, or SEPT7 proteins quantified by densitometry from 4 independent experiments. Student's t-test, ns = non-significant; ** = p<0.01, *** = p<0.001.E HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, labelled with MitoTracker Red CMXRos, then fixed and labelled for endogenous P-Drp1 for quantitative confocal microscopy. Representative images shown here. The scale bar represents 1 μm. The recruitment of P-Drp1 to mitochondria was significantly decreased as compared to control cells as measured by Pearson's correlation coefficient from at least 5 independent experiments (mean ± SEM, analysis of at least 250 cells per biological replicate). Student's t-test, * = p<0.05.F HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, labelled with MitoTracker Red CMXRos, then fixed and labelled for endogenous Mfn1 for quantitative confocal microscopy. Representative images shown here. The scale bar represents 1 μm. The recruitment of Mfn1 to mitochondria was significantly increased as compared to control cells as measured by Pearson's correlation coefficient from at least 5 independent experiments (mean ± SEM, analysis of at least 250 cells per biological replicate). Student's t-test, * = p<0.05.


Figure 6A HeLa cells stably expressing SEPT6 - GFP were treated with Drp1 siRNA for 72 h , transfected with mito - BFP 24 h , and then infected with S . flexneri - mCherry for 2 h for live confocal microscopy . Representative image shows interplay between two Shigella - septin cages and fused mitochondria . The scale bar represents 1 μm . See also Movie EV3 . B HeLa cells were treated with Drp1 siRNA for 72 h , and then infected with x - light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy . IPTG was added 30 min prior to fixation , and then samples were labelled with antibody for SEPT7 . Graph represents mean % ± SEM of Shigella responding to IPTG outside ( - ) or inside ( + ) SEPT7 cages from 3 independent experiments . Student ' s t - test , * * * = p < 0 . 001 . C HeLa cells were infected with S . flexneri M90T for 4 h 40 min , labelled with MitoTracker Red CMXRos , and fixed for quantitative confocal microscopy . Boxplots show the length of mitochondrial ( μm ; whiskers from min to max ) surrounding Shigella outside ( - ) or inside ( + ) SEPT7 cages from 3 independent experiments ( analysis of at least 130 measurements per biological replicate ) . Student ' s t - test ; * * * = p < 0 . 001 . D HeLa cells were either kept uninfected as a control or infected with S . flexneri M90T or S . flexneri ΔIcsA for 4 h 40 min . Samples were labelled with MitoTracker Red CMXRos , and fixed for quantitative confocal microscopy . Boxplots show length of mitochondria ( μm ; whiskers from min to max ) in uninfected cells ( CTRL ) , surrounding S . flexneri M90T ( + M90T ) or S . flexneri ΔIcsA ( + ΔIcsA ) from 3 independent experiments ( analysis of at least 250 measurements per biological replicate ) . Student ' s t - test ; * * * = p < 0 . 001 .

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Figure 6A HeLa cells stably expressing SEPT6-GFP were treated with Drp1 siRNA for 72 h, transfected with mito-BFP 24 h, and then infected with S. flexneri-mCherry for 2 h for live confocal microscopy. Representative image shows interplay between two Shigella-septin cages and fused mitochondria. The scale bar represents 1 μm. See also Movie EV3.B HeLa cells were treated with Drp1 siRNA for 72 h, and then infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for SEPT7. Graph represents mean % ± SEM of Shigella responding to IPTG outside (-) or inside (+) SEPT7 cages from 3 independent experiments. Student's t-test, *** = p<0.001.C HeLa cells were infected with S. flexneri M90T for 4 h 40 min, labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Boxplots show the length of mitochondrial (μm; whiskers from min to max) surrounding Shigella outside (-) or inside (+) SEPT7 cages from 3 independent experiments (analysis of at least 130 measurements per biological replicate). Student's t-test; *** = p<0.001.D HeLa cells were either kept uninfected as a control or infected with S. flexneri M90T or S. flexneri ΔIcsA for 4 h 40 min. Samples were labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Boxplots show length of mitochondria (μm; whiskers from min to max) in uninfected cells (CTRL), surrounding S. flexneri M90T (+M90T) or S. flexneri ΔIcsA (+ΔIcsA) from 3 independent experiments (analysis of at least 250 measurements per biological replicate). Student's t-test; *** = p<0.001.


Figure 1LC - MS / MS measurements of m6A levels in total RNA ( B ) or in poly ( A ) + RNA ( C ) upon KD of predicted methyltransferases in Drosophila S2R + cells . m6A abundance in total RNA is significantly reduced when Mettl5 is depleted , while its depletion has no effect on m6A level in mRNA . As expected , the KD of Mettl3 and Mettl14 reduce m6A levels in mRNA . Bar chart represents the mean ± standard deviation of three technical measurements from three biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( two - tailed t - test ) .

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Figure 1LC-MS/MS measurements of m6A levels in total RNA (B) or in poly(A)+ RNA (C) upon KD of predicted methyltransferases in Drosophila S2R+ cells. m6A abundance in total RNA is significantly reduced when Mettl5 is depleted, while its depletion has no effect on m6A level in mRNA. As expected, the KD of Mettl3 and Mettl14 reduce m6A levels in mRNA. Bar chart represents the mean ± standard deviation of three technical measurements from three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t-test).


Figure 2Subcellular localization of Mettl5 - FLAG ( purple ) and Trmt112 - eGFP ( green ) expressed under the control of their own promoter in S2R + cells . 63X magnified merge of immunofluorescently - labelled S2R + cells . Scale bar : 4 . 47 μm . Developmental expression of Mettl5 transcript assayed by RT - qPCR analysis . The figure shows mean ± standard deviation of three technical measurements from three biological replicates . In situ hybridization of Mettl5 transcript at different embryonic stages . The central nervous system ( CNS ) is highlighted in the schematics . Data retrieved from FlyExpress 7 . ( http : / / www . flyexpress . net / search . php ? type = image & search = FBgn0036856 ) .

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Figure 2Subcellular localization of Mettl5-FLAG (purple) and Trmt112-eGFP (green) expressed under the control of their own promoter in S2R+ cells. 63X magnified merge of immunofluorescently-labelled S2R+ cells. Scale bar: 4.47 μm.Developmental expression of Mettl5 transcript assayed by RT-qPCR analysis. The figure shows mean ± standard deviation of three technical measurements from three biological replicates.In situ hybridization of Mettl5 transcript at different embryonic stages. The central nervous system (CNS) is highlighted in the schematics. Data retrieved from FlyExpress 7. (http://www.flyexpress.net/search.php?type=image&search=FBgn0036856).


Figure 3LC - MS / MS measurements of m6A levels in total RNA of WT and Mettl5 mutant flies . Bars represent mean ± standard deviation of measurements of three biological replicates . * * * P < 0 . 001 ( two - tailed t - test ) . Purified 18S rRNA analyzed for its m6A content by quantitative HPLC . The 18S rRNA was extracted from 40S subunits isolated on sucrose gradients . The calibration control is a commercial source of m6A ( in grey ) . m6A elutes at 48 min . Mature rRNA analysis on ethidium - stained denaturing agarose gels . The same amounts of total RNA extracted from the indicated flies , and from S2R + cells depleted or not of Mettl5 , were loaded . The 28S / 18S ratio was established by densitometry . Analysis of 18S rRNA maturation in WT and mutant flies ( Mettl5fs ) . Total RNA extracted from the indicated animals was resolved on denaturing agarose gels and processed for northern blotting with specific probes ( complementary to ITS1 or ITS2 sequences ) . The pre - rRNAs detected accumulate to normal levels indicating that processing is unaffected .

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Figure 3LC-MS/MS measurements of m6A levels in total RNA of WT and Mettl5 mutant flies. Bars represent mean ± standard deviation of measurements of three biological replicates. ***P<0.001 (two-tailed t-test).Purified 18S rRNA analyzed for its m6A content by quantitative HPLC. The 18S rRNA was extracted from 40S subunits isolated on sucrose gradients. The calibration control is a commercial source of m6A (in grey). m6A elutes at 48 min.Mature rRNA analysis on ethidium-stained denaturing agarose gels. The same amounts of total RNA extracted from the indicated flies, and from S2R+ cells depleted or not of Mettl5, were loaded. The 28S/18S ratio was established by densitometry.Analysis of 18S rRNA maturation in WT and mutant flies (Mettl5fs). Total RNA extracted from the indicated animals was resolved on denaturing agarose gels and processed for northern blotting with specific probes (complementary to ITS1 or ITS2 sequences). The pre-rRNAs detected accumulate to normal levels indicating that processing is unaffected.


Figure 4Western blot validation of the co - immunoprecipitated proteins Mettl5 - FLAG - Myc and Trmt112 , bearing either a N - terminal HA tag ( top ) or a C - terminal GFP tag ( bottom ) , in S2R + cells . The arrows point at the indicated proteins . HA - Trmt12 migrates below the antibody light chains ( top ) . Trmt112 - eGFP migrates above the heavy chains indicated by an asterisk ( bottom ) . Trmt112 depletion reduces m6A levels in total RNA from Drosophila S2R + cells . Mean ± standard deviation of three technical measurements from three biological replicates . * P < 0 . 05 ( two - tailed t - test ) .

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Figure 4Western blot validation of the co-immunoprecipitated proteins Mettl5-FLAG-Myc and Trmt112, bearing either a N-terminal HA tag (top) or a C-terminal GFP tag (bottom), in S2R+ cells. The arrows point at the indicated proteins. HA-Trmt12 migrates below the antibody light chains (top). Trmt112-eGFP migrates above the heavy chains indicated by an asterisk (bottom).Trmt112 depletion reduces m6A levels in total RNA from Drosophila S2R+ cells. Mean ± standard deviation of three technical measurements from three biological replicates. *P < 0.05 (two-tailed t-test).


Figure 5Representative trajectories of WT and Mettl5 mutant flies analysed by the Buridan ' s Paradigm . The blue lines indicate when the fly stops and changes direction . Orientation evaluated with the help of the Buridan ' s paradigm for Mettl5 mutant flies . Orientation was measured as the angular deviation from the straight path needed to move from one landmark to another in the arena . Number of flies tested per genotype : 30 . Mean ± standard deviation . Shapiro - Wilk test was used to test for normal distribution in each group . Normally distributed groups were tested by t - test . Due to multiple comparison Bonferroni correction was applied . ( p < 0 . 05 = * ; p < 0 . 0001 = * * * * ) .

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Figure 5Representative trajectories of WT and Mettl5 mutant flies analysed by the Buridan's Paradigm. The blue lines indicate when the fly stops and changes direction. Orientation evaluated with the help of the Buridan's paradigm for Mettl5 mutant flies. Orientation was measured as the angular deviation from the straight path needed to move from one landmark to another in the arena. Number of flies tested per genotype: 30. Mean ± standard deviation. Shapiro-Wilk test was used to test for normal distribution in each group. Normally distributed groups were tested by t-test. Due to multiple comparison Bonferroni correction was applied. (p<0.05 = *; p<0.0001=****).


Figure 1 ( E - F ) Serum levels of T ( E ) and LH ( F ) in WT males , ARLmon / Y and AR - / Y mice at the age of 13 weeks .

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Figure 1(E-F) Serum levels of T (E) and LH (F) in WT males, ARLmon/Y and AR-/Y mice at the age of 13 weeks.


Figure 2 ( D ) RT - qPCR analyses of Rhox5 and Insl3 of testes from 13 - week old WT males , ARLmon / Y and AR - / Y . Expression levels are normalized to WT .

Figure 2(D) RT-qPCR analyses of Rhox5 and Insl3 of testes from 13-week old WT males, ARLmon/Y and AR-/Y. Expression levels are normalized to WT.


Figure 3 ( A ) Expression levels of genes involved in steroidogenesis normalized to WT . The bar graphs show means ± SEM ( data derived from RNA - seq analysis of testes , biological replicates , n = 5 , one - way ANOVA with Tukey ' s multiple comparisons test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant ) . ( B ) Serum levels of A - dione in WT males , ARLmon / Y and AR - / Y mice at the age of 13 weeks . ( D ) Reporter gene assays in Hela cells co - transfected with the mouse Insl3 promotor together with the WT or mutant mouse AR .

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Figure 3(A) Expression levels of genes involved in steroidogenesis normalized to WT. The bar graphs show means ± SEM (data derived from RNA-seq analysis of testes, biological replicates, n = 5, one-way ANOVA with Tukey's multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant).(B) Serum levels of A-dione in WT males, ARLmon/Y and AR-/Y mice at the age of 13 weeks.(D) Reporter gene assays in Hela cells co-transfected with the mouse Insl3 promotor together with the WT or mutant mouse AR.


Figure 4mRNA expression levels extracted from RNA - seq data for Fkbp5 ( D ) in kidneys used for RNA - seq . Expression levels are normalized to WT ORX . mRNA expression levels extracted from RNA - seq data for , Odc1 ( E ) and Kap ( F ) in kidneys used for RNA - seq . Expression levels are normalized to WT ORX .

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Figure 4mRNA expression levels extracted from RNA-seq data for Fkbp5 (D) in kidneys used for RNA-seq. Expression levels are normalized to WT ORX.mRNA expression levels extracted from RNA-seq data for , Odc1 (E) and Kap (F) in kidneys used for RNA-seq. Expression levels are normalized to WT ORX.