B ) Top , schematics of the HSP - 1 peptide sequence ( cyan ) aligned with the C - terminal sequence of full - length HSP - 1 ( amino acids 600 - 640 ) used in the ubiquitylation reaction . Auto - Ub of recombinant CHN - 1 was performed as indicated HSP - 1 ‑ derived peptide . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . Right , quantification of the CHN - 1 modifications ( unmodified , multi - monoubiquitylated , poly - ubiquitylated ) when CHN - 1 alone ( black ) or CHN - 1 + HSP - 1 peptide ( cyan ) . Plotted data are the mean of three technical replicates .

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

B) Top, schematics of the HSP-1 peptide sequence (cyan) aligned with the C-terminal sequence of full-length HSP-1 (amino acids 600-640) used in the ubiquitylation reaction. Auto-Ub of recombinant CHN-1 was performed as indicated HSP-1‑derived peptide. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Right, quantification of the CHN-1 modifications (unmodified, multi-monoubiquitylated, poly-ubiquitylated) when CHN-1 alone (black) or CHN-1+HSP-1 peptide (cyan). Plotted data are the mean of three technical replicates.


C ) Top , schematics of the UFD - 2 peptide sequences aligned with the C - terminal sequence of full - length UFD - 2 ( UFD - 2 amino acids 864 - 914 ) for PEPTIDE 1 ( yellow ) and PEPTIDE 2 ( cyan ) and ( UFD - 2 amino acids 648 - 678 ) for PEPTIDE 3 ( magenta ) used in the ubiquitylation reaction . Below , auto - Ub of recombinant CHN - 1 was performed as indicated in the presence of UFD - 2 ‑ derived peptides . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . Right , quantification of the CHN - 1 modifications ( unmodified , multi - monoubiquitylated , poly - ubiquitylated ) when CHN - 1 alone ( black ) , CHN - 1 + PEPTIDE 1 ( yellow ) , CHN - 1 + PEPTIDE 2 ( cyan ) , or CHN - 1 + PEPTIDE 3 ( magenta ) . Plotted data are the mean of three technical replicates .

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

C) Top, schematics of the UFD-2 peptide sequences aligned with the C-terminal sequence of full-length UFD-2 (UFD-2 amino acids 864-914) for PEPTIDE 1 (yellow) and PEPTIDE 2 (cyan) and (UFD-2 amino acids 648-678) for PEPTIDE 3 (magenta) used in the ubiquitylation reaction. Below, auto-Ub of recombinant CHN-1 was performed as indicated in the presence of UFD-2‑derived peptides. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Right, quantification of the CHN-1 modifications (unmodified, multi-monoubiquitylated, poly-ubiquitylated) when CHN-1 alone (black), CHN-1+PEPTIDE 1 (yellow), CHN-1+PEPTIDE 2 (cyan), or CHN-1+PEPTIDE 3 (magenta). Plotted data are the mean of three technical replicates.


D ) Auto - Ub of recombinant CHN - 1 was performed as indicated in the presence of recombinant HSP - 1 or HSP - 1EEYD . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies .

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

D) Auto-Ub of recombinant CHN-1 was performed as indicated in the presence of recombinant HSP-1 or HSP-1EEYD. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.


E ) Auto - Ub of recombinant CHN - 1 was performed as indicated alone or with UFD - 2 or UFD - 2EEVD . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies .

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

E) Auto-Ub of recombinant CHN-1 was performed as indicated alone or with UFD-2 or UFD-2EEVD. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.


F ) CHN - 1R230A auto - Ub was performed as indicated in the presence of recombinant UFD - 2 and HSP - 1 . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies .

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

F) CHN-1R230A auto-Ub was performed as indicated in the presence of recombinant UFD-2 and HSP-1. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.


A ) To detect CHN - 1 and UFD - 2 , indicated lysates of young adult worms were subjected to immunoblotting with anti - CHN - 1 and anti - UFD - 2 antibodies . Tubulin served as a loading control . Right , quantification of the CHN - 1 signals normalized to tubulin levels and plotted as N2 ( wild - type ; black ) , ufd - 2 ( tm1380 ) ( cyan ) , and UFD - 2P951A ( magenta ) . Plotted data are the mean of three biological replicates .

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

A) To detect CHN-1 and UFD-2, indicated lysates of young adult worms were subjected to immunoblotting with anti-CHN-1 and anti-UFD-2 antibodies. Tubulin served as a loading control. Right, quantification of the CHN-1 signals normalized to tubulin levels and plotted as N2 (wild-type; black), ufd-2(tm1380) (cyan), and UFD-2P951A (magenta). Plotted data are the mean of three biological replicates.


B ) CHN - 1 protein levels were determined in the indicated lysates of young adult worms treated with a proteasome inhibitor ( MG - 132 , 10µM ) and DUB inhibitor ( NEM , 100mM ) . Tubulin served as a loading control . Right , quantification of the CHN - 1 signals normalized to tubulin levels plotted as control ( black ) , or MG - 132 treated ( magenta ) in N2 ( wild - type ) and ufd - 2 ( tm1380 ) . Plotted data are the mean of three biological replicates .

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

B) CHN-1 protein levels were determined in the indicated lysates of young adult worms treated with a proteasome inhibitor (MG-132, 10µM) and DUB inhibitor (NEM, 100mM). Tubulin served as a loading control. Right, quantification of the CHN-1 signals normalized to tubulin levels plotted as control (black), or MG-132 treated (magenta) in N2 (wild-type) and ufd-2(tm1380). Plotted data are the mean of three biological replicates.


B ) Ubiquitylation of recombinant AHCY - 1 was performed as indicated . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - AHCY - 1 antibodies . Right , quantification of the AHCY - 1 modifications ( unmodified , oligo - monoubiquitylated , poly - ubiquitylated ) when CHN - 1 alone ( black ) , CHN - 1 - UFD - 2 ( magenta ) , or CHN - 1 - UFD - 2EEVD ( cyan ) . Plotted data are the mean of three technical replicates .

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

B) Ubiquitylation of recombinant AHCY-1 was performed as indicated. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-AHCY-1 antibodies. Right, quantification of the AHCY-1 modifications (unmodified, oligo-monoubiquitylated, poly-ubiquitylated) when CHN-1 alone (black), CHN-1-UFD-2 (magenta), or CHN-1-UFD-2EEVD (cyan). Plotted data are the mean of three technical replicates.


C ) Protein level of endogenous AHCY - 1 in N2 ( wild - type ) , chn - 1 ( by155 ) , CHN - 1 OE , and ufd - 2 ( tm1380 ) young adult worms treated with the proteasome inhibitor ( MG - 132 , 10 µM ) and DUB inhibitor ( NEM , 100 mM ) . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - AHCY - 1 antibodies . Tubulin served as a loading control . Right , quantification of the modified AHCY - 1 signals plotted as Ub - modified AHCY - 1 species normalized to unmodified endogenous AHCY - 1 signal and plotted for N2 ( wild - type ; black ) , chn - 1 ( by155 ) ( magenta ) , CHN - 1 OE ( yellow ) , and ufd - 2 ( tm1380 ) ( cyan ) . Plotted data are the mean of three biological replicates .

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

C) Protein level of endogenous AHCY-1 in N2 (wild-type), chn-1(by155), CHN-1 OE, and ufd-2(tm1380) young adult worms treated with the proteasome inhibitor (MG-132, 10 µM) and DUB inhibitor (NEM, 100 mM). Protein samples were resolved via SDS-PAGE and immunoblotted with anti-AHCY-1 antibodies. Tubulin served as a loading control. Right, quantification of the modified AHCY-1 signals plotted as Ub-modified AHCY-1 species normalized to unmodified endogenous AHCY-1 signal and plotted for N2 (wild-type; black), chn-1(by155) (magenta), CHN-1 OE (yellow), and ufd-2(tm1380) (cyan). Plotted data are the mean of three biological replicates.


D ) Representative images of GFP : : AHCY - 1 fluorescence in chn - 1 ( by155 ) , ufd - 2 ( tm1380 ) , and CHN - 1 OE background . Scale bar = 200µm . Below , quantification of the AHCY - 1 GFP signal plotted as fluorescence intensity for GFP : : AHCY - 1 expressing worms ( control ; black ) , chn - 1 ( by155 ) ( magenta ) , ufd - 2 ( tm1380 ) ( cyan ) or CHN - 1 OE ( yellow ) . Plotted data are the mean of three biological replicates .

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

D) Representative images of GFP::AHCY-1 fluorescence in chn-1(by155), ufd-2(tm1380), and CHN-1 OE background. Scale bar = 200µm. Below, quantification of the AHCY-1 GFP signal plotted as fluorescence intensity for GFP::AHCY-1 expressing worms (control; black), chn-1(by155) (magenta), ufd-2(tm1380) (cyan) or CHN-1 OE (yellow). Plotted data are the mean of three biological replicates.


a , Protein extracts from drpr null ( w ; drpr ∆ 5 / drpr ∆ 5 ) pupae at puparium formation ( 0 h ) and wild - type ( Canton - S ) salivary glands6 h , 12 h and 14 h after puparium formation , analysed by western blotting with anti - Drpr antibody .

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

a, Protein extracts from drpr null (w; drpr∆5/drpr∆5) pupae at puparium formation (0 h) and wild-type (Canton-S) salivary glands6 h, 12 h and 14 h after puparium formation, analysed by western blotting with anti-Drpr antibody.


b , Samples from control animals ( + / w ; + / drpr ∆ 5 ) , n = 12 , and drpr null mutants ( w ; drpr ∆ 5 / drpr ∆ 5 ) , n = 47 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation . c , Quantification of data from b and d . d , Samples from control animals ( + / w ; + / UAS - drprIR ) , n = 11 , and those with salivary gland - specific knockdown of drpr ( fkh - Gal4 / w ; UAS - drprIR / + ) , n = 19 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation .

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

b, Samples from control animals (+/w; +/drpr∆5), n = 12, and drpr null mutants (w; drpr∆5/drpr∆5), n = 47, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. c, Quantification of data from b and d. d, Samples from control animals (+/w; +/UAS-drprIR), n = 11, and those with salivary gland-specific knockdown of drpr (fkh-Gal4/w; UAS-drprIR/+), n = 19, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation.


e , Control animals ( + / w ; + / UAS - drpr - IIR ) , n = 9 , and those with salivary gland - specific knockdown of drpr - I ( fkh - Gal4 / w ; UAS - drpr - IIR / + ) , n = 20 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation . f , Quantification of data from e .

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

e, Control animals (+/w; +/UAS-drpr-IIR), n = 9, and those with salivary gland-specific knockdown of drpr-I (fkh-Gal4/w; UAS-drpr-IIR/+), n = 20, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. f, Quantification of data from e.


g , Samples from drpr null animals ( + / w ; + / UAS - Drpr - I ; drpr ∆ 5 / drpr ∆ 5 ) , n = 9 , and those with salivary gland - specific expression of Drpr - I ( fkh - Gal4 / w ; UAS - Drpr - I / + ; drpr ∆ 5 / drpr ∆ 5 ) , n = 20 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation . h , Quantification of data from g .

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

g, Samples from drpr null animals (+/w; +/UAS-Drpr-I; drpr∆5/drpr∆5), n = 9, and those with salivary gland-specific expression of Drpr-I (fkh-Gal4/w; UAS-Drpr-I/+; drpr∆5/drpr∆5), n = 20, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. h, Quantification of data from g.


a , Samples from animals with salivary gland - specific expression of p35 ( fkh - Gal4 / + ; UAS - p35 / + ) , n = 18 ( left ) , drpr null animals ( + / w ; + / UAS - p35 ; drpr ∆ 5 / drpr ∆ 5 ) , n = 10 ( middle ) , and drpr null animals with salivary gland - specific expression of p35 ( fkh - Gal4 / w ; UAS - p35 / + ; drpr ∆ 5 / drpr ∆ 5 ) , n = 16 ( right ) , analysed by histology for the presence of salivary gland material 24 h after puparium formation . Red circles , cell fragments ; yellow circle , gland fragments .

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

a, Samples from animals with salivary gland-specific expression of p35 (fkh-Gal4/+; UAS-p35/+), n = 18 (left), drpr null animals (+/w; +/UAS-p35; drpr∆5/drpr∆5), n = 10 (middle), and drpr null animals with salivary gland-specific expression of p35 (fkh-Gal4/w; UAS-p35/+; drpr∆5/drpr∆5), n = 16 (right), analysed by histology for the presence of salivary gland material 24 h after puparium formation. Red circles, cell fragments; yellow circle, gland fragments.


a , Samples from animals with salivary gland - specific expression of p35 ( fkh - Gal4 / + ; UAS - p35 / + ) , n = 18 ( left ) , drpr null animals ( + / w ; + / UAS - p35 ; drpr ∆ 5 / drpr ∆ 5 ) , n = 10 ( middle ) , and drpr null animals with salivary gland - specific expression of p35 ( fkh - Gal4 / w ; UAS - p35 / + ; drpr ∆ 5 / drpr ∆ 5 ) , n = 16 ( right ) , analysed by histology for the presence of salivary gland material 24 h after puparium formation . Red circles , cell fragments ; yellow circle , gland fragments . b , Quantification of data from a .

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

a, Samples from animals with salivary gland-specific expression of p35 (fkh-Gal4/+; UAS-p35/+), n = 18 (left), drpr null animals (+/w; +/UAS-p35; drpr∆5/drpr∆5), n = 10 (middle), and drpr null animals with salivary gland-specific expression of p35 (fkh-Gal4/w; UAS-p35/+; drpr∆5/drpr∆5), n = 16 (right), analysed by histology for the presence of salivary gland material 24 h after puparium formation. Red circles, cell fragments; yellow circle, gland fragments. b, Quantification of data from a.


c , Salivary glands dissected from animals expressing drprIR specifically in GFP - marked clone cells ( heat shock ( hs ) flp / w ; UAS - drprIR / + ; act , cd2 , FRT > Gal4 , UAS - GFP / + ) 6 h and 14 h after puparium formation . Stained with GFP antibody ( green ) , to label cells expressing drpr IR , and Lamin antibody ( red ) .

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

c, Salivary glands dissected from animals expressing drprIR specifically in GFP-marked clone cells (heat shock (hs)flp/w; UAS-drprIR/+; act,cd2, FRT > Gal4, UAS-GFP/+) 6 h and 14 h after puparium formation. Stained with GFP antibody (green), to label cells expressing drpr IR, and Lamin antibody (red).


a , Samples from animals with salivary gland - specific knockdown of Atg12 ( fkh - Gal4 / w ; UAS - Atg12IR / + ) , n = 21 , and those with salivary gland - specific knockdown of both Atg12 and drpr - I ( fkh - Gal4 / w ; UAS - Atg12IR / + ; UAS - drpr - IIR / + ) , n = 19 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation . b , Quantification of data from a .

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

a, Samples from animals with salivary gland-specific knockdown of Atg12 (fkh-Gal4/w; UAS-Atg12IR/+), n = 21, and those with salivary gland-specific knockdown of both Atg12 and drpr-I (fkh-Gal4/w; UAS-Atg12IR/+; UAS-drpr-IIR/+), n = 19, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. b, Quantification of data from a.


c , GFP - LC3 was expressed in salivary glands of control animals ( + / w ; UAS - GFP - LC3 / + ; fkh - Gal4 / + ) and those with salivary gland - specific knockdown of drpr ( w ; UAS - drprIR / UAS - GFP - LC3 ; fkh - Gal4 / + ) . Salivary glands were dissected 6 h and 14 h after puparium formation , imaged for GFP - LC3 , and LC3 puncta were quantified using Zeiss Automeasure software . d , Quantification of data from c . Error bars represent s . e . m . ; n ≥ 10 ; p 0 . 0000001 .

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

c, GFP-LC3 was expressed in salivary glands of control animals (+/w; UAS-GFP-LC3/+; fkh-Gal4/+) and those with salivary gland-specific knockdown of drpr (w; UAS-drprIR/UAS-GFP-LC3; fkh-Gal4/+). Salivary glands were dissected 6 h and 14 h after puparium formation, imaged for GFP-LC3, and LC3 puncta were quantified using Zeiss Automeasure software. d, Quantification of data from c. Error bars represent s.e.m.; n ≥ 10; p  0.0000001.


e , LAMP1 - GFP was expressed in control animals ( tub - LAMP - GFP / w ; + / fkh - Gal4 ) and those with salivary gland - specific knockdown of drpr ( tub - LAMP - GFP / w ; UAS - drprIR / + ; fkh - Gal4 / + ) . Salivary glands were dissected 14 h after puparium formation , imaged for LAMP - GFP , and LAMP puncta were quantified using Zeiss Automeasure software . f , Quantification of data from e . Error bars represent s . e . m . ; n ≥ 10 ; p 0 . 05 .

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

e, LAMP1-GFP was expressed in control animals (tub-LAMP-GFP/w; +/fkh-Gal4) and those with salivary gland-specific knockdown of drpr (tub-LAMP-GFP/w; UAS-drprIR/+; fkh-Gal4/+). Salivary glands were dissected 14 h after puparium formation, imaged for LAMP-GFP, and LAMP puncta were quantified using Zeiss Automeasure software. f, Quantification of data from e. Error bars represent s.e.m.; n ≥ 10; p  0.05.


g , Samples from drpr mutant animals ( + / w ; + / UAS - Atg16A ; drprDgr ; 5 / drprDgr ; 5 ) , n = 10 , and those with salivary gland - specific expression of Atg16A ( fkh - Gal4 / w ; UAS - Atg16A / + ; drprDgr ; 5 / drprDgr ; 5 ) , n = 16 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation . h , Quantification of data from g .

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

g, Samples from drpr mutant animals (+/w; +/UAS-Atg16A; drprDgr;5/drprDgr;5), n = 10, and those with salivary gland-specific expression of Atg16A (fkh-Gal4/w; UAS-Atg16A/+; drprDgr;5/drprDgr;5), n = 16, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. h, Quantification of data from g.


a , Protein extracts from drpr null ( w ; drprDgr ; 5 / drprDgr ; 5 ) pupae at puparium formation ( 0 h ) and from the fat bodies of wild - type ( Canton - S ) third - instar larvae were analysed by western blotting with anti - Drpr antibody . Third - instar larvae were either fed or starved for 4 h .

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

a, Protein extracts from drpr null (w; drprDgr;5/drprDgr;5) pupae at puparium formation (0 h) and from the fat bodies of wild-type (Canton-S) third-instar larvae were analysed by western blotting with anti-Drpr antibody. Third-instar larvae were either fed or starved for 4 h.


b , We either fed Wild - type ( Canton - S ) third - instar larvae or starved them for 4 h , and dissected fat bodies , stained them with anti - Drpr antibody , and imaged them for Drpr ( green ) . We stained nuclei with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) ( blue ) .

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

b, We either fed Wild-type (Canton-S) third-instar larvae or starved them for 4 h, and dissected fat bodies, stained them with anti-Drpr antibody, and imaged them for Drpr (green). We stained nuclei with 4′,6-diamidino-2-phenylindole (DAPI) (blue).


c , We starved third - instar larvae expressing GFP - Atg8a in all cells , and drprIR specifically in dsRed - marked clone cells ( hsflp / w ; UAS - drprIR / + ; hsGFPAtg8a , act , cd2 , FRT > Gal4 , UAS - dsRed / + ) , for 4 h . We dissected larval fat bodies and imaged them for GFP - Atg8a ( green ) and dsRed ( red ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, We starved third-instar larvae expressing GFP-Atg8a in all cells, and drprIR specifically in dsRed-marked clone cells (hsflp/w; UAS-drprIR/+; hsGFPAtg8a, act,cd2, FRT > Gal4, UAS-dsRed/+), for 4 h. We dissected larval fat bodies and imaged them for GFP-Atg8a (green) and dsRed (red).


d , Salivary glands of animals expressing GFP - Atg8a in all cells , and drprIR specifically in dsRed - marked clone cells ( hsflp / w ; UAS - drprIR / + ; hsGFPAtg8a , act , cd2 , FRT > Gal4 , UAS - dsRed / + ) , dissected 14 h after puparium formation . Imaged for GFP - Atg8a ( green ) and dsRed ( red ) . We stained nuclei with Hoechst ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Salivary glands of animals expressing GFP-Atg8a in all cells, and drprIR specifically in dsRed-marked clone cells (hsflp/w; UAS-drprIR/+; hsGFPAtg8a, act,cd2, FRT > Gal4, UAS-dsRed/+), dissected 14 h after puparium formation. Imaged for GFP-Atg8a (green) and dsRed (red). We stained nuclei with Hoechst (blue).


( a ) Effect of steroids on MERS - CoV replication . Vero cells were infected with MERS - CoV at MOI = 0 . 01 in the presence of steroids for 24 h . The viral titer in the cell supernatant was quantified by standard plaque assay using Vero / TMPRSS2 cells . Cell viability in the presence of steroids was quantified by WST assay at 24 hours post - infection ( hpi ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of steroids on MERS-CoV replication. Vero cells were infected with MERS-CoV at MOI = 0.01 in the presence of steroids for 24 h. The viral titer in the cell supernatant was quantified by standard plaque assay using Vero/TMPRSS2 cells. Cell viability in the presence of steroids was quantified by WST assay at 24 hours post-infection (hpi).


( b ) Ciclesonide escape mutant . Vero cells treated with ciclesonide were infected with parental MERS - CoV or ciclesonide escape mutant . Viral titer was measured as described in panel a .

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(b) Ciclesonide escape mutant. Vero cells treated with ciclesonide were infected with parental MERS-CoV or ciclesonide escape mutant. Viral titer was measured as described in panel a.


( c ) Vero cells were infected with the parental MERS - CoV / EMC strain ( Re - EMC / MERS ) or the recombinant mutant strain ( Re - Nsp15 - A25V ) with an amino acid substitution at A25V in NSP15 . Viral titer was measured as described in panel a .

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(c) Vero cells were infected with the parental MERS-CoV/EMC strain (Re-EMC/MERS) or the recombinant mutant strain (Re-Nsp15-A25V) with an amino acid substitution at A25V in NSP15. Viral titer was measured as described in panel a.


( a ) Intracellular SARS - CoV - 2 RNA ( 6 hpi ) . VeroE6 / TMPRSS2 cells were infected with SARS - CoV - 2 at MOI = 1 in the presence of 10 □ μM compounds for 6 h . Cellular viral RNA was quantified by real - time PCR using the E gene primer / probe set .

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

(a) Intracellular SARS-CoV-2 RNA (6 hpi). VeroE6/TMPRSS2 cells were infected with SARS-CoV-2 at MOI = 1 in the presence of 10□μM compounds for 6 h. Cellular viral RNA was quantified by real-time PCR using the E gene primer/probe set.


( b ) Culture Medium SARS - CoV - 2 RNA ( 27 hpi ) . VeroE6 / TMPRSS2 cells were infected with SARS - CoV - 2 at MOI = 0 . 01 in the presence of ciclesonide for 27 h . Viral RNA in culture medium was quantified by real - time PCR using the E gene primer / probe set . Cell viability in the presence of ciclesonide was quantified at 27 hpi by WST 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(b) Culture Medium SARS-CoV-2 RNA (27 hpi). VeroE6/TMPRSS2 cells were infected with SARS-CoV-2 at MOI = 0.01 in the presence of ciclesonide for 27 h. Viral RNA in culture medium was quantified by real-time PCR using the E gene primer/probe set. Cell viability in the presence of ciclesonide was quantified at 27 hpi by WST assay.


( B ) CRMP2 expression in adult cortex ( layers VI - IV ) , corpus callosum ( CC ) and hippocampal area CA1 . CRMP2 ( green ) is expressed throughout the cortex and hippocampus , CRMP2A ( red ) is particularly strongly expressed in a subset of callosal axons . Nuclei are counterstained with Hoechst 33342 . Scale bars : 50 μm .

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

(B) CRMP2 expression in adult cortex (layers VI - IV), corpus callosum (CC) and hippocampal area CA1. CRMP2 (green) is expressed throughout the cortex and hippocampus, CRMP2A (red) is particularly strongly expressed in a subset of callosal axons. Nuclei are counterstained with Hoechst 33342. Scale bars: 50 μm.


( C ) Whole - mount immunohistochemistry with anti - neurofilaments antibody for visualization of peripheral nerves . In crmp2 - / - embryos , the growth of ophthalmic branch of the trigeminal nerve is increased and axons are defasciculated ( first and second row , arrowheads ) . Similarly , we detected increased growth and branching of lateral branches of spinal nerves in crmp2 - / - embryos ( third row , arrowheads ) . Trigeminal nerve ( V ) and its branches ( Op - ophthalmic , Mx - maxillar , Md - mandibular ) are indicated . Scale bars : 500 μm .

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

(C) Whole-mount immunohistochemistry with anti-neurofilaments antibody for visualization of peripheral nerves. In crmp2-/- embryos, the growth of ophthalmic branch of the trigeminal nerve is increased and axons are defasciculated (first and second row, arrowheads). Similarly, we detected increased growth and branching of lateral branches of spinal nerves in crmp2-/- embryos (third row, arrowheads). Trigeminal nerve (V) and its branches (Op - ophthalmic, Mx - maxillar, Md - mandibular) are indicated. Scale bars: 500 μm.


( D , E ) Quantification of areas innervated by ophthalmic branches of trigeminal ganglion and spinal nerve , normalized to WT littermates ( each dot depicts one embryo ) . Area of ophthalmic branch in crmp2 - / - was increased by 66 % ( p < 0 . 05 , n = 10 , 3 litters ) . For spinal nerves , total area in knockouts was increased by 45 % ( p < 0 . 05 , WT n = 8 , crmp2 - / - n = 7 , 3 litters ) . Mean ± SD , * p < 0 . 05 , t - test .

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

(D, E) Quantification of areas innervated by ophthalmic branches of trigeminal ganglion and spinal nerve, normalized to WT littermates (each dot depicts one embryo). Area of ophthalmic branch in crmp2-/- was increased by 66% (p<0.05, n=10, 3 litters). For spinal nerves, total area in knockouts was increased by 45% (p<0.05, WT n=8, crmp2-/- n=7, 3 litters). Mean ± SD, * p<0.05, t-test.


( F ) Growth of motor neurons from E11 . 5 - E12 . 5 WT and crmp2 - / - spinal cord explants in microfluidic chambers . Excerpts from 14 hour time - lapse imaging are shown . Upon application of Sema3A into distal compartment , WT axons tend to stop or slow down their growth unlike crmp2 - / - axons . Purple lines highlight the growth path of individual axons in a distal chamber . Scale bars : 100 μm .

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

(F) Growth of motor neurons from E11.5 - E12.5 WT and crmp2-/- spinal cord explants in microfluidic chambers. Excerpts from 14 hour time-lapse imaging are shown. Upon application of Sema3A into distal compartment, WT axons tend to stop or slow down their growth unlike crmp2-/- axons. Purple lines highlight the growth path of individual axons in a distal chamber. Scale bars: 100 μm.


( G ) Quantification of the fraction of growing ( > 50 μm ) axons in one imaging field ( WT : control 76 . 8 ± 5 . 7 % , Sema3A 57 . 7 ± 10 . 6 % , p < 0 . 001 , crmp2 - / - : control 75 . 4 ± 10 . 4 % , Sema3A 75 . 4 ± 6 . 7 % , p = 0 . 99 ) , n = 3 experiments per genotype , 7 - 8 explants per condition . Mean ± SD . * * * p < 0 . 001 , 2 - way ANOVA with Bonferroni ' s multiple comparisons test .

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

(G) Quantification of the fraction of growing (>50 μm) axons in one imaging field (WT: control 76.8±5.7%, Sema3A 57.7±10.6%, p<0.001, crmp2-/-: control 75.4±10.4%, Sema3A 75.4±6.7%, p=0.99), n=3 experiments per genotype, 7-8 explants per condition. Mean ± SD. *** p<0.001, 2-way ANOVA with Bonferroni's multiple comparisons test.


( A ) CRMP2 deficiency leads to callosal hypoplasia . Shortening of corpus callosum ( arrowheads ) is apparent in both sagittal ( first row ) and coronal sections ( second row ) of adult brains . Scale bars : 1 mm .

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(A) CRMP2 deficiency leads to callosal hypoplasia. Shortening of corpus callosum (arrowheads) is apparent in both sagittal (first row) and coronal sections (second row) of adult brains. Scale bars: 1 mm.


( B ) Quantification of callosal length in 30 - days old mice ( P30 , n = 3 , crmp2 - / - is 80 . 8 ± 5 % of WT , p = 0 . 003 ) and adult mice ( n = 3 , crmp2 - / - is 80 . 5 ± 3 % of WT , p = 0 . 002 ) , mean ± SD , * p < 0 . 05 , t - test .

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

(B) Quantification of callosal length in 30-days old mice (P30, n=3, crmp2-/- is 80.8±5% of WT, p=0.003) and adult mice (n=3, crmp2-/- is 80.5±3% of WT, p=0.002), mean ± SD, * p<0.05, t-test.


( C ) Labeling of adult corpus callosum with anti - neurofilaments antibody in sagittal sections . Outline depicts missing posterior part of the tract in crmp2 - / - mice . Caudal part of the corpus callosum in WTs is located dorsally above the habenular commissure ( hbc ) , while in crmp2 - / - mice callosum terminates rostrally before reaching the hbc ( arrows ) . PC indicates posterior commissure . Scale bars : 500 µm .

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

(C) Labeling of adult corpus callosum with anti-neurofilaments antibody in sagittal sections. Outline depicts missing posterior part of the tract in crmp2-/- mice. Caudal part of the corpus callosum in WTs is located dorsally above the habenular commissure (hbc), while in crmp2-/- mice callosum terminates rostrally before reaching the hbc (arrows). PC indicates posterior commissure. Scale bars: 500 µm.


( D ) The growth of GFP - labeled callosal axons in the contralateral cortex at P6 ( embryos were electroporated at E15 . 5 ) . Note the disorganized paths of crmp2 - / - axons . Scale bars : 200 μm .

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

(D) The growth of GFP-labeled callosal axons in the contralateral cortex at P6 (embryos were electroporated at E15.5). Note the disorganized paths of crmp2-/- axons. Scale bars: 200 μm.


( F ) Quantification of tortuosity of axons ( WT n = 6 pups , crmp2 - / - n = 3 pups ) upon their exit from the callosal tract ( WT 1 . 016 ± 0 . 003 vs . crmp2 - / - 1 . 044 ± 0 . 012 , p < 0 . 001 ) , mean ± SD , * * * p < 0 . 001 , t - test .

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(F) Quantification of tortuosity of axons (WT n=6 pups, crmp2-/- n=3 pups) upon their exit from the callosal tract (WT 1.016±0.003 vs. crmp2-/- 1.044±0.012, p<0.001), mean ± SD, ***p<0.001, t-test.


( G ) DiI - labeled callosal axons from P9 oblique brain sections and their reconstruction in Neurolucida 360 ( WT n = 6 , crmp2 - / - n = 9 ) . Scale bars : 200 μm .

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(G) DiI-labeled callosal axons from P9 oblique brain sections and their reconstruction in Neurolucida 360 (WT n=6, crmp2-/- n=9). Scale bars: 200 μm.


( H ) Polar histograms of callosal axons reconstructed Note the broader range of axon growth angles in crmp2 - / - mice .

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

(H) Polar histograms of callosal axons reconstructed Note the broader range of axon growth angles in crmp2-/- mice.


( I ) Left : schematic representation of polar histogram analysis by clustering the traced axons ( WT n = 6 pups , crmp2 - / - n = 9 pups ) into 3 groups based on the growth angles . Right : proportion of axons growing in selected clusters ( WT , 0 ° - ± 20 ° : 67 . 3 ± 10 . 3 % ; ± 20 ° - ± 40 ° : 21 . 2 ± 5 . 3 % ; ± 40 ° - ± 60 ° : 6 . 22 ± 3 . 2 % ; crmp2 - / - , 58 . 5 ± 5 % , p = 0 . 047 , 27 . 7 ± 4 . 3 % , p = 0 . 022 , 8 . 64 ± 2 . 25 % , p = 0 . 11 ) , mean ± SD , * p < 0 . 05 , t - test .

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

(I) Left: schematic representation of polar histogram analysis by clustering the traced axons (WT n=6 pups, crmp2-/- n=9 pups) into 3 groups based on the growth angles. Right: proportion of axons growing in selected clusters (WT, 0°-±20°: 67.3±10.3%; ±20°-±40°: 21.2±5.3%; ±40°-±60°: 6.22±3.2%; crmp2-/-, 58.5±5%, p=0.047, 27.7±4.3%, p=0.022, 8.64±2.25%, p=0.11), mean ± SD, *p<0.05, t-test.


( A , B ) ( A ) Coronal sections of P14 brains stained for calbindin and VgluT1 . Infrapyramidal bundle ( IPB ) progresses into hippocampal CA3 region in both WT and crmp2 - / - mice . Nuclei are counterstained with Hoechst 33342 . ( B ) Details from depicted regions in ( A ) , arrowheads show the course of IPB . Scale bars : 100 μm .

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

(A, B) (A) Coronal sections of P14 brains stained for calbindin and VgluT1. Infrapyramidal bundle (IPB) progresses into hippocampal CA3 region in both WT and crmp2-/- mice. Nuclei are counterstained with Hoechst 33342. (B) Details from depicted regions in (A), arrowheads show the course of IPB. Scale bars: 100 μm.


( C , D ) ( C ) Staining for calbindin shows unpruned infrapyramidal bundle in adult crmp2 - / - mice . No VGluT2 signal in hippocampal CA3 region is present in WT and crmp2 - / - suggesting mature synapses . Scale bars : 500 μm . ( D ) Details from depicted regions in ( C ) , arrowheads show the course of IPB . Scale bars : 100 μm .

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

(C, D) (C) Staining for calbindin shows unpruned infrapyramidal bundle in adult crmp2-/- mice. No VGluT2 signal in hippocampal CA3 region is present in WT and crmp2-/- suggesting mature synapses. Scale bars: 500 μm. (D) Details from depicted regions in (C), arrowheads show the course of IPB. Scale bars: 100 μm.


( E , F ) Staining for calbindin and VgluT1 shows mature synapses in the main bundle in both genotypes and in the IPB in crmp2 - / - mice ( arrowheads ) . Scale bars : 100 μm .

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(E, F) Staining for calbindin and VgluT1 shows mature synapses in the main bundle in both genotypes and in the IPB in crmp2-/- mice (arrowheads). Scale bars: 100 μm.


( G ) Quantification of IPB index ( length of the IPB vs . main bundle , WT 0 . 43 ± 0 . 05 , crmp2 - / - 0 . 78 ± 0 . 05 , p < 0 . 001 ) counted from adult coronal sections ( WT n = 4 mice , crmp2 - / - n = 5 mice ) . Mean ± SD , * * * p < 0 . 001 , t - test .

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

(G) Quantification of IPB index (length of the IPB vs. main bundle, WT 0.43±0.05, crmp2-/- 0.78±0.05, p<0.001) counted from adult coronal sections (WT n=4 mice, crmp2-/- n=5 mice). Mean ± SD, ***p<0.001, t-test.


( A ) Upper row : DiI tracing of the visual cortex axons at P9 ( before pruning ) , sagittal sections . Branching point of the tract is shown . Lower row : visual cortex axons in adult mice after pruning period . Note significantly reduced number of axons continuing into pyramidal tract in WT ( dashed arrow ) . ( arrows - 2 branches of corticospinal visual axons ) . Maximum projections are shown . Scale bars : 200 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Upper row: DiI tracing of the visual cortex axons at P9 (before pruning), sagittal sections. Branching point of the tract is shown. Lower row: visual cortex axons in adult mice after pruning period. Note significantly reduced number of axons continuing into pyramidal tract in WT (dashed arrow). (arrows - 2 branches of corticospinal visual axons). Maximum projections are shown. Scale bars: 200 μm.


( C ) Quantification of VP ( visual pruning ) index ( fluorescence intensity of pyramidal axons after vs . before the branch point ) . In WT animals after the pruning period , only a minor part of axons descends towards the pyramidal tract ( WT : P9 0 . 65 ± 0 . 09 , adults 0 . 17 ± 0 . 04 , p = 0 . 006 ) . However , in crmp2 - / - mice , corticospinal axons are still largely present , and their VP indexes are not significantly different between adult and P9 stages ( crmp2 - / - : P9 0 . 54 ± 0 . 18 , adults 0 . 62 ± 0 . 27 , p = 0 . 24 ) . Mean ± SD , * * p < 0 . 01 , 2 - way ANOVA with Bonferroni ' s multiple comparisons test . P9 : n = 3 pups / genotype , adults : n = 5 mice / genotype )

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

(C) Quantification of VP (visual pruning) index (fluorescence intensity of pyramidal axons after vs. before the branch point). In WT animals after the pruning period, only a minor part of axons descends towards the pyramidal tract (WT: P9 0.65±0.09, adults 0.17±0.04, p=0.006). However, in crmp2-/- mice, corticospinal axons are still largely present, and their VP indexes are not significantly different between adult and P9 stages (crmp2-/-: P9 0.54±0.18, adults 0.62±0.27, p=0.24). Mean ± SD, **p<0.01, 2-way ANOVA with Bonferroni's multiple comparisons test. P9: n=3 pups/genotype, adults: n=5 mice/genotype)


( A ) Time - lapse imaging of DIV7 cultured hippocampal neurons before and after Semaphorin stimulation . Upper panel : Axon growth without Semaphorin stimulation . Middle panel : stimulation with Sema3A ( 0 hours ) ( 1nM , n = 669 axons for WT , 761 axons for knockout ) causes retraction of both WT and crmp2 - / - neurons . Lower panel : stimulation with Sema3F ( 5 nM , n = 602 axons for WT , 955 axons for knockout ) causes axon retraction in WT , but not in crmp2 - / - neurons . Red triangles depict growing axons , yellow retracting axons and blue asterisks indicate steady non - growing axons . Scale bars : 100 μm ( whole image field ) and 50 μm ( magnified ) .

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

(A) Time-lapse imaging of DIV7 cultured hippocampal neurons before and after Semaphorin stimulation. Upper panel: Axon growth without Semaphorin stimulation. Middle panel: stimulation with Sema3A (0 hours) (1nM, n=669 axons for WT, 761 axons for knockout) causes retraction of both WT and crmp2-/- neurons. Lower panel: stimulation with Sema3F (5 nM, n=602 axons for WT, 955 axons for knockout) causes axon retraction in WT, but not in crmp2-/- neurons. Red triangles depict growing axons, yellow retracting axons and blue asterisks indicate steady non-growing axons. Scale bars: 100 μm (whole image field) and 50 μm (magnified).


( C ) Quantification of retracting axons ( number of retracting vs . steady axons , 3 experiments ) . WT : control 13 . 4 ± 5 % , Sema3A 31 . 2 ± 6 % ( p < 0 . 001 ) , Sema3F 36 . 9 ± 8 . 7 % ( p < 0 . 001 ) ; crmp2 - / - : control 19 . 8 ± 1 . 8 % , Sema3A 28 . 5 ± 4 . 4 % ( p < 0 . 05 ) , Sema3F 22 . 4 ± 4 . 1 % ( p > 0 . 99 ) , mean ± SD are shown . * * * p < 0 . 001 , * p < 0 . 05 , 2 - way ANOVA with Bonferroni ' s multiple comparisons test .

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

(C) Quantification of retracting axons (number of retracting vs. steady axons, 3 experiments). WT: control 13.4±5%, Sema3A 31.2±6% (p<0.001), Sema3F 36.9±8.7% (p<0.001); crmp2-/-: control 19.8±1.8%, Sema3A 28.5±4.4% (p<0.05), Sema3F 22.4±4.1% (p>0.99), mean ± SD are shown. ***p<0.001, *p<0.05, 2-way ANOVA with Bonferroni's multiple comparisons test.


( A , B ) Dendritic spine density in DiOlistically labeled DG granule cells is similar in WT and crmp2 - / - mice at P30 . In adults , however , spine density in crmp2 - / - granule cells is increased comparing to WT . Scale bars : 5 μm . ( C ) Quantification of dendritic spine density in DG granule cells in the inner molecular layer ( 50 - 100 μm away from the soma ) . WT , P30 : 12 . 5 ± 2 . 2 spines / 10 μm , adults : 9 . 95 ± 2 . 3 ; crmp2 - / - , P30 : 12 . 86 ± 2 . 1 ( p > 0 . 99 ) , adults : 12 . 88 ± 2 . 9 ( p < 0 . 001 ) , mean ± SD , * * * p < 0 . 001 , 2 - way ANOVA with Bonferroni correction . P30 : 3 animals / genotype , WT = 61 dendrites , knockout = 64 dendrites ; adults : 3 animals / genotype , WT = 41 dendrites , knockout = 37 dendrites .

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

(A, B) Dendritic spine density in DiOlistically labeled DG granule cells is similar in WT and crmp2-/- mice at P30. In adults, however, spine density in crmp2-/- granule cells is increased comparing to WT. Scale bars: 5 μm. (C) Quantification of dendritic spine density in DG granule cells in the inner molecular layer (50 - 100 μm away from the soma). WT, P30: 12.5±2.2 spines/10 μm, adults: 9.95±2.3; crmp2-/-, P30: 12.86±2.1 (p>0.99), adults: 12.88±2.9 (p<0.001), mean ± SD, ***p<0.001, 2-way ANOVA with Bonferroni correction. P30: 3 animals/genotype, WT=61 dendrites, knockout=64 dendrites; adults: 3 animals/genotype, WT=41 dendrites, knockout=37 dendrites.


( D , E ) Analysis of branching of DiOlistically labeled DG granule cell dendrites in adult WT and crmp2 - / - mice ( n = 3 animals , ≥ 25 dendrites ) . Quantification of granule cell branching by Sholl analysis showed no significant differences ( p > 0 . 99 ) . Scale bars : 50 μm , mean ± SEM , 2 - way ANOVA with Bonferroni correction .

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

(D, E) Analysis of branching of DiOlistically labeled DG granule cell dendrites in adult WT and crmp2-/- mice (n=3 animals, ≥25 dendrites). Quantification of granule cell branching by Sholl analysis showed no significant differences (p>0.99). Scale bars: 50 μm, mean ± SEM, 2-way ANOVA with Bonferroni correction.


( F ) Defects in synapse elimination in the inner molecular layer revealed by double immunostaining with PSD95 and VGluT2 antibodies ( n = 5 mice / genotype ) . GL indicates granule cell layer , IML / OML indicate inner / outer molecular layer , respectively . Density of colocalized PSD95 / VGLuT2 puncta was counted . IML : WT 109 ± 21 , crmp2 - / - 156 ± 15 ( p = 0 . 004 ) . OML : WT 281 ± 38 , crmp2 - / - 297 ± 80 ( p = 0 . 7 ) . Scale bars : 10 μm , mean ± SD , * p < 0 . 05 , t - test .

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

(F) Defects in synapse elimination in the inner molecular layer revealed by double immunostaining with PSD95 and VGluT2 antibodies (n=5 mice/genotype). GL indicates granule cell layer, IML/OML indicate inner/outer molecular layer, respectively. Density of colocalized PSD95/VGLuT2 puncta was counted. IML: WT 109±21, crmp2-/- 156±15 (p=0.004). OML: WT 281±38, crmp2-/- 297±80 (p=0.7). Scale bars: 10 μm, mean ± SD, *p<0.05, t-test.


( G ) Spine density in DiOlistically labeled prefrontal cortex ( PFC ) is similar in WT and crmp2 - / - mice in both juvenile and adult mice . Scale bars : 5 μm . ( H ) Quantification of dendritic spine density of PFC pyramidal neurons , basal dendrites ( WT , juvenile : 8 . 63 ± 1 . 44 spines / 10 μm , adults : 7 . 3 ± 1 . 4 ; crmp2 - / - , juvenile : 9 . 05 ± 1 . 73 , p = 0 . 97 , adults : 6 . 98 ± 1 . 53 , p > 0 . 99 ) , mean ± SD , 2 - way ANOVA with Bonferroni ' s multiple comparisons test . P25 : n = 3 , ≥ 50 dendrites ; adults : n = 3 , ≥ 50 dendrites .

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

(G) Spine density in DiOlistically labeled prefrontal cortex (PFC) is similar in WT and crmp2-/- mice in both juvenile and adult mice. Scale bars: 5 μm. (H) Quantification of dendritic spine density of PFC pyramidal neurons, basal dendrites (WT, juvenile: 8.63±1.44 spines/10 μm, adults: 7.3±1.4; crmp2-/-, juvenile: 9.05±1.73, p=0.97, adults: 6.98±1.53, p>0.99), mean ± SD, 2-way ANOVA with Bonferroni's multiple comparisons test. P25: n=3, ≥50 dendrites; adults: n=3, ≥50 dendrites.


( A - B ) Ultrasonic vocalization was measured at P6 , P8 and P12 ( WT n = 14 pups , crmp2 - / - n = 13 pups ) . In crmp2 - / - mice , there is a significant decrease in the rate and duration of calls at P8 ( call rate , WT 352 ± 58 / 5 min , crmp2 - / - 164 ± 41 / 5 min , p = 0 . 015 ; call duration , WT 9 . 52 ± 2 ms , crmp2 - / - 3 . 36 ± 1 ms , p = 0 . 011 ) and P12 ( call rate , WT 46 ± 16 / 5 min crmp2 - / - 1 . 54 ± 1 . 24 / 5 min , p = 0 . 002 ; call duration , WT 0 . 68 ± 0 . 35 ms , crmp2 - / - 0 . 007 ± 0 . 006 ms , p = 0 . 002 ) . Mean ± SEM , * p < 0 . 05 , t - test ( P6 , P8 ) , Mann - Whitney test ( P12 ) . ( C ) Representative sonograms of the P8 mice .

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

(A - B) Ultrasonic vocalization was measured at P6, P8 and P12 (WT n=14 pups, crmp2-/- n=13 pups). In crmp2-/- mice, there is a significant decrease in the rate and duration of calls at P8 (call rate, WT 352±58/5 min, crmp2-/- 164±41/5 min, p=0.015; call duration, WT 9.52±2 ms, crmp2-/- 3.36±1 ms, p=0.011) and P12 (call rate, WT 46±16/5 min crmp2-/- 1.54±1.24/5 min, p=0.002; call duration, WT 0.68±0.35 ms, crmp2-/- 0.007±0.006 ms, p=0.002). Mean ± SEM, *p<0.05, t-test (P6, P8), Mann-Whitney test (P12). (C) Representative sonograms of the P8 mice.


( D ) 3 - chamber test ( WT n = 11 mice , crmp2 - / - n = 13 mice ) . In sociability phase , WT mice spent significantly more time with a social partner ( stranger 1 ) , unlike knockouts . ( WT - stranger 1 : 0 . 14 ± 0 . 01 , object : 0 . 07 ± 0 . 007 ( p = 0 . 0001 ) ; crmp2 - / - - stranger 1 : 0 . 07 ± 0 . 01 , object : 0 . 04 ± 0 . 006 ( p = 0 . 07 ) ) . In social novelty phase , when an object was substituted with a second social partner ( stranger 2 ) , both WTs and knockouts preferred novel mice to known mice . ( WT - stranger 1 : 0 . 03 ± 0 . 006 , stranger 2 : 0 . 09 ± 0 . 008 ( p < 0 . 0001 ) ; crmp2 - / - - stranger 1 : 0 . 02 ± 0 . 004 , stranger 2 : 0 . 05 ± 0 . 01 ( p = 0 . 02 ) ) . Mean ± SEM , * p < 0 . 05 , * * * p < 0 . 001 , t - test .

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

(D) 3-chamber test (WT n=11 mice, crmp2-/- n=13 mice). In sociability phase, WT mice spent significantly more time with a social partner (stranger 1), unlike knockouts. (WT - stranger 1: 0.14±0.01, object: 0.07±0.007 (p=0.0001); crmp2-/- - stranger 1: 0.07±0.01, object: 0.04±0.006 (p=0.07)). In social novelty phase, when an object was substituted with a second social partner (stranger 2), both WTs and knockouts preferred novel mice to known mice. (WT - stranger 1: 0.03±0.006, stranger 2: 0.09±0.008 (p<0.0001); crmp2-/- - stranger 1: 0.02±0.004, stranger 2: 0.05±0.01 (p=0.02)). Mean ± SEM, *p<0.05, ***p<0.001, t-test.


Elevated plus maze test ( n = 10 mice / genotype ) . ( E ) Total distance walked is similar in WT and crmp2 - / - ( WT 7 . 9 ± 0 . 45 m , crmp2 - / - 8 . 6 ± 0 . 5 m , p = 0 . 3 ) . ( F ) Frequency of open arm ( OA ) visits is increased in crmp2 - / - mice suggesting decreased anxiety . CA denotes closed arms , MID denotes the transition zone between arms , Total arm visits represents a sum of visits in all four arms . ( CA frequency : WT 13 . 7 ± 0 . 9 / 5 min , crmp2 - / - 10 . 5 ± 1 . 1 / 5 min , p = 0 . 04 ; OA frequency : WT 7 . 7 ± 0 . 8 / 5 min , crmp2 - / - 14 . 6 ± 1 . 7 / 5 min , p = 0 . 002 ; MID frequency : WT 20 . 6 ± 1 / 5 min , crmp2 - / - 24 . 6 ± 1 . 6 / 5 min , p = 0 . 06 mean ± SEM , * p < 0 . 05 , * * * p < 0 . 001 , t - test .

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

Elevated plus maze test (n=10 mice/genotype). (E) Total distance walked is similar in WT and crmp2-/- (WT 7.9±0.45 m, crmp2-/- 8.6±0.5 m, p=0.3). (F) Frequency of open arm (OA) visits is increased in crmp2-/- mice suggesting decreased anxiety. CA denotes closed arms, MID denotes the transition zone between arms, Total arm visits represents a sum of visits in all four arms. (CA frequency: WT 13.7±0.9/5 min, crmp2-/- 10.5±1.1/5 min, p=0.04; OA frequency: WT 7.7±0.8/5 min, crmp2-/- 14.6±1.7/5 min, p=0.002; MID frequency: WT 20.6±1/5 min, crmp2-/- 24.6±1.6/5 min, p=0.06 mean ± SEM, *p<0.05, ***p<0.001, t-test.


Elevated plus maze test ( n = 10 mice / genotype ) . ( G ) duration of open arm ( OA ) visits is increased in crmp2 - / - mice suggesting decreased anxiety . CA denotes closed arms , MID denotes the transition zone between arms , Total arm visits represents a sum of visits in all four arms . Total arm visits : WT 21 . 4 ± 1 / 5 min , crmp2 - / - 25 . 1 ± 1 . 5 / 5 min , p = 0 . 07 ; CA duration : WT 177 ± 17 s . , crmp2 - / - 75 . 3 ± 12 . 6 s , p < 0 . 001 ; OA duration : WT 42 ± 12 . 6 s , crmp2 - / - 144 . 4 ± 21 s , p < 0 . 001 ; MID duration : WT 80 . 26 ± 11 . 5 s . , crmp2 - / - 80 . 17 ± 12 . 6 s . , p = 0 . 99 ) , mean ± SEM , * p < 0 . 05 , * * * p < 0 . 001 , t - test .

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

Elevated plus maze test (n=10 mice/genotype). (G) duration of open arm (OA) visits is increased in crmp2-/- mice suggesting decreased anxiety. CA denotes closed arms, MID denotes the transition zone between arms, Total arm visits represents a sum of visits in all four arms. Total arm visits: WT 21.4±1/5 min, crmp2-/- 25.1±1.5/5 min, p=0.07; CA duration: WT 177±17 s., crmp2-/- 75.3±12.6 s, p<0.001; OA duration: WT 42±12.6 s, crmp2-/- 144.4±21 s, p<0.001; MID duration: WT 80.26±11.5 s., crmp2-/- 80.17±12.6 s., p=0.99), mean ± SEM, *p<0.05, ***p<0.001, t-test.


( H , I ) Y maze test ( WT n = 9 mice , crmp2 - / - n = 8 mice ) . Decreased alternations between arms of the maze indicates impaired working memory ( number of visits , WT : 23 . 1 ± 1 . 9 , crmp2 - / - 30 . 4 ± 3 . 8 , p = 0 . 12 ; alternations , WT 68 . 8 ± 2 . 5 % , crmp2 - / - 57 . 3 ± 2 . 5 % , p = 0 . 006 ) , mean ± SEM , * p < 0 . 05 , t - test .

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(H, I) Y maze test (WT n=9 mice, crmp2-/- n=8 mice). Decreased alternations between arms of the maze indicates impaired working memory (number of visits, WT: 23.1±1.9, crmp2-/- 30.4±3.8, p=0.12; alternations, WT 68.8±2.5%, crmp2-/- 57.3±2.5%, p=0.006), mean ± SEM, *p<0.05, t-test.


( a ) MN - 1 cells were transfected with mCherry - EGFP - LC3 vector , fixed and imaged after 24 h . Numbers of yellow puncta ( autophagosomes ) and red puncta ( autolysosomes ) per cell were counted . Autophagosomes : n = 3 independent experiments , F = 3 . 56 ; autolysosomes : n = 3 independent experiments , F = 20 . 13 . One - way ANOVA with post hoc Tukey test . * P 0 . 05 , * * * P 0 . 001 . n = 34 cells per genotype .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) MN-1 cells were transfected with mCherry-EGFP-LC3 vector, fixed and imaged after 24 h. Numbers of yellow puncta (autophagosomes) and red puncta (autolysosomes) per cell were counted. Autophagosomes: n = 3 independent experiments, F = 3.56; autolysosomes: n = 3 independent experiments, F = 20.13. One-way ANOVA with post hoc Tukey test. *P 0.05, ***P 0.001. n = 34 cells per genotype.


( b ) We transfected MEFs stably expressing mCherry - EGFP - LC3 with BFP - tagged AR25Q or AR125Q . Cells were imaged after 24 h , and yellow puncta ( autophagosomes ) and red puncta ( autolysosomes ) in AR - expressing ( blue ) cells were counted . Autophagosomes : n = 3 independent experiments , F = 20 . 65 ; autolysosomes : n = 3 independent experiments , F = 4 . 49 . One - way ANOVA with post hoc Tukey test . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 . n = 33 cells per genotype .

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

(b) We transfected MEFs stably expressing mCherry-EGFP-LC3 with BFP-tagged AR25Q or AR125Q. Cells were imaged after 24 h, and yellow puncta (autophagosomes) and red puncta (autolysosomes) in AR-expressing (blue) cells were counted. Autophagosomes: n = 3 independent experiments, F = 20.65; autolysosomes: n = 3 independent experiments, F = 4.49. One-way ANOVA with post hoc Tukey test. *P 0.05, **P 0.01, ***P 0.001. n = 33 cells per genotype.


( c ) MN - 1 cells were immunoblotted for LC3 in the presence or absence of ammonium chloride to evaluate autophagic flux . A representative LC3 western blot is shown . All ratios were normalized to MN - 1 WT cells at baseline , which was set to 1 . ( d ) The ratio of LC3 - II : actin for c determined by densitometry analysis using ImageJ . n = 3 independent experiments , F = 0 . 4501 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 .

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

(c) MN-1 cells were immunoblotted for LC3 in the presence or absence of ammonium chloride to evaluate autophagic flux. A representative LC3 western blot is shown. All ratios were normalized to MN-1 WT cells at baseline, which was set to 1. (d) The ratio of LC3-II:actin for c determined by densitometry analysis using ImageJ. n = 3 independent experiments, F = 0.4501, one-way ANOVA with post hoc Tukey test. *P 0.05.


( e ) MN - 1 cells were treated with ammonium chloride to block lysosomal activity , and then ammonium chloride was removed and cells were allowed to degrade accumulated autophagy cargo during a 6 - h recovery period . We performed p62 western blot analysis on MN - 1 cells at baseline ( - ) , after ammonium chloride treatment ( + ) and after the recovery period ( Rec ) . All ratios were normalized to MN - 1 WT cells at baseline , which was set to 1 . ( f ) On the basis of the p62 values obtained in e , we calculated the efficiency of degraded accumulated autophagic cargo by dividing p62 at baseline by p62 at recovery and normalized the baseline / recovery values to baseline / recovery for MN - 1 WT cells . n = 3 independent experiments , F = 5 . 45 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 , * * P 0 . 01 . Data are presented as mean ± s . e . m . Scale bar , 20 μm . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

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

(e) MN-1 cells were treated with ammonium chloride to block lysosomal activity, and then ammonium chloride was removed and cells were allowed to degrade accumulated autophagy cargo during a 6-h recovery period. We performed p62 western blot analysis on MN-1 cells at baseline (-), after ammonium chloride treatment (+) and after the recovery period (Rec). All ratios were normalized to MN-1 WT cells at baseline, which was set to 1. (f) On the basis of the p62 values obtained in e, we calculated the efficiency of degraded accumulated autophagic cargo by dividing p62 at baseline by p62 at recovery and normalized the baseline/recovery values to baseline/recovery for MN-1 WT cells. n = 3 independent experiments, F = 5.45, one-way ANOVA with post hoc Tukey test. *P 0.05, ** P 0.01. Data are presented as mean ± s.e.m. Scale bar, 20 μm. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( a , b ) Electron micrographs of motor neuron perinuclear regions from age - matched non - transgenic ( Nt ) , YAC AR20 and YAC AR100 transgenic mice before disease onset ( a , 6 months of age ) and after prominent neuromuscular and molecular pathology is apparent ( b , 14 months of age ) . ( a ) At 6 months of age , occasional autophagosomes ( yellow arrowheads ) are noted in Nt , YAC AR20 and YAC AR100 at roughly equivalent frequency . Autolysosomes ( red arrowheads ) are much more common and are present in higher numbers in YAC AR20 and YAC AR100 motor neurons . ( b ) At 14 months of age , when YAC AR100 mice display signs of motor neuronopathy and molecular pathology , we observed many YAC AR100 motor neuron micrographs with frequent autophagosomes ( yellow arrowheads ) . Despite this increase in autophagosomes , autolysosomes were fewer in number at this age in YAC AR100 motor neurons , such that autophagosome numbers approached autolysosome numbers in YAC AR100 motor neurons . This was never the case for Nt motor neurons or YAC AR20 motor neurons . Main panels are at original magnification 2 , 200 × and insets are at original magnification 3 , 700 × .

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

(a,b) Electron micrographs of motor neuron perinuclear regions from age-matched non-transgenic (Nt), YAC AR20 and YAC AR100 transgenic mice before disease onset (a, 6 months of age) and after prominent neuromuscular and molecular pathology is apparent (b, 14 months of age). (a) At 6 months of age, occasional autophagosomes (yellow arrowheads) are noted in Nt, YAC AR20 and YAC AR100 at roughly equivalent frequency. Autolysosomes (red arrowheads) are much more common and are present in higher numbers in YAC AR20 and YAC AR100 motor neurons. (b) At 14 months of age, when YAC AR100 mice display signs of motor neuronopathy and molecular pathology, we observed many YAC AR100 motor neuron micrographs with frequent autophagosomes (yellow arrowheads). Despite this increase in autophagosomes, autolysosomes were fewer in number at this age in YAC AR100 motor neurons, such that autophagosome numbers approached autolysosome numbers in YAC AR100 motor neurons. This was never the case for Nt motor neurons or YAC AR20 motor neurons. Main panels are at original magnification 2,200× and insets are at original magnification 3,700×.


( a ) Mean number of autophagosomes per motor neuron field in electron micrographs from non - transgenic ( Nt ) , YAC AR20 and YAC AR100 transgenic mice at 6 and 14 months of age , ± s . e . m . 6 months : n = 3 independent experiments , F = 0 . 63 , one - way ANOVA with post hoc Tukey test . P = 0 . 534 . 14 months : n = 3 independent experiments , F = 4 . 32 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 . ( b ) Mean number of autolysosomes per motor neuron field in electron micrographs from Nt , YAC AR20 and YAC AR100 transgenic mice at 6 and 14 months of age , ± s . e . m . 6 months : n = 3 independent experiments , F = 4 . 18 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 . 14 months : n = 3 independent experiments , F = 4 . 56 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 . ( c ) Mean autophagy index , dividing number of autolysosome by number of autophagosomes for each motor neuron field , ± s . e . m . At 6 months , YAC AR100 motor neurons displayed a markedly increased autophagy index ; n = 3 independent experiments , F = 129 . 81 , one - way ANOVA with post hoc Tukey test . * * P 0 . 01 . However , once YAC AR100 mice develop disease pathology at 14 months , the autophagy index for YAC AR100 motor neurons is significantly decreased ; n = 3 independent experiments , F = 82 . 82 , one - way ANOVA with post hoc Tukey test . * * P 0 . 01 . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mean number of autophagosomes per motor neuron field in electron micrographs from non-transgenic (Nt), YAC AR20 and YAC AR100 transgenic mice at 6 and 14 months of age, ± s.e.m. 6 months: n = 3 independent experiments, F = 0.63, one-way ANOVA with post hoc Tukey test. P = 0.534. 14 months: n = 3 independent experiments, F = 4.32, one-way ANOVA with post hoc Tukey test. *P 0.05. (b) Mean number of autolysosomes per motor neuron field in electron micrographs from Nt, YAC AR20 and YAC AR100 transgenic mice at 6 and 14 months of age, ± s.e.m. 6 months: n = 3 independent experiments, F = 4.18, one-way ANOVA with post hoc Tukey test. *P 0.05. 14 months: n = 3 independent experiments, F = 4.56, one-way ANOVA with post hoc Tukey test. *P 0.05. (c) Mean autophagy index, dividing number of autolysosome by number of autophagosomes for each motor neuron field, ± s.e.m. At 6 months, YAC AR100 motor neurons displayed a markedly increased autophagy index; n = 3 independent experiments, F = 129.81, one-way ANOVA with post hoc Tukey test. **P 0.01. However, once YAC AR100 mice develop disease pathology at 14 months, the autophagy index for YAC AR100 motor neurons is significantly decreased; n = 3 independent experiments, F = 82.82, one-way ANOVA with post hoc Tukey test. **P 0.01. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( a ) HEK293 cells were transfected with TFEB and AR25Q or AR125Q , as indicated . Immunoprecipitation ( IP ) was performed with anti - TFEB antibody , followed by immunoblotting ( IB ) for TFEB and AR . Untransfected HEK293 cells served as a negative control , and no AR was observed with IgG antibody only ( not shown ) .

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

(a) HEK293 cells were transfected with TFEB and AR25Q or AR125Q, as indicated. Immunoprecipitation (IP) was performed with anti-TFEB antibody, followed by immunoblotting (IB) for TFEB and AR. Untransfected HEK293 cells served as a negative control, and no AR was observed with IgG antibody only (not shown).


( b ) We transfected MN - 1 cells with the 4X - CLEAR luciferase reporter and treated cells with sucrose to induce TFEB activation . Untreated : n = 3 independent experiments , F = 3 . 48 , one - way ANOVA with post hoc Tukey test . * * P 0 . 01 . Sucrose treatment : n = 3 independent experiments , * * P 0 . 01 , t - test , WT t ( 10 ) = 8 . 133 ; * * * P 0 . 001 , t - test , AR 24Q t ( 10 ) = 6 . 11 ; P not significant ( n . s . ) , AR 65Q t ( 10 ) = 0 . 644 . Sucrose - treated MN - 1 AR24Q cells displayed significantly higher 4X - CLEAR luciferase activity : n = 3 independent experiments , F = 2 . 49 , one - way ANOVA with post hoc Tukey test . * * * P 0 . 001 . All ratios were normalized to untreated MN - 1 WT cells , whose value was set to 1 .

O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) We transfected MN-1 cells with the 4X-CLEAR luciferase reporter and treated cells with sucrose to induce TFEB activation. Untreated: n = 3 independent experiments, F = 3.48, one-way ANOVA with post hoc Tukey test. **P 0.01. Sucrose treatment: n = 3 independent experiments, **P 0.01, t-test, WT t(10) = 8.133; ***P 0.001, t-test, AR 24Q t(10) = 6.11; P not significant (n.s.), AR 65Q t(10) = 0.644. Sucrose-treated MN-1 AR24Q cells displayed significantly higher 4X-CLEAR luciferase activity: n = 3 independent experiments, F = 2.49, one-way ANOVA with post hoc Tukey test. ***P 0.001. All ratios were normalized to untreated MN-1 WT cells, whose value was set to 1.


( c ) We exposed MN - 1 cells to ammonium chloride , an inhibitor of lysosomal activity , to determine the capacity for TFEB induction . RT - PCR analysis revealed lack of TFEB target gene induction in MN - 1 AR65Q cells for four gene targets : Lamp1 ( lysosomal - associated membrane protein 1 ) , F = 37 . 51 ; Atp6v1h ( vesicular ATPase V1 subunit H ) , F = 57 . 26 ; Mcoln1 ( mucolipin 1 ) , F = 251 . 0 ; and Gla ( galactosidase - α ) , F = 75 . 51 ; n = 3 independent experiments , * P 0 . 05 , * * P 0 . 01 , ANOVA with post hoc Tukey test . For Lamp1 and Mcoln1 , MN - 1 AR24Q cells yielded significantly higher expression . Differences in the expression of TFEB target genes at baseline were negligible .

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

(c) We exposed MN-1 cells to ammonium chloride, an inhibitor of lysosomal activity, to determine the capacity for TFEB induction. RT-PCR analysis revealed lack of TFEB target gene induction in MN-1 AR65Q cells for four gene targets: Lamp1 (lysosomal-associated membrane protein 1), F = 37.51; Atp6v1h (vesicular ATPase V1 subunit H), F = 57.26; Mcoln1 (mucolipin 1), F = 251.0; and Gla (galactosidase-α), F = 75.51; n = 3 independent experiments, *P 0.05, **P 0.01, ANOVA with post hoc Tukey test. For Lamp1 and Mcoln1, MN-1 AR24Q cells yielded significantly higher expression. Differences in the expression of TFEB target genes at baseline were negligible.


( d ) We measured expression levels of TFEB target genes in E13 motor neurons derived from non - transgenic ( Nt ) , YAC AR20 and YAC AR100 mice by RT - PCR analysis : Ctsf ( cathepsin F ) , F = 4 . 63 ; Mcoln1 , F = 6 . 64 ; Atp6v1h , F = 5 . 6 ; and Gla , F = 5 . 65 ; n = 3 independent experiments , * P 0 . 05 , ANOVA with post hoc Tukey test . Data are presented as mean ± s . e . m . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

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

(d) We measured expression levels of TFEB target genes in E13 motor neurons derived from non-transgenic (Nt), YAC AR20 and YAC AR100 mice by RT-PCR analysis: Ctsf (cathepsin F), F = 4.63; Mcoln1, F = 6.64; Atp6v1h, F = 5.6; and Gla, F = 5.65; n = 3 independent experiments, *P 0.05, ANOVA with post hoc Tukey test. Data are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( a ) We transfected MN - 1 cells with the 4X - CLEAR luciferase reporter and with BFP - tagged TFEB or BFP empty vector , and then measured luciferase activity . Marked induction of luciferase activity occurred with BFP - TFEB ; n = 3 independent experiments , * * * P 0 . 001 , t - test , WT t ( 4 ) = 48 . 79 ; * * * P 0 . 001 , t - test , AR 24Q t ( 4 ) = 22 . 91 ; * * * P 0 . 001 , t - test , AR 65Q t ( 4 ) = 14 . 02 . Results for each line were normalized to the BFP - empty luciferase activity .

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

(a) We transfected MN-1 cells with the 4X-CLEAR luciferase reporter and with BFP-tagged TFEB or BFP empty vector, and then measured luciferase activity. Marked induction of luciferase activity occurred with BFP-TFEB; n = 3 independent experiments, ***P 0.001, t-test, WT t(4) = 48.79; ***P 0.001, t-test, AR 24Q t(4) = 22.91; ***P 0.001, t-test, AR 65Q t(4) = 14.02. Results for each line were normalized to the BFP-empty luciferase activity.


( b ) We transfected MN - 1 AR65Q cells with the 4X - CLEAR luciferase reporter and with BFP - TFEB or BFP empty vector and subjected the MN - 1 AR65Q cells to conditions that promote TFEB activation , as shown . MN - 1 WT cells were also transfected with the 4X - CLEAR luciferase reporter and BFP empty vector and subjected to the identical treatments for control purposes . Results are shown normalized to untreated MN - 1 WT cells expressing BFP - empty . Untreated , F = 182 . 7 ; starvation , F = 63 . 64 ; NH4Cl , F = 291 . 9 ; Rapamycin , F = 3128 ; ANOVA with post hoc Tukey test . * * P 0 . 01 , * * * P 0 . 001 .

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

(b) We transfected MN-1 AR65Q cells with the 4X-CLEAR luciferase reporter and with BFP-TFEB or BFP empty vector and subjected the MN-1 AR65Q cells to conditions that promote TFEB activation, as shown. MN-1 WT cells were also transfected with the 4X-CLEAR luciferase reporter and BFP empty vector and subjected to the identical treatments for control purposes. Results are shown normalized to untreated MN-1 WT cells expressing BFP-empty. Untreated, F = 182.7; starvation, F = 63.64; NH4Cl, F = 291.9; Rapamycin, F = 3128; ANOVA with post hoc Tukey test. **P 0.01, ***P 0.001.


( c ) We transfected MN - 1 AR65Q cells with the mCherry - EGFP - LC3 construct and with either BFP - empty or BFP - TFEB . Autophagic vesicles forming in BFP - expressing cells were then detected as red , green or yellow puncta , as shown . Scale bar , 20 μm . ( d ) Left , quantification of autophagic vesicle type . Note marked reduction in autophagosomes in MN - 1 AR65Q cells expressing BFP - TFEB ; n = 3 independent experiments , F = 16 . 596 , ANOVA with post hoc Tukey test . * * P 0 . 01 , * * * P 0 . 001 . Right , calculation of the autophagy index ; n = 3 independent experiments , F = 24 . 43 , ANOVA with post hoc Tukey test . * * P 0 . 01 . Data are presented as mean ± s . e . m . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

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

(c) We transfected MN-1 AR65Q cells with the mCherry-EGFP-LC3 construct and with either BFP-empty or BFP-TFEB. Autophagic vesicles forming in BFP-expressing cells were then detected as red, green or yellow puncta, as shown. Scale bar, 20 μm. (d) Left, quantification of autophagic vesicle type. Note marked reduction in autophagosomes in MN-1 AR65Q cells expressing BFP-TFEB; n = 3 independent experiments, F = 16.596, ANOVA with post hoc Tukey test. **P 0.01, ***P 0.001. Right, calculation of the autophagy index; n = 3 independent experiments, F = 24.43, ANOVA with post hoc Tukey test. **P 0.01. Data are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( b ) HEK293 cells were transfected with TFEB and influenza hemagglutinin ( HA ) - tagged full - length AR25 ( FL ) or a C - terminal AR mutant ( m1 , m2 , m3 , m4 ) . Immunoprecipitation ( IP ) was performed with anti - TFEB antibody , followed by immunoblotting ( IB ) for TFEB and AR . Untransfected HEK293 cells served as a negative control , and no AR was observed with IgG antibody only ( not shown ) .

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

(b) HEK293 cells were transfected with TFEB and influenza hemagglutinin (HA)-tagged full-length AR25 (FL) or a C-terminal AR mutant (m1, m2, m3, m4). Immunoprecipitation (IP) was performed with anti-TFEB antibody, followed by immunoblotting (IB) for TFEB and AR. Untransfected HEK293 cells served as a negative control, and no AR was observed with IgG antibody only (not shown).


( c ) HEK293 cells were transfected with TFEB and HA - tagged N - terminal AR mutant containing a 19Q or 112Q tract ( NT19Q , NT112Q ) . Immunoprecipitation was performed as in b . with anti - TFEB antibody , followed by immunoblotting ( IB ) for TFEB and AR . Untransfected HEK293 cells served as a negative control , and no AR was observed with IgG antibody only ( not shown ) .

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

(c) HEK293 cells were transfected with TFEB and HA-tagged N-terminal AR mutant containing a 19Q or 112Q tract (NT19Q, NT112Q). Immunoprecipitation was performed as in b. with anti-TFEB antibody, followed by immunoblotting (IB) for TFEB and AR. Untransfected HEK293 cells served as a negative control, and no AR was observed with IgG antibody only (not shown).


( d ) MN - 1 WT , MN - 1 AR24Q and MN - 1 AR65Q cells were cultured in standard medium ( Ctl ) or in medium supplemented with R1881 and then immunostained with antibodies to AR and to TFEB . Cells were scored for nuclear or cytosolic localization of TFEB . Scale bars , 20 μm . ( e ) Quantification of d . Untreated , F = 0 . 56 ; R1881 , F = 5 . 51 ; ANOVA with post hoc Tukey test . * * P 0 . 01 .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O 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) MN-1 WT, MN-1 AR24Q and MN-1 AR65Q cells were cultured in standard medium (Ctl) or in medium supplemented with R1881 and then immunostained with antibodies to AR and to TFEB. Cells were scored for nuclear or cytosolic localization of TFEB. Scale bars, 20 μm. (e) Quantification of d. Untreated, F = 0.56; R1881, F = 5.51; ANOVA with post hoc Tukey test. **P 0.01.


( f ) We transfected MN - 1 WT cells with the 4X - CLEAR luciferase reporter and either BFP - empty or AR25Q - BFP , under standard culture conditions ( control ) , ammonium chloride treatment or rapamycin treatment . Control , t ( 3 ) = 15 . 73 ; NH4Cl , t ( 4 ) = 4 . 108 ; rapamycin . t ( 4 ) = 4 . 58 ; t - test . * * P 0 . 01 .

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

(f) We transfected MN-1 WT cells with the 4X-CLEAR luciferase reporter and either BFP-empty or AR25Q-BFP, under standard culture conditions (control), ammonium chloride treatment or rapamycin treatment. Control, t(3) = 15.73; NH4Cl, t(4) = 4.108; rapamycin. t(4) = 4.58; t-test. **P 0.01.


( g ) We transfected MN - 1 AR24Q cells with either scrambled shRNA control vector or AR shRNA vector and measured 4X - CLEAR luciferase reporter activity under baseline conditions ( control ) or after rapamycin . Control , t ( 4 ) = 0 . 51 ; rapamycin , t ( 4 ) = 7 . 15 ; t - test . * * P 0 . 01 .

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

(g) We transfected MN-1 AR24Q cells with either scrambled shRNA control vector or AR shRNA vector and measured 4X-CLEAR luciferase reporter activity under baseline conditions (control) or after rapamycin. Control, t(4) = 0.51; rapamycin, t(4) = 7.15; t-test. **P 0.01.


( h ) RT - PCR analysis of MN - 1 AR24Q cells transfected with scrambled shRNA control or AR shRNA vector : Gla , t ( 4 ) = 4 . 77 ; Atp6v1h , t ( 4 ) = 3 . 75 ; Mcoln1 , t ( 4 ) = 13 . 74 ; Ctsd ( cathepsin D ) , t ( 4 ) = 7 . 91 ; and Lamp1 , t ( 4 ) = 6 . 46 ; n = 3 independent experiments , * P 0 . 05 , * * P 0 . 01 , t - test . Data are presented as mean ± s . e . m . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-PCR analysis of MN-1 AR24Q cells transfected with scrambled shRNA control or AR shRNA vector: Gla, t(4) = 4.77; Atp6v1h, t(4) = 3.75; Mcoln1, t(4) = 13.74; Ctsd (cathepsin D), t(4) = 7.91; and Lamp1, t(4) = 6.46; n = 3 independent experiments, *P 0.05, **P 0.01, t-test. Data are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( a ) We performed filter - trap assays to determine the extent of accumulation of insoluble AR protein in NPC lines from three different controls and three different SBMA patients . Significantly higher levels of insoluble AR protein were present in the SBMA NPC lines , as based on densitometry ; n = 3 independent experiments , P 0 . 05 , t - test , t ( 4 ) = 4 . 026 . AU , arbitrary units .

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

(a) We performed filter-trap assays to determine the extent of accumulation of insoluble AR protein in NPC lines from three different controls and three different SBMA patients. Significantly higher levels of insoluble AR protein were present in the SBMA NPC lines, as based on densitometry; n = 3 independent experiments, P 0.05, t-test, t(4) = 4.026. AU, arbitrary units.


( b ) NPCs from three different controls and three different SBMA patients were cultured in the presence of JC - 1 dye . Results are presented as a Tukey box plots with box corresponding to first to third quartiles from the median and whiskers corresponding to 1 . 5 quartiles from the median ; n = 3 independent experiments , * P 0 . 05 , t - test , t ( 4 ) = 3 . 983 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NPCs from three different controls and three different SBMA patients were cultured in the presence of JC-1 dye. Results are presented as a Tukey box plots with box corresponding to first to third quartiles from the median and whiskers corresponding to 1.5 quartiles from the median; n = 3 independent experiments, *P 0.05, t-test, t(4) = 3.983.


( c ) We transfected NPC lines derived from SBMA patients and controls ( three different NPC lines per patient ) with the mCherry - EGFP - LC3 vector . Autophagic vesicles were detected as colored puncta , as shown . Scale bar , 10 μm . ( d ) Quantification of autophagic vesicle type from c . SBMA NPCs exhibit a significant increase in autophagosomes ; n = 18 different samples : 3 clones per line , 3 lines per group , * P 0 . 05 , t - test ; t ( 18 ) = 2 . 3799 ( autophagosomes ) .

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

(c) We transfected NPC lines derived from SBMA patients and controls (three different NPC lines per patient) with the mCherry-EGFP-LC3 vector. Autophagic vesicles were detected as colored puncta, as shown. Scale bar, 10 μm. (d) Quantification of autophagic vesicle type from c. SBMA NPCs exhibit a significant increase in autophagosomes; n = 18 different samples: 3 clones per line, 3 lines per group, *P 0.05, t-test; t(18) = 2.3799 (autophagosomes).


( e ) We isolated RNAs from SBMA and control NPC lines ( three different NPC lines per human subject ) and then measured the expression levels of TFEB target genes including CYCS ( cytochrome c ) , t ( 4 ) = 2 . 844 ; MCOLN1 , t ( 4 ) = 3 . 246 ; GLA , t ( 4 ) = 3 . 174 ; ACADM ( acyl - CoA dehydrogenase ) , t ( 4 ) = 4 . 405 ; COX6A1 ( cytochrome oxidase 6A1 ) , t ( 4 ) = 5 . 584 ; and the TFEB gene itself , t ( 4 ) = 0 . 593 . Significant expression reductions were noted in SBMA NPC lines for all tested TFEB target genes . * P 0 . 05 , * * P 0 . 01 . Data in d , e are presented as mean ± s . e . m . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

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

(e) We isolated RNAs from SBMA and control NPC lines (three different NPC lines per human subject) and then measured the expression levels of TFEB target genes including CYCS (cytochrome c), t(4) = 2.844; MCOLN1, t(4) = 3.246; GLA, t(4) = 3.174; ACADM (acyl-CoA dehydrogenase), t(4) = 4.405; COX6A1 (cytochrome oxidase 6A1), t(4) = 5.584; and the TFEB gene itself, t(4) = 0.593. Significant expression reductions were noted in SBMA NPC lines for all tested TFEB target genes. *P 0.05, **P 0.01. Data in d,e are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( a ) Control and SBMA NPC lines were maintained in the presence or absence of the AR ligand dihydrotestosterone ( DHT ) and then harvested for immunoblotting analysis after TFEB antibody immunoprecipitation ( IP ) . Immunoblotting ( IB ) for AR confirmed expression of AR in control ( Ctrl ) and SBMA NPCs , and immunoblotting for TFEB confirmed the interaction of AR with TFEB in both control and SBMA NPCs .

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

(a) Control and SBMA NPC lines were maintained in the presence or absence of the AR ligand dihydrotestosterone (DHT) and then harvested for immunoblotting analysis after TFEB antibody immunoprecipitation (IP). Immunoblotting (IB) for AR confirmed expression of AR in control (Ctrl) and SBMA NPCs, and immunoblotting for TFEB confirmed the interaction of AR with TFEB in both control and SBMA NPCs.