B ) CHN - 1 auto - Ub was performed as indicated using increasing molar concentrations ( 0 . 5 , 1 , 2 , 3 , 4 µM ) of UBE2D1 without with a complexing equimolar ( 1 : 1 ) of recombinant CHN - 1 UFD - 2P951A ( 1 . 5 µM CHN - 1 with 1 . 5 µM of recombinant UFD - 2P951A ) . samples were resolved via SDS - PAGE immunoblotted with anti - CHN - 1 antibodies . Right , a graph representing nmol of ubiquitylated CHN - 1 vs . UBE2D1 ( µM ) for CHN - 1 alone ( black ) or CHN - 1 + UFD - 2P951A ( cyan ) . Plotted data are mean from three technical replicates . Error bars represent ; statistical significance was determined using Pearson correlation coefficients which define statistical relation between two continuous variables [ nmol of ubiquitylated CHN - 1 vs UBE2D1 ( µM ) in presence of CHN - 1 or CHN - 1 + UFD - 2P951A CHN - 1 vs CHN - 1 + UFD - 2P951A with increasing UBE2D1 ( µM ) ] .

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

B) CHN-1 auto-Ub was performed as indicated using increasing molar concentrations (0.5, 1, 2, 3, 4 µM) of UBE2D1 without and with a complexing equimolar concentration (1:1) of recombinant CHN-1 and UFD-2P951A (1.5 µM CHN-1 with 1.5 µM of recombinant UFD-2P951A). Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Right, a graph representing the nmol of ubiquitylated CHN-1 vs. UBE2D1 (µM) for CHN-1 alone (black) or CHN-1+UFD-2P951A (cyan). Plotted data are the mean from the three technical replicates. Error bars represent the SEM; statistical significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [nmol of ubiquitylated CHN-1 vs UBE2D1(µM) in the presence of CHN-1 or CHN-1+UFD-2P951A and CHN-1 vs CHN-1+UFD-2P951A with increasing UBE2D1(µM)].


C ) Discharging of Ub - charged UBE2D1 was carried out in presence of CHN - 1 or CHN - 1 - UFD - 2P951A . experimental sample was run together with control with without a reducing agent ( 50 mM DTT ) . reaction was stopped after 30 min by boiling samples in 1x SDS sample buffer . were resolved via SDS - PAGE immunoblotted with anti - UBE2D1 antibodies . Below , of uncharged UBE2D1 plotted as uncharged UBE2D1 fraction vs . UBE2D1 - Ub ( µM ) . graph representing UBE2D1 fraction vs . UBE2D1 - Ub ( µM ) for CHN - 1 alone ( black ) or CHN - 1 + UFD - 2P951A ( cyan ) Plotted data are mean of three technical replicates . Significance was determined using Pearson correlation coefficients which define statistical relation between two continuous variables [ UBE2D1 vs UBE2D1 - Ub ( µM ) in presene of CHN - 1 or CHN - 1 + UFD - 2P951A CHN - 1 vs CHN - 1 + UFD - 2P951A with increasing UBE2D1 - Ub ( µM ) ] .

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

C) Discharging assay of Ub-charged UBE2D1 was carried out in the presence of CHN-1 or CHN-1-UFD-2P951A. The experimental sample was run together with the control with and without a reducing agent (50 mM DTT). The reaction was stopped after 30 min by boiling the samples in 1x SDS sample buffer. Proteins were resolved via SDS-PAGE and immunoblotted with anti-UBE2D1 antibodies. Below, quantification of uncharged UBE2D1 plotted as the uncharged UBE2D1 fraction vs. UBE2D1-Ub (µM). The graph representing the UBE2D1 fraction vs. UBE2D1-Ub (µM) for CHN-1 alone (black) or CHN-1+UFD-2P951A (cyan) and Plotted data are the mean of three technical replicates. Significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [UBE2D1 vs UBE2D1-Ub(µM) in the presene of CHN-1 or CHN-1+UFD-2P951A and CHN-1 vs CHN-1+UFD-2P951A with increasing UBE2D1-Ub(µM)].


D ) CHN - 1 auto - Ub was performed as indicated in presence of Ube2W - Ub or Ube2W - UbFLAG without with equimolar of recombinant CHN - 1 UFD - 2P951A . samples were resolved via SDS - PAGE immunoblotted with anti - CHN - 1 antibodies . For each sample , quantified relative signal after probing blot using anti - CHN - 1 antibodies is shown as a above respective signal . Below , a schematic of CHN - 1 - UbFLAG ( cyan ) , CHN - 1 - Ub ( magenta ) , unmodified CHN - 1 signal ( black ) presented as signal fold change between CHN - 1 CHN - 1 - UFD - 2P951A .

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

D) CHN-1 auto-Ub was performed as indicated in the presence of Ube2W-Ub or Ube2W-UbFLAG without and with equimolar concentration of recombinant CHN-1 and UFD-2P951A. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. For each sample, the quantified relative signal after probing the blot using anti-CHN-1 antibodies is shown as a ratio above the respective signal. Below, a schematic of the CHN-1-UbFLAG (cyan), CHN-1-Ub (magenta), and unmodified CHN-1 signal (black) presented as the ratio and the signal fold change between CHN-1 and CHN-1-UFD-2P951A.


A ) Auto - Ub of recombinant CHN - 1 using UBE2D1 was performed alone or complexed with UFD - 2 in presence of recombinant DAF - 21 , DAF - 21ΔEEVD , HSP - 1 , or HSP - 1ΔEEVD . samples were resolved via SDS - PAGE immunoblotted with anti - CHN - 1 antibodies .

A) Auto-Ub of recombinant CHN-1 using UBE2D1 was performed alone or complexed with UFD-2 in the presence of recombinant DAF-21, DAF-21ΔEEVD, HSP-1, or HSP-1ΔEEVD. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.


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

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 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-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 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-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

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 UFD - 2 peptide sequences aligned with C - terminal of full - UFD - 2 ( UFD - 2 864 - 914 ) for PEPTIDE 1 ( yellow ) PEPTIDE 2 ( cyan ) ( UFD - 2 648 - 678 ) for PEPTIDE 3 ( magenta ) used in ubiquitylation reaction . Below , auto - Ub of recombinant CHN - 1 was performed as indicated in presence of UFD - 2 ‑ derived peptides . samples were resolved via SDS - PAGE immunoblotted with anti - CHN - 1 antibodies . Right , of 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 mean of three technical replicates .

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 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 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 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 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 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

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 presence of recombinant HSP - 1 or HSP - 1EEYD . samples were resolved via SDS - PAGE 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 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 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 . samples were resolved via SDS - PAGE 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 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 presence of recombinant UFD - 2 HSP - 1 . samples were resolved via SDS - PAGE 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 UFD - 2 , indicated lysates of young adult worms were subjected to immunoblotting with anti - CHN - 1 anti - UFD - 2 antibodies . Tubulin served as a loading control . Right , of CHN - 1 signals normalized to tubulin plotted as N2 ( wild - type ; black ) , ufd - 2 ( tm1380 ) ( cyan ) , UFD - 2P951A ( magenta ) . Plotted data are mean of three biological replicates .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-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 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 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 were determined in indicated lysates of young adult worms treated with a proteasome inhibitor ( MG - 132 , 10µM ) DUB inhibitor ( NEM , 100mM ) . Tubulin served as a loading control . Right , of CHN - 1 signals normalized to tubulin plotted as control ( black ) , or MG - 132 treated ( magenta ) in N2 ( wild - type ) ufd - 2 ( tm1380 ) . Plotted data are 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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 . samples were resolved via SDS - PAGE immunoblotted with anti - AHCY - 1 antibodies . Right , of 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 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 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 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

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

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 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 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 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 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 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

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

O O 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 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 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 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

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

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

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 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 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

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

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 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

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

O O O O B-CONTROLLED_VAR 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 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

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

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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 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

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

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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 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

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 dissected from expressing drprIR specifically in GFP - marked clone ( heat shock ( hs ) flp / w ; UAS - drprIR / + ; act , cd2 , FRT > Gal4 , UAS - GFP / + ) 6 h 14 h after puparium formation . Stained with GFP antibody ( green ) , to label expressing drpr IR , 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 B-MEASURED_VAR 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 with salivary - specific knockdown of Atg12 ( fkh - Gal4 / w ; UAS - Atg12IR / + ) , n = 21 , those with salivary - specific knockdown of both Atg12 drpr - I ( fkh - Gal4 / w ; UAS - Atg12IR / + ; UAS - drpr - IIR / + ) , n = 19 , analysed by histology for presence of salivary material ( red circles ) 24 h after puparium formation . b , 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 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

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 of control ( + / w ; UAS - GFP - LC3 / + ; fkh - Gal4 / + ) those with salivary - specific knockdown of drpr ( w ; UAS - drprIR / UAS - GFP - LC3 ; fkh - Gal4 / + ) . Salivary were dissected 6 h 14 h after puparium formation , imaged for GFP - LC3 , LC3 were quantified using Zeiss Automeasure software . d , 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 B-MEASURED_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 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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ( tub - LAMP - GFP / w ; + / fkh - Gal4 ) those with salivary - specific knockdown of drpr ( tub - LAMP - GFP / w ; UAS - drprIR / + ; fkh - Gal4 / + ) . Salivary were dissected 14 h after puparium formation , imaged for LAMP - GFP , LAMP were quantified using Zeiss Automeasure software . f , 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 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

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

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

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 , extracts from drpr null ( w ; drprDgr ; 5 / drprDgr ; 5 ) pupae at puparium formation ( 0 h ) from 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 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 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 , dissected fat bodies , stained them with anti - Drpr antibody , 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 B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

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 , drprIR specifically in dsRed - marked clone ( hsflp / w ; UAS - drprIR / + ; hsGFPAtg8a , act , cd2 , FRT > Gal4 , UAS - dsRed / + ) , for 4 h . We dissected larval fat bodies imaged them for GFP - Atg8a ( green ) 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

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 of expressing GFP - Atg8a in , drprIR specifically in dsRed - marked clone ( hsflp / w ; UAS - drprIR / + ; hsGFPAtg8a , act , cd2 , FRT > Gal4 , UAS - dsRed / + ) , dissected 14 h after puparium formation . Imaged for GFP - Atg8a ( green ) 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

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 were infected with MERS - CoV at MOI = 0 . 01 in presence of steroids for 24 h . viral titer in supernatant was quantified by standard plaque using Vero / TMPRSS2 . viability in presence of steroids was quantified by WST 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

(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 treated with ciclesonide were infected with parental MERS - CoV or ciclesonide escape mutant . Viral titer was measured as described in panel 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

(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 were infected with parental MERS - CoV / EMC ( Re - EMC / MERS ) or recombinant mutant ( Re - Nsp15 - A25V ) with an acid substitution at A25V in NSP15 . Viral titer was measured as described in panel 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 O O O O O O

(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 ( 6 hpi ) . VeroE6 / TMPRSS2 were infected with SARS - CoV - 2 at MOI = 1 in presence of 10 □ μM compounds for 6 h . viral was quantified by real - time PCR using 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 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 ( 27 hpi ) . VeroE6 / TMPRSS2 were infected with SARS - CoV - 2 at MOI = 0 . 01 in presence of ciclesonide for 27 h . Viral in culture medium was quantified by real - time PCR using E gene primer / probe set . viability in presence of ciclesonide was quantified at 27 hpi by WST .

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

(b) 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 ) hippocampal CA1 . CRMP2 ( green ) is expressed throughout cortex 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 B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 - / - , growth of ophthalmic branch of trigeminal nerve is increased axons are defasciculated ( first second row , arrowheads ) . Similarly , we detected increased growth branching of lateral branches of spinal nerves in crmp2 - / - ( third row , arrowheads ) . Trigeminal nerve ( V ) 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 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

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

O O O O O O O O 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 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

(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 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 growth path of 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 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

(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 ) of 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 .

O O O O O O O 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

(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 ) coronal sections ( second row ) of adult brains . Scale bars : 1 mm .

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

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

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(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 tract in crmp2 - / - mice . Caudal part of corpus callosum in WTs is located dorsally above habenular commissure ( hbc ) , while in crmp2 - / - mice callosum terminates rostrally before reaching hbc ( arrows ) . PC indicates posterior commissure . Scale bars : 500 µm .

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

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

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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 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 broader range of axon growth angles in crmp2 - / - mice .

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(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 by clustering traced axons ( WT n = 6 pups , crmp2 - / - n = 9 pups ) into 3 groups on 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 - .

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

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

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(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 ) for calbindin VgluT1 shows mature synapses in main bundle in both genotypes in 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 ) of IPB index ( of 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 - .

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(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 visual cortex axons at P9 ( before pruning ) , sagittal sections . Branching point of 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 .

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(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 ) of VP ( visual pruning ) index ( of pyramidal axons after vs . before branch point ) . In WT after pruning period , only a minor part of axons descends towards 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 , their VP indexes are not significantly different between adult 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 . P9 : n = 3 pups / genotype , adults : n = 5 mice / genotype )

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(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 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 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 blue asterisks indicate steady non - growing axons . Scale bars : 100 μm ( whole image field ) 50 μm ( magnified ) .

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(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 ) 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 .

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(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 is similar in WT crmp2 - / - mice at P30 . In adults , however , spine density in crmp2 - / - granule is increased comparing to WT . Scale bars : 5 μm . ( C ) of dendritic spine density in DG granule in inner molecular layer ( 50 - 100 μm away from 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 / genotype , WT = 61 dendrites , knockout = 64 dendrites ; adults : 3 / genotype , WT = 41 dendrites , knockout = 37 dendrites .

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

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(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 inner molecular layer revealed by double immunostaining with PSD95 VGluT2 antibodies ( n = 5 mice / genotype ) . GL indicates granule layer , IML / OML indicate inner / outer molecular layer , respectively . Density of colocalized PSD95 / VGLuT2 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 - .

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(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 crmp2 - / - mice in both juvenile adult mice . Scale bars : 5 μm . ( H ) 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 . P25 : n = 3 , ≥ 50 dendrites ; adults : n = 3 , ≥ 50 dendrites .

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(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 P12 ( WT n = 14 pups , crmp2 - / - n = 13 pups ) . In crmp2 - / - mice , there is a significant decrease in rate 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 ) 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 ± , * p < 0 . 05 , t - ( P6 , P8 ) , Mann - Whitney ( P12 ) . ( C ) Representative sonograms of P8 mice .

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(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 ( 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 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 ± , * p < 0 . 05 , * * * p < 0 . 001 , t - .

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 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

(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 ( n = 10 mice / genotype ) . ( E ) Total walked is similar in WT 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 transition zone between arms , Total arm visits represents a sum of visits in 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 ± , * p < 0 . 05 , * * * p < 0 . 001 , t - .

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 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

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 ( 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 transition zone between arms , Total arm visits represents a sum of visits in 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 ± , * p < 0 . 05 , * * * p < 0 . 001 , t - .

O O O 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 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

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 ( WT n = 9 mice , crmp2 - / - n = 8 mice ) . Decreased alternations between arms of 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 ± , * p < 0 . 05 , t - .

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 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

(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 were transfected with mCherry - EGFP - LC3 vector , fixed imaged after 24 h . Numbers of yellow ( autophagosomes ) red ( autolysosomes ) per 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 . * P 0 . 05 , * * * P 0 . 001 . n = 34 per genotype .

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

(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 . were imaged after 24 h , yellow ( autophagosomes ) red ( autolysosomes ) in AR - expressing ( blue ) 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 . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 . n = 33 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 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

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

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 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

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

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

(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 from non - transgenic ( Nt ) , YAC AR20 YAC AR100 transgenic mice at 6 14 months of , ± s . e . m . 6 months : n = 3 independent experiments , F = 0 . 63 , one - way ANOVA with post hoc Tukey . P = 0 . 534 . 14 months : n = 3 independent experiments , F = 4 . 32 , one - way ANOVA with post hoc Tukey . * P 0 . 05 . ( b ) Mean number of autolysosomes per motor neuron field in electron from Nt , YAC AR20 YAC AR100 transgenic mice at 6 14 months of , ± s . e . m . 6 months : n = 3 independent experiments , F = 4 . 18 , one - way ANOVA with post hoc Tukey . * P 0 . 05 . 14 months : n = 3 independent experiments , F = 4 . 56 , one - way ANOVA with post hoc Tukey . * 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 . * * P 0 . 01 . However , once YAC AR100 mice develop disease pathology at 14 months , autophagy index for YAC AR100 motor neurons is significantly decreased ; n = 3 independent experiments , F = 82 . 82 , one - way ANOVA with post hoc Tukey . * * P 0 . 01 . P values degrees of freedom are available in 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 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

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

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 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

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

(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 to ammonium chloride , an inhibitor of lysosomal activity , to determine capacity for TFEB induction . RT - PCR revealed lack of TFEB target gene induction in MN - 1 AR65Q for four gene targets : Lamp1 ( lysosomal - associated 1 ) , F = 37 . 51 ; Atp6v1h ( vesicular ATPase V1 subunit H ) , F = 57 . 26 ; Mcoln1 ( mucolipin 1 ) , F = 251 . 0 ; Gla ( galactosidase - α ) , F = 75 . 51 ; n = 3 independent experiments , * P 0 . 05 , * * P 0 . 01 , ANOVA with post hoc Tukey . For Lamp1 Mcoln1 , MN - 1 AR24Q yielded significantly higher expression . Differences in 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 B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O 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 O O O O O O O O O O O B-MEASURED_VAR I-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

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

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

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

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

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

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-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-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

(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 were transfected with TFEB influenza hemagglutinin ( HA ) - tagged full - 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 AR . Untransfected HEK293 served as a negative control , 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 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 I-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

(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 were transfected with TFEB 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 AR . Untransfected HEK293 served as a negative control , no AR was observed with IgG antibody only ( not shown ) .

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

(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 MN - 1 AR65Q were cultured in standard medium ( Ctl ) or in medium supplemented with R1881 then immunostained with antibodies to AR to TFEB . were scored for or localization of TFEB . Scale bars , 20 μm . ( e ) of d . Untreated , F = 0 . 56 ; R1881 , F = 5 . 51 ; ANOVA with post hoc Tukey . * * 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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O 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 with 4X - CLEAR luciferase reporter 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 - . * * P 0 . 01 .

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

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

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 O O 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 of MN - 1 AR24Q 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 ; Lamp1 , t ( 4 ) = 6 . 46 ; n = 3 independent experiments , * P 0 . 05 , * * P 0 . 01 , t - . Data are presented as mean ± s . e . m . P values degrees of freedom are available in Supplementary Methods Checklist .

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 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

(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 extent of accumulation of insoluble AR in NPC lines from three different controls three different SBMA . Significantly higher of insoluble AR were present in SBMA NPC lines , as on densitometry ; n = 3 independent experiments , P 0 . 05 , t - , t ( 4 ) = 4 . 026 . AU , arbitrary units .

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 O 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 three different SBMA were cultured in presence of JC - 1 dye . Results are presented as a Tukey box plots with box corresponding to first to third quartiles from median whiskers corresponding to 1 . 5 quartiles from median ; n = 3 independent experiments , * P 0 . 05 , t - , 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

(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.