( G ) Differential analysis of leukocyte populations in the BM . R26 - CreERki / + Adar1fl / + ( control ; n = 3 ) and R26 - CreERki / + Adar1fl / P195A ( P195A ; n = 4 ) . All BM analysis at day 28 post tamoxifen

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Differential analysis of leukocyte populations in the BM. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). All BM analysis at day 28 post tamoxifen treatment


( H ) BM erythroid cells . R26 - CreERki / + Adar1fl / + ( control ; n = 3 ) and R26 - CreERki / + Adar1fl / P195A ( P195A ; n = 4 ) . All BM , analysis at day 28 post tamoxifen

O O O O O O O O O O O O O O O O O O O O O O O O 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) BM erythroid cells. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). All BM, analysis at day 28 post tamoxifen treatment


( J ) Differential analysis of leukocyte populations in the spleen . R26 - CreERki / + Adar1fl / + ( control ; n = 3 ) and R26 - CreERki / + Adar1fl / P195A ( P195A ; n = 4 ) . spleen analysis at day 28 post tamoxifen treatment ;

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

(J) Differential analysis of leukocyte populations in the spleen. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). spleen analysis at day 28 post tamoxifen treatment;


( K ) Thymic cellularity . ( L ) Differential analysis of thymocyte populations in the thymus . ( R26 - CreERki / + Adar1fl / + ( control ; n = 3 ) and R26 - CreERki / + Adar1fl / P195A ( P195A ; n = 4 ) . thymic analysis at day 28 post tamoxifen

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

(K) Thymic cellularity. (L) Differential analysis of thymocyte populations in the thymus. ( R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). thymic analysis at day 28 post tamoxifen treatment


( N ) qPCR ( SYBR green ) based analysis of indicated gene expression in BM . Data expressed as mean + / - SEM gene expression related to Ppia expression . R26 - CreERki / + Adar1fl / + ( control ; n = 3 ) and R26 - CreERki / + Adar1fl / P195A ( P195A ; n = 4 ) .

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

(N) qPCR (SYBR green) based analysis of indicated gene expression in BM. Data expressed as mean +/- SEM gene expression related to Ppia expression. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4).


, ( C ) survival analysis of mice of the indicated genotypes .

, (C) survival analysis of mice of the indicated genotypes.


( E ) survival of mice from inbreeding to generate P195A / - animals .

(E) survival of mice from inbreeding to generate P195A/- animals.


( H ) survival of pups derived from breeding of a Adar1P195A / P195A to a Adar1 + / - animal ; number as indicated for each genotype .

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

(H) survival of pups derived from breeding of a Adar1P195A/P195A to a Adar1+/- animal; number as indicated for each genotype.


( K ) survival of pups derived from breeding an Adar1P195A / - to an Adar1P195A / + genotype ; number indicated for each genotype . Inset photo : 35 - day - old Adar1P195A / - male bred from Adar1P195A / P195A x Adar1 + / - parents ( data in Panel F ) ; sibling 29 - day - old Adar1P195A / + and Adar1P195A / - male bred from an Adar1P195A / - x Adar1P195A / + ( data in Panel I ) .

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

(K) survival of pups derived from breeding an Adar1P195A/- to an Adar1P195A/+ genotype; number indicated for each genotype. Inset photo: 35-day-old Adar1P195A/- male bred from Adar1P195A/P195A x Adar1+/- parents (data in Panel F); sibling 29-day-old Adar1P195A/+ and Adar1P195A/- male bred from an Adar1P195A/- x Adar1P195A/+ (data in Panel I).


( B ) survival of Adar1P195A / E861AIfih1 - / - and littermates ( all Ifih1 - / - ) .

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

(B) survival of Adar1P195A/E861AIfih1-/- and littermates (all Ifih1-/-).


( D ) survival of Adar1P195A / - Ifih1 - / - and littermates ( all Ifih1 - / - ) .

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

(D) survival of Adar1P195A/-Ifih1-/- and littermates (all Ifih1-/-).


( F ) Individual plots of the normalised expression of ISGs and ISR genes in the kidney of the indicated genotypes using Taqman - based qPCR .

O O O 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) Individual plots of the normalised expression of ISGs and ISR genes in the kidney of the indicated genotypes using Taqman-based qPCR.


( G ) Western blot of PKR , phospho - eIF2α and total eIF2α levels in whole kidney lysates derived from the indicated genotypes ; same mice as used for panel F . Relative phospho - eIF2α expression was calculated against total eIF2α normalised to the average of P195A / + samples using ImageJ Fiji . All images are from the same gel and re - probed for different antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot of PKR, phospho-eIF2α and total eIF2α levels in whole kidney lysates derived from the indicated genotypes; same mice as used for panel F. Relative phospho-eIF2α expression was calculated against total eIF2α normalised to the average of P195A/+ samples using ImageJ Fiji. All images are from the same gel and re-probed for different antibodies.


( E ) Viability of cell lines with and without tamoxifen treatment ( isogenic pairs ) over 14 days of treatment ( n = 3 independent lines per genotype ; biological replicates ) .

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

(E) Viability of cell lines with and without tamoxifen treatment (isogenic pairs) over 14 days of treatment (n=3 independent lines per genotype; biological replicates).


( F ) qPCR ( SYBR green ) analysis of Ifit1 ( left ) and Irf7 ( right ) expression on day 14 of analysis .

O O 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) qPCR (SYBR green) analysis of Ifit1 (left) and Irf7 (right) expression on day 14 of analysis.


( G ) Cell viability at IFNβ dosages 0 , 50 and 1000U / mL after 24 hours . Statistical analysis was Two - way ANOVA with multiple comparisons correction .

O O O O O O O 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) Cell viability at IFNβ dosages 0, 50 and 1000U/mL after 24 hours. Statistical analysis was Two-way ANOVA with multiple comparisons correction.


( H ) qPCR ( SYBR green ) of Ifit1 ( left ) and Irf7 ( right ) expression in the cell lines collected at 3 or 24 hours post - treatment with 50 or 1000 U / mL of IFNβ . No statistical difference between genotypes in Ifit1 ( left ) and Irf7 ( right ) expression .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR (SYBR green) of Ifit1 (left) and Irf7 (right) expression in the cell lines collected at 3 or 24 hours post-treatment with 50 or 1000 U/mL of IFNβ. No statistical difference between genotypes in Ifit1 (left) and Irf7 (right) expression.


The reversal potential of currents was positively shifted when a long voltage ramp pulse followed the depolarizing steps . ( top panel ) The voltage protocol used for the experiment . ( bottom panel ) Representative traces of currents elicited by voltage ramp pulses are shown . ( top panel ) . The red line represents the arithmetical difference between currents elicited by 500 - ms ( 2 ) and 2 - ms pulses ( 1 ) . B . The summary bar graph represents the mean values of the reversal potentials of currents . ( 9 cells from 3 mice , paired Student t - test ; data are presented as mean values ± s . e . m ) .

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

The reversal potential of currents was positively shifted when a long voltage ramp pulse followed the depolarizing steps. (top panel) The voltage protocol used for the experiment. (bottom panel) Representative traces of currents elicited by voltage ramp pulses are shown. (top panel). The red line represents the arithmetical difference between currents elicited by 500-ms (2) and 2-ms pulses (1). B. The summary bar graph represents the mean values of the reversal potentials of currents. (9 cells from 3 mice, paired Student t-test; data are presented as mean values ± s.e.m).


Tail currents elicited by the hyperpolarizing voltage steps following the depolarizing steps of variable durations . Representative traces of currents elicited by the voltage protocol with a pipette solution containing 150 mM Cl - and 0 . 2 mM EGTA ( top panel ) , 150 mM gluconate ( middle panel ) , or 2 mM BAPTA ( bottom panel ) .

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

Tail currents elicited by the hyperpolarizing voltage steps following the depolarizing steps of variable durations. Representative traces of currents elicited by the voltage protocol with a pipette solution containing 150 mM Cl- and 0.2 mM EGTA (top panel), 150 mM gluconate (middle panel), or 2 mM BAPTA (bottom panel).


A summary graph of averaged tail current amplitudes as a function of the depolarizing step duration ( the numbers on the right indicate the numbers of recorded cells , data are presented as mean values ± s . e . m . )

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

A summary graph of averaged tail current amplitudes as a function of the depolarizing step duration (the numbers on the right indicate the numbers of recorded cells, data are presented as mean values ± s.e.m.)


A bar graph of averaged values of the tail current amplitudes summarizing the results of ( c ) at 500 ms . ( The numbers inside each bar indicate the numbers of recorded cells from at least 3 mice , unpaired Student t - test ; data are presented as mean values ± s . e . m . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 bar graph of averaged values of the tail current amplitudes summarizing the results of (c) at 500 ms. (The numbers inside each bar indicate the numbers of recorded cells from at least 3 mice, unpaired Student t-test; data are presented as mean values ± s.e.m.).


A summary bar graph showing the quantitative real - time PCR data for TMEM16A , TMEM16B , and BEST1 with cDNA samples prepared from the mHb and hippocampus of adult mouse brains ( N = 3 , n = 3 each , unpaired Student t - test , data are presented as mean values ± s . e . m . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 summary bar graph showing the quantitative real-time PCR data for TMEM16A, TMEM16B, and BEST1 with cDNA samples prepared from the mHb and hippocampus of adult mouse brains (N=3, n=3 each, unpaired Student t-test, data are presented as mean values ± s.e.m.).


RNAscope ISH showed an apparent expression of TMEM16A mRNA co - localized with ChAT and Tac1 signals in the mHb compared to the ones in lHb . Enlarged images showed that signals of TMEM16A and Tac1 in the dorsal mHb or ChAT in the ventral mHb reside together at the single cell level .

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

RNAscope ISH showed an apparent expression of TMEM16A mRNA co-localized with ChAT and Tac1 signals in the mHb compared to the ones in lHb. Enlarged images showed that signals of TMEM16A and Tac1 in the dorsal mHb or ChAT in the ventral mHb reside together at the single cell level.


Immunohistochemical staining of 40 - µm coronal brain sections with anti - TMEM16A antibody . In the mHb , TMEM16A signals colocalize with the cholinergic marker ChAT ( arrows ) . High magnifications of the indicated areas are shown in the merged panel .

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

Immunohistochemical staining of 40-µm coronal brain sections with anti-TMEM16A antibody. In the mHb, TMEM16A signals colocalize with the cholinergic marker ChAT (arrows). High magnifications of the indicated areas are shown in the merged panel.


Immunohistochemical staining of 40 - µm coronal brain sections with an anti - TMEM16A antibody . The TMEM16A signals in the mHb of TMEM16A cKO mice were compared to the ones of littermate controls . TMEM16A colocalized with the cholinergic marker ChAT in the mHb . The specific deletion of TMEM16A in the mHb is evident .

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

Immunohistochemical staining of 40-µm coronal brain sections with an anti-TMEM16A antibody. The TMEM16A signals in the mHb of TMEM16A cKO mice were compared to the ones of littermate controls. TMEM16A colocalized with the cholinergic marker ChAT in the mHb. The specific deletion of TMEM16A in the mHb is evident.


Representative traces demonstrating the spontaneous activity of the mHb neurons in a cell - attached mode from TMEM16A cKO mice compared to the one from littermate controls . D . A summary bar graph showing the frequencies of spontaneous firing of the mHb neurons in TMEM16A cKO ( 4 mice ) and littermate controls ( The numbers inside each bar indicate the numbers of recorded cells from 4 mice each , paired Student t - test , data are presented as mean values ± s . e . m . ) .

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Representative traces demonstrating the spontaneous activity of the mHb neurons in a cell-attached mode from TMEM16A cKO mice compared to the one from littermate controls. D. A summary bar graph showing the frequencies of spontaneous firing of the mHb neurons in TMEM16A cKO (4 mice) and littermate controls (The numbers inside each bar indicate the numbers of recorded cells from 4 mice each, paired Student t-test, data are presented as mean values ± s.e.m.).


Representative averaged traces of sAPs of the mHb neurons with the pipette solution ( 40 mM Cl - and 0 . 5 mM EGTA ) from TMEM16A cKO mice and littermate controls .

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

Representative averaged traces of sAPs of the mHb neurons with the pipette solution (40 mM Cl- and 0.5 mM EGTA) from TMEM16A cKO mice and littermate controls.


Summary bar graphs showing the frequency , half - width , and AHP slope of the sAPs of mHb neurons from TMEM16A cKO mice and littermate controls ( The numbers inside each bar indicate the numbers of recorded cells , unpaired Student t - test , data are presented as mean values ± s . e . m . ) .

Summary bar graphs showing the frequency, half-width, and AHP slope of the sAPs of mHb neurons from TMEM16A cKO mice and littermate controls (The numbers inside each bar indicate the numbers of recorded cells, unpaired Student t-test, data are presented as mean values ± s.e.m.).


Immunohistochemical images of the IPN region from control and TMEM16A cKO mice . The c - Fos signal was greatly reduced in TMEM16A cKO mice . Scale bar = 200 μm .

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

Immunohistochemical images of the IPN region from control and TMEM16A cKO mice. The c-Fos signal was greatly reduced in TMEM16A cKO mice. Scale bar = 200 μm.


A summary bar graph showing reduced c - Fos expression in the IPN in TMEM16A cKO mice . ( NCTL = 7 ( n = 42 ) , NcKO = 6 ( n = 36 ) , unpaired Student t - test , data are presented as mean values ± s . e . m . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 summary bar graph showing reduced c-Fos expression in the IPN in TMEM16A cKO mice. (NCTL=7 (n=42), NcKO=6 (n=36), unpaired Student t-test, data are presented as mean values ± s.e.m.).


Representative traces of sEPSCs of neurons in the medial part of the IPN showing a reduced frequency in TMEM16A cKO mice compared to that of littermate controls .

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

Representative traces of sEPSCs of neurons in the medial part of the IPN showing a reduced frequency in TMEM16A cKO mice compared to that of littermate controls.


Summary bar graphs of the frequency and amplitude of the sEPSCs of IPN neurons in TMEM16A cKO ( cKO ) mice and littermate controls ( CTL ) . Data are presented as mean values ± s . e . m . ( paired Student t - test ) . The numbers inside each bar indicate the numbers of recorded cells .

Summary bar graphs of the frequency and amplitude of the sEPSCs of IPN neurons in TMEM16A cKO (cKO) mice and littermate controls (CTL). Data are presented as mean values ± s.e.m. (paired Student t-test). The numbers inside each bar indicate the numbers of recorded cells.


In the EPM task , the time spent in the open arms was significantly decreased in cKO mice compared to that in the control ( CTL ) group ( nCTL = 20 , ncKO = 12 , unpaired Student t - test ) . B . In the LDB task , the time spent in the light zone by cKO mice was significantly decreased compared to that spent by the CTL mice ( nCTL = 20 , ncKO = 12 , unpaired Student t - test ) . C . In the PA task , the entry latency of cKO mice was significantly decreased compared to that of the CTL mice ( nCTL = 20 , ncKO = 12 , unpaired Student t - test ) . . D . Nestlet shredding behaviors of the cKO group were also decreased compared to those of the CTL group . The weight of the shredded nestlets of the cKO group was significantly lower than that of the CTL group ( nCTL = 20 , ncKO = 12 , unpaired Student t - test ) .

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

In the EPM task, the time spent in the open arms was significantly decreased in cKO mice compared to that in the control (CTL) group (nCTL=20, ncKO=12, unpaired Student t-test). B. In the LDB task, the time spent in the light zone by cKO mice was significantly decreased compared to that spent by the CTL mice (nCTL=20, ncKO=12, unpaired Student t-test). C. In the PA task, the entry latency of cKO mice was significantly decreased compared to that of the CTL mice (nCTL=20, ncKO=12, unpaired Student t-test)..D. Nestlet shredding behaviors of the cKO group were also decreased compared to those of the CTL group. The weight of the shredded nestlets of the cKO group was significantly lower than that of the CTL group (nCTL=20, ncKO=12, unpaired Student t-test).


In the 3 - chamber test , the cKO mice exhibited a decreased interest for the stranger mouse ( S1 ) against objects ( O ) compared to the CTL group ( nCTL = 12 , ncKO = 14 , unpaired Student t - test ) . F . When cKO mice were exposed to a new stranger mouse ( S2 ) against the familiar S1 mouse , they also showed a decreased interest for the S2 mouse compared to that of the CTL group . The preference index showed the decreased sociability of the cKO group in both sessions ( nCTL = 12 , ncKO = 14 , unpaired Student t - test ) . Data information : All data are presented as mean values ± s . e . m .

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

In the 3-chamber test, the cKO mice exhibited a decreased interest for the stranger mouse (S1) against objects (O) compared to the CTL group (nCTL=12, ncKO=14, unpaired Student t-test). F. When cKO mice were exposed to a new stranger mouse (S2) against the familiar S1 mouse, they also showed a decreased interest for the S2 mouse compared to that of the CTL group. The preference index showed the decreased sociability of the cKO group in both sessions (nCTL=12, ncKO=14, unpaired Student t-test). Data information: All data are presented as mean values ± s.e.m.


Representative traces showing that the spontaneous firing of mHb neurons from ChAT ( + ) - cre : : hM4D ( Gi ) mice were reduced when CNO ( 10 μM ) was applied in the ACSF .

O O O 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

Representative traces showing that the spontaneous firing of mHb neurons from ChAT(+)-cre::hM4D(Gi) mice were reduced when CNO (10 μM) was applied in the ACSF.


A summary bar graph showing the reduction of normalized frequencies of spontaneous firing of the mHb neurons transfected with AAV - hM4Di - mCherry before and during CNO treatment ( The numbers inside each bar indicate the numbers of recorded cells ) .

O O O 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

A summary bar graph showing the reduction of normalized frequencies of spontaneous firing of the mHb neurons transfected with AAV-hM4Di-mCherry before and during CNO treatment (The numbers inside each bar indicate the numbers of recorded cells).


In the EPM task , the time spent in the open arms of CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice was significantly decreased compared to that of the PBS - treated controls ( CTL ) . ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ) . E . In the LDB task , the time spent in the light zone by CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice was significantly decreased compared to that spent by the PBS - treated control mice . ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ) . F . In the PA task , the entry latency of CNO - treated mice was significantly decreased compared to that of the CTL group . ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ) . G . Nestlet shredding behaviors of the CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice were also decreased . ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ; ) .

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

In the EPM task, the time spent in the open arms of CNO-treated ChAT(+)-cre::hM4D(Gi) mice was significantly decreased compared to that of the PBS-treated controls (CTL). (nCTL=6, ncKO=9, unpaired Student t-test). E. In the LDB task, the time spent in the light zone by CNO-treated ChAT(+)-cre::hM4D(Gi) mice was significantly decreased compared to that spent by the PBS-treated control mice. (nCTL=6, ncKO=9, unpaired Student t-test). F. In the PA task, the entry latency of CNO-treated mice was significantly decreased compared to that of the CTL group. (nCTL=6, ncKO=9, unpaired Student t-test). G. Nestlet shredding behaviors of the CNO-treated ChAT(+)-cre::hM4D(Gi) mice were also decreased. (nCTL=6, ncKO=9, unpaired Student t-test;).


In the 3 - chamber test , the subject mouse of the PBS - treated mice and the CNO - treated mice explored freely the three test chambers . The time spent in each chamber during two sessions was measured . The CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice exhibited a decreased interest for a stranger mouse ( S1 ) against objects ( O ) compared to the PBS - treated group ( left two panels ) ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ) . I . When CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice were exposed to a new stranger mouse ( S2 ) against the familiar S1 mouse , they also showed a decreased interest for the S2 mouse compared to the PBS - treated control group ( right two panels ) . The preference index showed decreased sociability of the CNO - treated group in both sessions ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ; ) .

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 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 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

In the 3-chamber test, the subject mouse of the PBS-treated mice and the CNO-treated mice explored freely the three test chambers. The time spent in each chamber during two sessions was measured. The CNO-treated ChAT(+)-cre::hM4D(Gi) mice exhibited a decreased interest for a stranger mouse (S1) against objects (O) compared to the PBS-treated group (left two panels) (nCTL=6, ncKO=9, unpaired Student t-test). I. When CNO-treated ChAT(+)-cre::hM4D(Gi) mice were exposed to a new stranger mouse (S2) against the familiar S1 mouse, they also showed a decreased interest for the S2 mouse compared to the PBS-treated control group (right two panels). The preference index showed decreased sociability of the CNO-treated group in both sessions (nCTL=6, ncKO=9, unpaired Student t-test;).


A . Western blot analysis of reporter HslO - FLAG protein produced with a custom - made PURExpress ® coupled in vitro transcription - translation system supplemented with the indicated EF - G proteins and incubated for the indicated times . Blot was developed using anti - FLAG antibodies . Shown is a representative from at least two independent experiments . Eco EF - G : E . coli EF - G .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Western blot analysis of reporter HslO-FLAG protein produced with a custom-made PURExpress® coupled in vitro transcription-translation system supplemented with the indicated EF-G proteins and incubated for the indicated times. Blot was developed using anti-FLAG antibodies. Shown is a representative from at least two independent experiments. Eco EF-G: E. coli EF-G.


B . Western blot analysis of reporter HslO - FLAG protein synthesized in vitro using a custom - made PURExpress ® system with EF - G1 , EF - G2 , and two engineered EF - G2 variants : one missing the 26 - amino acid insert , EF - G2 ( ∆ 514 - 539 ) and one missing the beta hairpin in the insert , EF - G2 ( ∆ 516 - 527 ) . Shown is a representative experiment from at least two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Western blot analysis of reporter HslO-FLAG protein synthesized in vitro using a custom-made PURExpress® system with EF-G1, EF-G2, and two engineered EF-G2 variants: one missing the 26-amino acid insert, EF-G2 (∆514-539) and one missing the beta hairpin in the insert, EF-G2 (∆516-527). Shown is a representative experiment from at least two independent experiments.


D . SDS - PAGE analysis of co - sedimentation assays monitoring factor ( EF - G1 EF - G2 or EF - G2 variant EF - G2 ( ∆ 516 - 527 ) or EF - G2 ( ∆ 514 - 539 ) ) binding to E . coli 70S ribosomes . Binding reactions were centrifuged through a sucrose cushion to recover ribosome and associated factor in the pellets . The positions of EF - G proteins and ribosomal proteins and molecular weight markers are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SDS-PAGE analysis of co-sedimentation assays monitoring factor (EF-G1 EF-G2 or EF-G2 variant EF-G2 (∆516-527) or EF-G2 (∆514-539)) binding to E. coli 70S ribosomes. Binding reactions were centrifuged through a sucrose cushion to recover ribosome and associated factor in the pellets. The positions of EF-G proteins and ribosomal proteins and molecular weight markers are indicated.


E . Western blot analysis of reporter HisC - FLAG protein synthesized in vitro using a custom - made PURExpress ® system with the EF - G1 , EF - G2 or EF - G2 ( S84H ) proteins . Shown is a representative from at least two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Western blot analysis of reporter HisC-FLAG protein synthesized in vitro using a custom-made PURExpress® system with the EF-G1, EF-G2 or EF-G2(S84H) proteins. Shown is a representative from at least two independent experiments.


Western blot analysis of B . thetaiotaomicron strains expressing C - terminally FLAG - tagged EF - G1 ( WH405 ) ( A ) , or EF - G2 ( GT1301 ) ( B ) , from their normal promoters and chromosomal locations , grown in Tryptone Yeast Extract Glucose liquid medium ( TYG ) and sampled at the indicated times .

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

Western blot analysis of B. thetaiotaomicron strains expressing C-terminally FLAG-tagged EF-G1 (WH405) (A), or EF-G2 (GT1301) (B), from their normal promoters and chromosomal locations, grown in Tryptone Yeast Extract Glucose liquid medium (TYG) and sampled at the indicated times.


E - F . Western blot of crude extracts from wild - type B . thetaiotaomicron harvested from the ceca of mice ( representative blot for 2 out of 4 mice tested ) or corresponding to stationary phase cultures harvested following growth in TYG for 24 h or purified EF - G1 and EF - G2 proteins at the indicated amounts ( E ) . Blots were developed with anti - EF - G1 or EF - G2 polyclonal antibodies ; anti - GroEL antibodies were used as loading controls . Western blot quantifications of all four biological replicates and their average are shown in ( F ) , where EF - G1 and EF - G2 abundances were estimated by normalizing the signal of bacterial sample to the closest signal of known amount of purified protein , and the derived absolute amount ( pmol ) was normalized to the signal of GroEL .

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

E-F. Western blot of crude extracts from wild-type B. thetaiotaomicron harvested from the ceca of mice (representative blot for 2 out of 4 mice tested) or corresponding to stationary phase cultures harvested following growth in TYG for 24 h or purified EF-G1 and EF-G2 proteins at the indicated amounts (E). Blots were developed with anti-EF-G1 or EF-G2 polyclonal antibodies; anti-GroEL antibodies were used as loading controls. Western blot quantifications of all four biological replicates and their average are shown in (F), where EF-G1 and EF-G2 abundances were estimated by normalizing the signal of bacterial sample to the closest signal of known amount of purified protein, and the derived absolute amount (pmol) was normalized to the signal of GroEL.


( A ) Dynamic regulation of fluorescently tagged Beta - catenin / Armadillo ( Arm - GFP ) during mitosis in the growing fly wing epithelium . Notice down - regulation of Arm - GFP as cells round up for mitosis ( arrow ) . Scale bar ~ 2µm . n > 10 independent biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dynamic regulation of fluorescently tagged Beta-catenin/Armadillo (Arm-GFP) during mitosis in the growing fly wing epithelium. Notice down-regulation of Arm-GFP as cells round up for mitosis (arrow). Scale bar ~2µm. n>10 independent biological replicates.


( B ) Dynamic regulation of fluorescently tagged E - cadherin ( E - cad - GFP ) during mitosis in the growing fly wing epithelium . Notice down - regulation of E - cad - GFP as cells round up for mitosis ( arrow ) . Scale bar ~ 2µm . n > 10 independent biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dynamic regulation of fluorescently tagged E-cadherin (E-cad-GFP) during mitosis in the growing fly wing epithelium. Notice down-regulation of E-cad-GFP as cells round up for mitosis (arrow). Scale bar ~2µm. n>10 independent biological replicates.


( C ) E - cad - GFP remains downregulated in cells arrested in mitosis with Colchicine . Scale bar ~ 2µm . n > 10 independent biological replicates .

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

(C) E-cad-GFP remains downregulated in cells arrested in mitosis with Colchicine. Scale bar ~2µm. n>10 independent biological replicates.


( D ) Quantification of the degree of down - regulation of Arm - GFP and E - cad - GFP at the junction between mitotic cells and their interphase neighbours ( n > 10 independent samples per cell cycle stage ) . Mean + / - 1 S . D . shown .

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

(D) Quantification of the degree of down-regulation of Arm-GFP and E-cad-GFP at the junction between mitotic cells and their interphase neighbours (n > 10 independent samples per cell cycle stage). Mean +/- 1 S.D. shown.


( E ) Two adjacent mitotic cells show no detectable Arm - GFP at the junction between them . Scale bar ~ 2µm .

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) Two adjacent mitotic cells show no detectable Arm-GFP at the junction between them. Scale bar ~2µm.


( F - I ) Electron microscopy cross - sections of wing epithelial cells at interphase , prophase , prometaphase and telophase . Notice adherens junctions ( dark staining , highlighted in insets ) are downregulated as cells round up for mitosis . Scale bars ~ 1µm ( low mag . ) and ~ 10nm ( high mag . ) . n > 10 independent biological replicates . Asterisks indicate a single mitotic cell . Green arrowheads indicate adherens junctions .

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

(F-I) Electron microscopy cross-sections of wing epithelial cells at interphase, prophase, prometaphase and telophase. Notice adherens junctions (dark staining, highlighted in insets) are downregulated as cells round up for mitosis. Scale bars ~1µm (low mag.) and ~10nm (high mag.). n>10 independent biological replicates. Asterisks indicate a single mitotic cell. Green arrowheads indicate adherens junctions.


( A ) Phosphorylated Myosin - II Regulatory Light Chain ( p - MLC ; purple ) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild - type wing epithelial cells in the anterior ( A ) compartment , but not in Rho - kinase ( Rok ) RNAi expressing cells of the posterior ( P ) compartment ( GFP - positive ; green ) . Scale bar ~ 10µm . n > 10 independent biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; purple) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild-type wing epithelial cells in the anterior (A) compartment, but not in Rho-kinase (Rok) RNAi expressing cells of the posterior (P) compartment (GFP-positive; green). Scale bar ~10µm. n>10 independent biological replicates.


( B ) High - magnification view of single mitotic cell stained for p - MLC ( purple ) and E - cadherin ( green ) , with metaphase plate chromosomes stained by DAPI ( blue ) . Scale bar ~ 1µm . n > 10 independent biological replicates . ( C ) High - magnification view of single UAS . Rok - RNAi expressing mitotic cell stained for p - MLC ( purple ) and E - cadherin ( green ) , with metaphase plate chromosomes stained by DAPI ( blue ) . Note loss of cortical p - MLC staining ( purple ) . Scale bar ~ 1µm . n > 10 independent biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) High-magnification view of single mitotic cell stained for p-MLC (purple) and E-cadherin (green), with metaphase plate chromosomes stained by DAPI (blue). Scale bar ~1µm. n>10 independent biological replicates. (C) High-magnification view of single UAS.Rok-RNAi expressing mitotic cell stained for p-MLC (purple) and E-cadherin (green), with metaphase plate chromosomes stained by DAPI (blue). Note loss of cortical p-MLC staining (purple). Scale bar ~1µm. n>10 independent biological replicates.


( D ) Expression of constitutively active Rho - kinase ( UAS . Rok - CA ) in the posterior compartment of the wing epithelium is sufficient to elevate p - MLC and reduce E - cadherin immunostaining . Zoom ( right ) shows high p - MLC and low E - cad in both a mitotic cell and its neighbours . Scale bar ~ 10µm . n > 10 independent biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of constitutively active Rho-kinase (UAS.Rok-CA) in the posterior compartment of the wing epithelium is sufficient to elevate p-MLC and reduce E-cadherin immunostaining. Zoom (right) shows high p-MLC and low E-cad in both a mitotic cell and its neighbours. Scale bar ~10µm. n>10 independent biological replicates.


( E ) MyoII RLC - GFP ( MyoII - GFP ) accumulates at the cortex as cells round up for mitosis and later appears at the cleavage furrow during cytokinesis ( basal section , diagrammed left ) . Treatment with the Rho - kinase inhibitor Y - 27632 inhibits mitotic accumulation of MyoII - GFP . Quantification shown right ( n = 8 independent samples per genotype ) . Scale bar ~ 1µm . Mean + / - 1 S . D . shown .

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

(E) MyoII RLC-GFP (MyoII-GFP) accumulates at the cortex as cells round up for mitosis and later appears at the cleavage furrow during cytokinesis (basal section, diagrammed left). Treatment with the Rho-kinase inhibitor Y-27632 inhibits mitotic accumulation of MyoII-GFP. Quantification shown right (n = 8 independent samples per genotype). Scale bar ~1µm. Mean +/- 1 S.D. shown.


( F ) E - cad - GFP is downregulated as cells round up for mitosis ( apical section , diagrammed left ) . Treatment with the Rho - kinase inhibitor Y - 27632 inhibits mitotic downregulation of E - cad - GFP . Extended treatment with Y - 27632 leads to a general defect in all interphase and mitotic cells in which apical surface areas enlarge and E - cad - GFP is generally lost . Arrow indicates mitotic cell cortex at maximal rounding . Quantification shown right ( n = 6 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bar ~ 1µm .

O O O O O O O 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-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 O O O O O O O O O O O O O O O O O O O O O

(F) E-cad-GFP is downregulated as cells round up for mitosis (apical section, diagrammed left). Treatment with the Rho-kinase inhibitor Y-27632 inhibits mitotic downregulation of E-cad-GFP. Extended treatment with Y-27632 leads to a general defect in all interphase and mitotic cells in which apical surface areas enlarge and E-cad-GFP is generally lost. Arrow indicates mitotic cell cortex at maximal rounding. Quantification shown right (n = 6 independent samples per genotype; mean +/- 1 S.D. shown). Scale bar ~1µm.


( A ) Nuclear localised Pbl - GFP becomes cytoplasmic in mitotic cells , co - incident with Rok - GFP relocalisation to the plasma membrane . Scale bar ~ 1µm . n > 5 independent biological replicates .

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

(A) Nuclear localised Pbl-GFP becomes cytoplasmic in mitotic cells, co-incident with Rok-GFP relocalisation to the plasma membrane. Scale bar ~1µm. n > 5 independent biological replicates.


( B ) Phosphorylated Myosin - II Regulatory Light Chain ( p - MLC ; purple ) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild - type wing epithelial cells , but not those expressing UAS . pbl - RNAi . Phospho - Histone H3 ( pH3 ) staining ( green ) marks mitotic cell chromosomes . Quantification shown right ( n > 10 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bar ~ 1µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; purple) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild-type wing epithelial cells, but not those expressing UAS.pbl-RNAi. Phospho-Histone H3 (pH3) staining (green) marks mitotic cell chromosomes. Quantification shown right (n > 10 independent samples per genotype; mean +/- 1 S.D. shown). Scale bar ~1µm.


( C ) A stripe of expression of ptc . Gal4 UAS . pbl - RNAi in the wing disc leads to both enlarged cells and many basally extruded cells that undergo apoptosis , marked by pyknotic nuclei and loss of aPKC / Dlg staining . Scale bars ~ 20µm . n > 6 independent biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A stripe of expression of ptc.Gal4 UAS.pbl-RNAi in the wing disc leads to both enlarged cells and many basally extruded cells that undergo apoptosis, marked by pyknotic nuclei and loss of aPKC/Dlg staining. Scale bars ~20µm. n > 6 independent biological replicates.


( A ) Nuclear localised Tum - GFP becomes cytoplasmic in mitotic cells , including a distinct plasma membrane localisation ( arrows ) . Note also localisation to the cytokinetic ring ( asterisk ) and remnant mid - bodies / ring canals in interphase cells . Quantification shown on the right ( n > 7 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bars ~ 2µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Nuclear localised Tum-GFP becomes cytoplasmic in mitotic cells, including a distinct plasma membrane localisation (arrows). Note also localisation to the cytokinetic ring (asterisk) and remnant mid-bodies/ring canals in interphase cells. Quantification shown on the right (n > 7 independent samples per genotype; mean +/- 1 S.D. shown). Scale bars ~2µm.


( B ) A stripe of expression of ptc . Gal4 UAS . tum - RNAi in the wing disc leads to basally extruded cells that undergo apoptosis , marked by Dcp1 immunostaining , similar to UAS . pbl - RNAi . Quantification shown on the right ( n > 10 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bars ~ 20µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A stripe of expression of ptc.Gal4 UAS.tum-RNAi in the wing disc leads to basally extruded cells that undergo apoptosis, marked by Dcp1 immunostaining, similar to UAS.pbl-RNAi. Quantification shown on the right (n > 10 independent samples per genotype; mean +/- 1 S.D. shown). Scale bars ~20µm.


( C ) Phosphorylated Myosin - II Regulatory Light Chain ( p - MLC ; red ) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild - type wing epithelial cells , but not those cells homozygous mutant for tum347 , marked by expression of GFP in single - celled clones ( MARCM system ) . pH3 staining ( blue ) marks mitotic cell chromosomes . Arrow indicates a GFP - positive single cell tum347 mutant clone . Quantification shown on the right ( n > 3 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bars ~ 20µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; red) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild-type wing epithelial cells, but not those cells homozygous mutant for tum347, marked by expression of GFP in single-celled clones (MARCM system). pH3 staining (blue) marks mitotic cell chromosomes. Arrow indicates a GFP-positive single cell tum347 mutant clone. Quantification shown on the right (n > 3 independent samples per genotype; mean +/- 1 S.D. shown). Scale bars ~20µm.


( A ) Phosphorylated Myosin - II Regulatory Light Chain ( p - MLC ; purple ) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells ( pH3 , red ) in wild - type wing discs , and aurA homozygous mutant wing discs , but not upon inhibition of both AurA and AurB by treatment with VX - 680 . Note increased numbers of mitotic cells in aurA homozygous mutant wing discs , due to delayed mitotic progression . Scale bars ~ 20µm ( low mag . ) and ~ 1µm ( high mag . ) . n > 15 independent biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; purple) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells (pH3, red) in wild-type wing discs, and aurA homozygous mutant wing discs, but not upon inhibition of both AurA and AurB by treatment with VX-680. Note increased numbers of mitotic cells in aurA homozygous mutant wing discs, due to delayed mitotic progression. Scale bars ~20µm (low mag.) and ~1µm (high mag.). n > 15 independent biological replicates.


( B ) Live - imaging of MyoII - GFP reveals delayed mitosis in aurA homozygous mutant wing discs , while inhibition of both AurA and AurB by treatment with VX - 680 leads to complete loss of mitotic rounding and MyoII - GFP membrane localisation . Asterisks indicate a single mitotic cell . Scale bars ~ 1µm . Quantification shown on the bottom right ( n = 5 independent samples per genotype ) .

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

(B) Live-imaging of MyoII-GFP reveals delayed mitosis in aurA homozygous mutant wing discs, while inhibition of both AurA and AurB by treatment with VX-680 leads to complete loss of mitotic rounding and MyoII-GFP membrane localisation. Asterisks indicate a single mitotic cell. Scale bars ~1µm. Quantification shown on the bottom right (n = 5 independent samples per genotype).


( C ) Live - imaging of E - cad - GFP reveals delayed mitosis in aurA homozygous mutant wing discs , cytokinesis failure in UAS . aurB - RNAi expressing wing discs , and complete failure of mitotic rounding and E - cad - GFP downregulation upon inhibition of both AurA and AurB by treatment with VX - 680 . Asterisks indicate a single mitotic cell . Scale bars ~ 1µm . Quantification shown on the bottom right ( n = 9 independent samples per genotype ) .

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

(C) Live-imaging of E-cad-GFP reveals delayed mitosis in aurA homozygous mutant wing discs, cytokinesis failure in UAS.aurB-RNAi expressing wing discs, and complete failure of mitotic rounding and E-cad-GFP downregulation upon inhibition of both AurA and AurB by treatment with VX-680. Asterisks indicate a single mitotic cell. Scale bars ~1µm. Quantification shown on the bottom right (n = 9 independent samples per genotype).


( A ) Striped ptc . Gal4 - driven expression of UAS . GFP in a control wing imaginal disc . Scale bars ~ 20µm . n > 6 independent biological replicates . ( B ) Striped ptc . Gal4 - driven expression of UAS . GFP in an aurA homozygous mutant wing imaginal disc leads to increased numbers of pH3 positive mitotic cells . n > 7 independent biological replicates . ( C ) Striped ptc . Gal4 - driven expression of UAS . GFP and UAS . aurB - RNAi in a control wing imaginal disc leads to enlarged cells with large nuclei ( marked by DAPI ; white arrowhead ) . n > 6 independent biological replicates . ( D ) Striped ptc . Gal4 - driven expression of UAS . GFP and UAS . aurB - RNAi in an aurA homozygous mutant wing imaginal disc leads to extrusion and apoptosis of cells ( marked by Dcp1 and pyknotic nuclei ) . n > 8 independent biological replicates . ( E ) Striped ptc . Gal4 - driven expression of UAS . GFP and UAS . pbl - RNAi in the wing imaginal disc leads to extrusion and apoptosis of cells ( marked by Dcp1 and pyknotic nuclei ) . n > 8 independent biological replicates . ( F ) ptc . Gal4 - driven expression of UAS . aurB - RNAi in an aurA homozygous mutant endoreplicating salivary gland has no phenotypic consequence . n > 6 independent biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Striped ptc.Gal4-driven expression of UAS.GFP in a control wing imaginal disc. Scale bars ~20µm. n > 6 independent biological replicates. (B) Striped ptc.Gal4-driven expression of UAS.GFP in an aurA homozygous mutant wing imaginal disc leads to increased numbers of pH3 positive mitotic cells. n > 7 independent biological replicates. (C) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.aurB-RNAi in a control wing imaginal disc leads to enlarged cells with large nuclei (marked by DAPI; white arrowhead). n > 6 independent biological replicates. (D) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.aurB -RNAi in an aurA homozygous mutant wing imaginal disc leads to extrusion and apoptosis of cells (marked by Dcp1 and pyknotic nuclei). n > 8 independent biological replicates. (E) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.pbl-RNAi in the wing imaginal disc leads to extrusion and apoptosis of cells (marked by Dcp1 and pyknotic nuclei). n > 8 independent biological replicates. (F) ptc.Gal4-driven expression of UAS.aurB-RNAi in an aurA homozygous mutant endoreplicating salivary gland has no phenotypic consequence. n > 6 independent biological replicates.


( G ) MARCM clones ( GFP - positive ) expressing UAS . aurB - RNAi in an aurA homozygous mutant cells result in single cell clones ( i . e . : no cell division ) , while individual loss of AurA or AurB allows some cell proliferation . Quantification shown on the right ( n > 7 independent samples per genotype ; Mean + / - 1 S . D . shown ) . Scale bar ~ 20µm .

(G) MARCM clones (GFP-positive) expressing UAS.aurB-RNAi in an aurA homozygous mutant cells result in single cell clones (i.e.: no cell division), while individual loss of AurA or AurB allows some cell proliferation. Quantification shown on the right (n > 7 independent samples per genotype; Mean +/- 1 S.D. shown). Scale bar ~20µm.


( A ) Live - imaging of MyoII - GFP in a control wing disc epithelium . Asterisk indicates the location of a single mitotic cell about to initiate rounding . Scale bar ~ 1µm . n > 5 independent biological replicates . ( B ) Live - imaging of MyoII - GFP upon treatment with the dual AurA / B inhibitor VX - 680 after initiation of mitotic rounding , which rapidly arrests progression of rounding and prevents cytokinesis . Scale bar ~ 1µm . n > 9 independent biological replicates .

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

(A) Live-imaging of MyoII-GFP in a control wing disc epithelium. Asterisk indicates the location of a single mitotic cell about to initiate rounding. Scale bar ~1µm. n > 5 independent biological replicates. (B) Live-imaging of MyoII-GFP upon treatment with the dual AurA/B inhibitor VX-680 after initiation of mitotic rounding, which rapidly arrests progression of rounding and prevents cytokinesis. Scale bar ~1µm. n > 9 independent biological replicates.


( C ) Live - imaging of E - cad - GFP upon treatment with the dual AurA / B inhibitor VX - 680 after initiation of mitotic rounding , which rapidly arrests progression of rounding and prevents cytokinesis . Scale bar ~ 1µm . n > 6 independent biological replicates .

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

(C) Live-imaging of E-cad-GFP upon treatment with the dual AurA/B inhibitor VX-680 after initiation of mitotic rounding, which rapidly arrests progression of rounding and prevents cytokinesis. Scale bar ~1µm. n > 6 independent biological replicates.


( A ) Early stage Drosophila egg chambers showing giant germ - line nurse cells surrounded by E - cadherin - positive follicle cell epithelium . A follicle cell undergoing mitotic rounding is highlighted with an asterisk ( * ) . Scale bar ~ 3µm . n > 3 independent biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Early stage Drosophila egg chambers showing giant germ-line nurse cells surrounded by E-cadherin-positive follicle cell epithelium. A follicle cell undergoing mitotic rounding is highlighted with an asterisk (*). Scale bar ~3µm. n > 3 independent biological replicates.


( B ) High resolution imaging of the F - actin cortex , E - cadherin , and phospho - MyoII in a mitotically rounded follicle cell . Note maintenance of adherens junctions including both E - cad and the contractile actomyosin ring during mitotic rounding . Asterisks or pH3 staining indicate a single mitotic cell . Scale bar ~ 3µm . n > 4 independent biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) High resolution imaging of the F-actin cortex, E-cadherin, and phospho-MyoII in a mitotically rounded follicle cell. Note maintenance of adherens junctions including both E-cad and the contractile actomyosin ring during mitotic rounding. Asterisks or pH3 staining indicate a single mitotic cell. Scale bar ~3µm. n > 4 independent biological replicates.


( B ) RAW 264 . 7 cells transfected for 48 h with control ( sc ) , MTMR6 or Jumpy siRNA , were pretreated for 30 min with 100 nM Bafilomycin A1 ( BafA1 ) , 10 μg / ml E64d and 10 μg / ml pepstatin , then incubated for 30 min in full or starvation media in the presence of BafA1 , E64d and pepstatin . Cells were lysed and analysed by immunoblotting with anti - LC3 or anti ‐ actin . Densitometric LC3 ‐ II / actin ratios are shown underneath the blot . Graph : LC3 ‐ II / actin ratio for Jumpy siRNA in full medium is equal to or exceeds LC3 ‐ II / actin ratio for control siRNA ( sc ) in starvation medium .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RAW 264.7 cells transfected for 48 h with control (sc), MTMR6 or Jumpy siRNA, were pretreated for 30 min with 100 nM Bafilomycin A1 (BafA1), 10 μg/ml E64d and 10 μg/ml pepstatin, then incubated for 30 min in full or starvation media in the presence of BafA1, E64d and pepstatin. Cells were lysed and analysed by immunoblotting with anti-LC3 or anti‐actin. Densitometric LC3‐II/actin ratios are shown underneath the blot. Graph: LC3‐II/actin ratio for Jumpy siRNA in full medium is equal to or exceeds LC3‐II/actin ratio for control siRNA (sc) in starvation medium.


( C , D ) RAW 264 . 7 cells were transfected for 36 h with control ( scramble ) or Jumpy siRNA , transfected once more with corresponding siRNA and mRFP ‐ GFP ‐ LC3 DNA construct overnight and incubated for 2 h in full or starvation media . Cells were fixed and LC3 puncta analysed by confocal fluorescence microscopy . ( C ) Representative confocal images of RAW 264 . 7 cells in full or starvation media after transfection with control ( scramble ) or Jumpy siRNA ( siJumpy ) . Red and yellow arrows indicate GFP − RFP + and GFP + RFP + puncta , respectively . ( D ) Quantitation of number of LC3 puncta per cell , total puncta per cell ( GFP + RFP + and GFP − RFP + puncta ) GFP + RFP + puncta per cell and GFP − RFP + puncta per cell . Data , mean ± s . e . m . n = 5 independent experiments , 30 cells per experiments . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ( t ‐ test ) . Scale bars , 5 μm .

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

(C, D) RAW 264.7 cells were transfected for 36 h with control (scramble) or Jumpy siRNA, transfected once more with corresponding siRNA and mRFP‐GFP‐LC3 DNA construct overnight and incubated for 2 h in full or starvation media. Cells were fixed and LC3 puncta analysed by confocal fluorescence microscopy. (C) Representative confocal images of RAW 264.7 cells in full or starvation media after transfection with control (scramble) or Jumpy siRNA (siJumpy). Red and yellow arrows indicate GFP−RFP+ and GFP+RFP+ puncta, respectively. (D) Quantitation of number of LC3 puncta per cell, total puncta per cell (GFP+RFP+ and GFP−RFP+ puncta) GFP+RFP+ puncta per cell and GFP−RFP+ puncta per cell. Data, mean±s.e.m. n=5 independent experiments, 30 cells per experiments. *P0.05, **P0.01, ***P0.001 (t‐test). Scale bars, 5 μm.


( E , F ) C2C12 myoblasts were transfected for 48 h with control ( scramble ) or Jumpy siRNA , followed by a second transfection overnight with corresponding siRNA and mRFP ‐ GFP ‐ LC3 DNA construct . Cells were fixed and LC3 puncta were counted by confocal fluorescence microscopy . ( E ) Representative confocal images of C2C12 cells in full media after transfection with control ( scramble ) or Jumpy siRNA ( siJumpy ) . ( F ) Quantification of the number of LC3 puncta per cell . Data , mean ± SEM for n = 3 ( independent experiments ) , 30 cells per experiments . * P 0 . 05 ( t - test ) . Scale bars , 10 μm .

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

(E, F) C2C12 myoblasts were transfected for 48 h with control (scramble) or Jumpy siRNA, followed by a second transfection overnight with corresponding siRNA and mRFP‐GFP‐LC3 DNA construct. Cells were fixed and LC3 puncta were counted by confocal fluorescence microscopy. (E) Representative confocal images of C2C12 cells in full media after transfection with control (scramble) or Jumpy siRNA (siJumpy). (F) Quantification of the number of LC3 puncta per cell. Data, mean±SEM for n=3 (independent experiments), 30 cells per experiments. *P 0.05 (t-test). Scale bars, 10 μm.


( G , H ) C2C12 cells were transfected for 48 h with control ( sc ) ( lanes 1 , 2 , 5 , 6 ) or Atg5 siRNA ( lanes 3 , 4 , 6 , 7 ) followed by a second transfection for 48 h with same siRNA and control ( lanes 1 , 3 , 5 , 7 ) or Jumpy siRNA ( lanes 2 , 4 , 6 , 8 ) . Cells were incubated for 1 h with or without 100 nM Baf A1 , 10 μg / ml E64d and 10 μg / ml pepstatin in full media , lysed and analysed by immunoblotting with anti ‐ LC3 or anti ‐ actin ( G ) . ( H ) Densitometric LC3 ‐ II / actin ratios for samples treated with BafA1 and protease inhibitors from G ( lanes 5 - 8 ) . Inset shows Atg5 knock ‐ down by immunoblotting .

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

(G, H) C2C12 cells were transfected for 48 h with control (sc) (lanes 1, 2, 5, 6) or Atg5 siRNA (lanes 3, 4, 6, 7) followed by a second transfection for 48 h with same siRNA and control (lanes 1, 3, 5, 7) or Jumpy siRNA (lanes 2, 4, 6, 8). Cells were incubated for 1 h with or without 100 nM Baf A1, 10 μg/ml E64d and 10 μg/ml pepstatin in full media, lysed and analysed by immunoblotting with anti‐LC3 or anti‐actin (G). (H) Densitometric LC3‐II/actin ratios for samples treated with BafA1 and protease inhibitors from G (lanes 5-8). Inset shows Atg5 knock‐down by immunoblotting.


( A ) Proteolysis of long ‐ lived proteins in C2C12 myoblasts . C2C12 cells were transfected with control ( scramble ) or Jumpy siRNA ( siJumpy ) , labelled overnight in media containing [ 3H ] leucine , washed , incubated for 2 h in complete media ( containing cold leucine ) and incubated for 4 h in full or starvation media . Leucine release was calculated from radioactivity in the tricarboxylic acid ‐ soluble form relative to total cell radioactivity . Results shown represent mean ± s . e . m . for combined data from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Proteolysis of long‐lived proteins in C2C12 myoblasts. C2C12 cells were transfected with control (scramble) or Jumpy siRNA (siJumpy), labelled overnight in media containing [3H] leucine, washed, incubated for 2 h in complete media (containing cold leucine) and incubated for 4 h in full or starvation media. Leucine release was calculated from radioactivity in the tricarboxylic acid‐soluble form relative to total cell radioactivity. Results shown represent mean±s.e.m. for combined data from three independent experiments.


( B ) Quantitation of p62 protein levels . C2C12 cells were transfected for 48 h with control ( sc ) or Jumpy siRNA and p62 levels analysed by immunoblotting , quantitated by densitometry and represented as a percentage of control . Data are mean ± s . e . m . ( n = 4 independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitation of p62 protein levels. C2C12 cells were transfected for 48 h with control (sc) or Jumpy siRNA and p62 levels analysed by immunoblotting, quantitated by densitometry and represented as a percentage of control. Data are mean±s.e.m. (n=4 independent experiments).


( C ) C2C12 cells were transfected for 48 h with control ( sc ) or Jumpy siRNA , incubated for 4 h with or without 100 nM Bafilomycin A1 in full media , lysed , probed for endogenous p62 and actin by immunoblotting and percentage of p62 were quantitated ( mean ± s . e . m . , n = 3 ) .

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

(C) C2C12 cells were transfected for 48 h with control (sc) or Jumpy siRNA, incubated for 4 h with or without 100 nM Bafilomycin A1 in full media, lysed, probed for endogenous p62 and actin by immunoblotting and percentage of p62 were quantitated (mean±s.e.m., n=3).


( D ) C2C12 cells were transfected for 48 h with control ( sc ) ( lanes 1 , 2 ) or Beclin siRNA ( lanes 3 , 4 ) followed by a second transfection for 48 h with same siRNA and control ( lanes 1 , 3 ) or Jumpy siRNA ( lanes 2 , 4 ) . Cells were lysed , probed for p62 , Beclin and actin by immunoblotting and percentage of p62 were quantitated ( mean ± s . e . m . , n = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) C2C12 cells were transfected for 48 h with control (sc) (lanes 1, 2) or Beclin siRNA (lanes 3, 4) followed by a second transfection for 48 h with same siRNA and control (lanes 1, 3) or Jumpy siRNA (lanes 2, 4). Cells were lysed, probed for p62, Beclin and actin by immunoblotting and percentage of p62 were quantitated (mean±s.e.m., n=5).


( E - M ) C2C12 cells were transfected for 48 h with GFP , GFP ‐ Jumpy ( Jumpy ) , GFP ‐ MTM1 ( MTM1 ) or GFP ‐ MTMR2 ( MTMR2 ) , fixed and immunostained with anti ‐ p62 antibody ( red ) . p62 puncta were quantitated by confocal fluorescence microscopy . Representative confocal images of GFP ( E ) , GFP ‐ Jumpy ( F ) , GFP ‐ MTM1 ( G ) , GFP ‐ MTMR2 ( H ) transfected cells , immunostained for p62 ( I ) , ( J ) , ( K ) and ( L ) , respectively . Scale bars , 5 μm . White lines represent outline of the cells . Quantitation of p62 puncta per cell ( M ) . Bars , SEM ( n = 3 independent experiments , with an average of 30 cells per experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-M) C2C12 cells were transfected for 48 h with GFP, GFP‐Jumpy (Jumpy), GFP‐MTM1 (MTM1) or GFP‐MTMR2 (MTMR2), fixed and immunostained with anti‐p62 antibody (red). p62 puncta were quantitated by confocal fluorescence microscopy. Representative confocal images of GFP (E), GFP‐Jumpy (F), GFP‐MTM1 (G), GFP‐MTMR2 (H) transfected cells, immunostained for p62 (I), (J), (K) and (L), respectively. Scale bars, 5 μm. White lines represent outline of the cells. Quantitation of p62 puncta per cell (M). Bars, SEM (n=3 independent experiments, with an average of 30 cells per experiments).


( N , O ) C2C12 cells were transfected for 48 h with GFP , GFP ‐ Jumpy ( Jumpy ) , GFP ‐ MTM1 ( MTM1 ) or GFP ‐ MTMR2 ( MTMR2 ) , lysed , analysed for p62 , GFP and actin by immunoblotting ( N ) and percentage of p62 were quantitated ( mean ± s . e . m . , n = 3 ) ( O ) .

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

(N, O) C2C12 cells were transfected for 48 h with GFP, GFP‐Jumpy (Jumpy), GFP‐MTM1 (MTM1) or GFP‐MTMR2 (MTMR2), lysed, analysed for p62, GFP and actin by immunoblotting (N) and percentage of p62 were quantitated (mean±s.e.m., n=3) (O).


( P , Q ) C2C12 cells were transfected for 48 h with GFP or GFP ‐ Jumpy ( Jumpy ) , incubated with or without 100 nM Bafilomycin A1 ( BafA1 ) for 4 h , lysed , analysed for p62 , GFP and actin by immunoblotting ( P ) and percentage of p62 were quantitated ( mean ± s . e . m . , n = 4 ) ( Q ) . * P0 . 05 , * * * P0 . 001 , † ns ( t ‐ test ) .

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(P, Q) C2C12 cells were transfected for 48 h with GFP or GFP‐Jumpy (Jumpy), incubated with or without 100 nM Bafilomycin A1 (BafA1) for 4 h, lysed, analysed for p62, GFP and actin by immunoblotting (P) and percentage of p62 were quantitated (mean±s.e.m., n=4) (Q). *P0.05, ***P0.001, †ns (t‐test).


( A ) Transient association of Jumpy WT with LC3 + organelles . Time lapse sequence of C2C12 myoblasts transfected for 24 h with GFP ‐ Jumpy WT and Cherry ‐ LC3 and analysed by live confocal microscopy in a 5LIVE Zeiss microscope . EBSS was added to the cells and z ‐ stacks collected at 3 ‐ min intervals for a total of 45 min . The collected images were processed to generate a maximum projection ( collapsing a 3D image into an x - y projection ) for each time point . Arrows indicate colocalization of GFP ‐ Jumpy WT with LC3 . Graph : Plots of relative fluorescence intensity ( GFP fluorescence pixel intensity ) over time of GFP ‐ Jumpy WT colocalizing with Cherry ‐ LC3 + ‐ positive puncta indicated by arrows .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Transient association of Jumpy WT with LC3+ organelles. Time lapse sequence of C2C12 myoblasts transfected for 24 h with GFP‐Jumpy WT and Cherry‐LC3 and analysed by live confocal microscopy in a 5LIVE Zeiss microscope. EBSS was added to the cells and z‐stacks collected at 3‐min intervals for a total of 45 min. The collected images were processed to generate a maximum projection (collapsing a 3D image into an x-y projection) for each time point. Arrows indicate colocalization of GFP‐Jumpy WT with LC3. Graph: Plots of relative fluorescence intensity (GFP fluorescence pixel intensity) over time of GFP‐Jumpy WT colocalizing with Cherry‐LC3+‐positive puncta indicated by arrows.


( B - I ) Association of Jumpy CS mutant with autophagic organelles . C2C12 cells were transfected for 24 h with GFP ‐ Jumpy C330S ( Jumpy CS ) and tdTomato ‐ LC3 ( LC3 ) , fixed and immunostained with anti ‐ G58K ( Alexa 648 ‐ labelled secondary antibody ) . Jumpy CS ( green ) , LC3 ( red ) and G58K ( white in D , H or blue in E , I ) . Boxed areas ( B - E ) are shown at higher magnification in the corresponding panel below ( F - I ) . Scale bars , 2 μm . White arrows indicate colocalization between Jumpy CS , LC3 but not G58K , blue arrow indicates colocalization between Jumpy CS and G58K but not LC3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I) Association of Jumpy CS mutant with autophagic organelles. C2C12 cells were transfected for 24 h with GFP‐Jumpy C330S (Jumpy CS) and tdTomato‐LC3 (LC3), fixed and immunostained with anti‐G58K (Alexa 648‐labelled secondary antibody). Jumpy CS (green), LC3 (red) and G58K (white in D, H or blue in E, I). Boxed areas (B-E) are shown at higher magnification in the corresponding panel below (F-I). Scale bars, 2 μm. White arrows indicate colocalization between Jumpy CS, LC3 but not G58K, blue arrow indicates colocalization between Jumpy CS and G58K but not LC3.


( A - H ) C2C12 cells were transfected for 24 h with GFP ‐ Jumpy C330S ( Jumpy CS ) and tdTomato ‐ LC3 ( LC3 ) , fixed and immunostained with anti ‐ Atg16 ( Alexa 648 ‐ labelled secondary antibody ) . Jumpy CS ( green ) , LC3 ( red ) and Atg16 ( white in B and F or blue in D and H ) . Boxed areas ( A - D ) are shown at higher magnification in the corresponding panel below ( E - H ) . White arrows indicate colocalization among Jumpy CS , LC3 and Atg16 , yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg16 , blue arrow indicates colocalization between Jumpy CS and Atg16 but not LC3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) C2C12 cells were transfected for 24 h with GFP‐Jumpy C330S (Jumpy CS) and tdTomato‐LC3 (LC3), fixed and immunostained with anti‐Atg16 (Alexa 648‐labelled secondary antibody). Jumpy CS (green), LC3 (red) and Atg16 (white in B and F or blue in D and H). Boxed areas (A-D) are shown at higher magnification in the corresponding panel below (E-H). White arrows indicate colocalization among Jumpy CS, LC3 and Atg16, yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg16, blue arrow indicates colocalization between Jumpy CS and Atg16 but not LC3.


( I , J ) C2C12 cells were transfected for 24 h with GFP ( I ) , fixed and immunostained with anti ‐ Atg16 ( J ) .

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

(I, J) C2C12 cells were transfected for 24 h with GFP (I), fixed and immunostained with anti‐Atg16 (J).


( K - R ) C2C12 cells were transfected for 24 h with GFP ‐ Jumpy C330S ( Jumpy CS ) and tdTomato ‐ LC3 ( LC3 ) , fixed and immunostained with anti ‐ Atg12 ( M ) antibody . Jumpy CS ( green ) , LC3 ( red ) and Atg12 ( white in L , P or blue in N , R ) . Boxed areas ( K - N ) are shown at higher magnification in the corresponding panel below ( O - R ) . White arrows indicate colocalization among Jumpy CS , LC3 and Atg12 , yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg12 , blue arrow indicates colocalization between Jumpy CS and Atg12 but not LC3 .

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

(K-R) C2C12 cells were transfected for 24 h with GFP‐Jumpy C330S (Jumpy CS) and tdTomato‐LC3 (LC3), fixed and immunostained with anti‐Atg12(M) antibody. Jumpy CS (green), LC3 (red) and Atg12 (white in L, P or blue in N, R). Boxed areas (K-N) are shown at higher magnification in the corresponding panel below (O-R). White arrows indicate colocalization among Jumpy CS, LC3 and Atg12, yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg12, blue arrow indicates colocalization between Jumpy CS and Atg12 but not LC3.


( S - ZA ) C2C12 cells were transfected for 24 h with Jumpy CS and Cherry ‐ LC3 ( LC3 ) , fixed and immunostained with anti ‐ lamp1 . Jumpy CS ( green ) , LC3 ( red ) and Lamp1 ( white in T , X or blue in V , ZA ) . Boxed areas ( S - V ) are shown at higher magnification in the corresponding panel below ( W - ZA ) . Yellow arrows indicate colocalization between Jumpy CS and LC3 but not Lamp1 . Scale bars , 2 μm .

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

(S-ZA) C2C12 cells were transfected for 24 h with Jumpy CS and Cherry‐LC3 (LC3), fixed and immunostained with anti‐lamp1. Jumpy CS (green), LC3 (red) and Lamp1 (white in T, X or blue in V, ZA). Boxed areas (S-V) are shown at higher magnification in the corresponding panel below (W-ZA). Yellow arrows indicate colocalization between Jumpy CS and LC3 but not Lamp1. Scale bars, 2 μm.


( A - C ) C2C12 were transfected for 48 h with Jumpy siRNA , followed by a second transfection overnight with Jumpy siRNA and GFP ‐ WIPI ‐ 1 , incubated for 4 h in starvation media , fixed and immunostained with anti ‐ Atg16 antibody ( red ) . GFP ‐ WIPI1 ‐ transfected cell ( A ) , endogenous Atg16 immunostaining ( B ) , colocalization of GFP ‐ WIPI ‐ 1 and Atg16 is indicated in yellow in merge image ( C ) . Boxed areas are shown at higher magnification as inset . Scale bars , 10 and 5 μm in insets .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) C2C12 were transfected for 48 h with Jumpy siRNA, followed by a second transfection overnight with Jumpy siRNA and GFP‐WIPI‐1, incubated for 4 h in starvation media, fixed and immunostained with anti‐Atg16 antibody (red). GFP‐WIPI1‐transfected cell (A), endogenous Atg16 immunostaining (B), colocalization of GFP‐WIPI‐1 and Atg16 is indicated in yellow in merge image (C). Boxed areas are shown at higher magnification as inset. Scale bars, 10 and 5 μm in insets.


( D - I ) C2C12 cells were transfected for 48 h with control ( scramble ) ( D , E ) or Jumpy siRNA ( F - H ) , followed by a second transfection overnight with corresponding siRNA and GFP ‐ WIPI ‐ 1 . Cells were incubated for 4 h in full ( D , G ) or starvation media ( E , F , H ) in presence ( F ) or absence ( E - H ) of 100 nM wortmannin , fixed and analysed by confocal fluorescence microscopy . Quantitation of WIPI ‐ 1 puncta per cell ( I ) . Bars , s . e . m . ( n = 3 independent experiments , with an average of 30 cells per experiment ) . * P0 . 05 , * * P0 . 01 ( t ‐ test ) .

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

(D-I) C2C12 cells were transfected for 48 h with control (scramble) (D, E) or Jumpy siRNA (F-H), followed by a second transfection overnight with corresponding siRNA and GFP‐WIPI‐1. Cells were incubated for 4 h in full (D, G) or starvation media (E, F, H) in presence (F) or absence (E-H) of 100 nM wortmannin, fixed and analysed by confocal fluorescence microscopy. Quantitation of WIPI‐1 puncta per cell (I). Bars, s.e.m. (n=3 independent experiments, with an average of 30 cells per experiment). *P0.05, **P0.01 (t‐test).


( A - T ) C2C12 cells were transfected for 24 h with GFP , GFP ‐ Jumpy WT or GFP ‐ Jumpy C330S ( Jumpy CS ) and Tomato ‐ LC3 ( LC3 ) and HA ‐ Atg9 , fixed and immunostained with anti ‐ HA . GFP ( I ) , Jumpy WT ( M ) , Jumpy CS ( green ) ( A , E , Q ) , LC3 ( red ) ( C , G , J , N , R ) and Atg9 ( white in B , F , K , O , S or blue in D , H , I , P , T ) . Boxed areas ( A - D ) are shown at higher magnification in the corresponding panel below ( E - H ) . White arrows indicate colocalization between Jumpy CS , LC3 and Atg9 , blue arrow indicates colocalization between Jumpy CS and Atg9 but not LC3 and yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg9 . White boxes ( I - T ) show LC3 puncta location . Scale bars , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-T) C2C12 cells were transfected for 24 h with GFP, GFP‐Jumpy WT or GFP‐Jumpy C330S (Jumpy CS) and Tomato‐LC3 (LC3) and HA‐Atg9, fixed and immunostained with anti‐HA. GFP (I), Jumpy WT (M), Jumpy CS (green) (A, E, Q), LC3 (red) (C, G, J, N, R) and Atg9 (white in B, F, K, O, S or blue in D, H, I, P, T). Boxed areas (A-D) are shown at higher magnification in the corresponding panel below (E-H). White arrows indicate colocalization between Jumpy CS, LC3 and Atg9, blue arrow indicates colocalization between Jumpy CS and Atg9 but not LC3 and yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg9. White boxes (I-T) show LC3 puncta location. Scale bars, 5 μm.


( U ) Percentage of LC3 puncta colocalizing with Atg9 . n , number of puncta counted .

(U) Percentage of LC3 puncta colocalizing with Atg9. n, number of puncta counted.


( A - K ) C2C12 cells were transfected for 48 h with YFP , YFP ‐ Jumpy WT ( Jumpy WT ) , YFP ‐ Jumpy C330S ( Jumpy CS ) , YFP ‐ Jumpy Y462C ( Jumpy YC ) or YFP ‐ Jumpy R336Q ( Jumpy RQ ) , fixed and immunostained with anti ‐ p62 antibody ( red ) . p62 puncta were quantitated by confocal fluorescence microscopy . Representative confocal images of YFP ( A ) , YFP ‐ Jumpy WT ( B ) , YFP ‐ Jumpy C330S ( C ) , YFP ‐ Jumpy Y462C ( D ) and YFP ‐ Jumpy R336Q ( E ) transfected cells , immunostained for p62 ( F ) , ( G ) , ( H ) , ( I ) and ( J ) , respectively . Scale bars , 5 μm . Quantitation of p62 puncta per cell ( K ) . Bars , s . e . m . ( n = 3 independent experiments , 30 cells per experiments ) . * P0 . 05 ( t ‐ test ) , ns : nonsignificant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K) C2C12 cells were transfected for 48 h with YFP, YFP‐Jumpy WT (Jumpy WT), YFP‐Jumpy C330S (Jumpy CS), YFP‐Jumpy Y462C (Jumpy YC) or YFP‐Jumpy R336Q (Jumpy RQ), fixed and immunostained with anti‐p62 antibody (red). p62 puncta were quantitated by confocal fluorescence microscopy. Representative confocal images of YFP (A), YFP‐Jumpy WT (B), YFP‐Jumpy C330S (C), YFP‐Jumpy Y462C (D) and YFP‐Jumpy R336Q (E) transfected cells, immunostained for p62 (F), (G), (H), (I) and (J), respectively. Scale bars, 5 μm. Quantitation of p62 puncta per cell (K). Bars, s.e.m. (n=3 independent experiments, 30 cells per experiments). *P0.05 (t‐test), ns: nonsignificant.


( L ) HeLa cells were transfected for 24 h with YFP , YFP ‐ Jumpy WT ( Jumpy WT ) or YFP ‐ Jumpy R336Q ( Jumpy RQ ) , incubated with or without 50 ng / μl rapamycin ( BafA1 ( 100 nM ) was present in both control and rapamycin treated cells ) for 2 h , lysed and analysed for LC3 , YFP and actin by immunoblotting . Densitometric LC3 ‐ II / actin ratios are shown underneath the blot .

O O O O O O O O O O O O O O O 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-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

(L) HeLa cells were transfected for 24 h with YFP, YFP‐Jumpy WT (Jumpy WT) or YFP‐Jumpy R336Q (Jumpy RQ), incubated with or without 50 ng/μl rapamycin (BafA1 (100 nM) was present in both control and rapamycin treated cells) for 2 h, lysed and analysed for LC3, YFP and actin by immunoblotting. Densitometric LC3‐II/actin ratios are shown underneath the blot.


( a ) MEFs stably expressing EGFP - Parkin were exposed to CCCP ( 30 μM ) for the indicated times and then subjected to immunoblot analysis ( IB ) with antibodies to the indicated proteins . The asterisk indicates a nonspecific band .

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

(a) MEFs stably expressing EGFP-Parkin were exposed to CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis (IB) with antibodies to the indicated proteins. The asterisk indicates a nonspecific band.


( b ) SH - SY5Y cells were exposed to CCCP ( 10 μM ) for the indicated times and then subjected to immunoblot analysis .

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) SH-SY5Y cells were exposed to CCCP (10 μM) for the indicated times and then subjected to immunoblot analysis.


( c ) MEFs stably expressing EGFP - Parkin were treated with CCCP ( 30 μM ) for the indicated times , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c and with a confocal microscope . The fluorescence of EGFP - Parkin was monitored directly . ( d ) Higher - magnification views of the boxed areas in c . Scale bar ( c , d ) , 10 μm .

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

(c) MEFs stably expressing EGFP-Parkin were treated with CCCP (30 μM) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c and with a confocal microscope. The fluorescence of EGFP-Parkin was monitored directly. (d) Higher-magnification views of the boxed areas in c. Scale bar (c,d), 10 μm.


( a , c ) MEFs ( a ) stably expressing FLAG - Parkin and either EGFP - VAP - A or HA - VAP - A were treated with CCCP ( 30 and 10 μM , respectively ) for the indicated times , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 , to HA or to FLAG ( M2 ) . The fluorescence of EGFP - VAP - A was also monitored directly . ( b , d ) Higher - magnification views of the boxed areas in a and c , respectively .

O O O O O O 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

(a,c) MEFs (a) stably expressing FLAG-Parkin and either EGFP-VAP-A or HA-VAP-A were treated with CCCP (30 and 10 μM, respectively) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to HA or to FLAG (M2). The fluorescence of EGFP-VAP-A was also monitored directly. (b,d) Higher-magnification views of the boxed areas in a and c, respectively.


( a , c ) HeLa cells ( c ) stably expressing FLAG - Parkin and either EGFP - VAP - A or HA - VAP - A were treated with CCCP ( 30 and 10 μM , respectively ) for the indicated times , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 , to HA or to FLAG ( M2 ) . The fluorescence of EGFP - VAP - A was also monitored directly . ( b , d ) Higher - magnification views of the boxed areas in a and c , respectively .

O O O O O O O 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

(a,c) HeLa cells (c) stably expressing FLAG-Parkin and either EGFP-VAP-A or HA-VAP-A were treated with CCCP (30 and 10 μM, respectively) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to HA or to FLAG (M2). The fluorescence of EGFP-VAP-A was also monitored directly. (b,d) Higher-magnification views of the boxed areas in a and c, respectively.


( e ) NIH 3T3 cells stably expressing EGFP - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , after which whole - cell homogenates as well as cytosolic , ER and mitochondrial fractions were prepared and subjected to immunoblot analysis . Scale bar ( a - d ) , 10 μm .

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

(e) NIH 3T3 cells stably expressing EGFP-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, after which whole-cell homogenates as well as cytosolic, ER and mitochondrial fractions were prepared and subjected to immunoblot analysis. Scale bar (a-d), 10 μm.


( a ) MEFs stably expressing EGFP - tagged wild - type ( WT ) or T240R mutant forms of Parkin as well as FLAG - tagged VAP - A were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 , to FLAG ( M2 ) and to cytochrome c . The fluorescence of EGFP was also monitored directly .

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

(a) MEFs stably expressing EGFP-tagged wild-type (WT) or T240R mutant forms of Parkin as well as FLAG-tagged VAP-A were incubated in the absence or presence of CCCP (30 μM) for 24 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to FLAG (M2) and to cytochrome c. The fluorescence of EGFP was also monitored directly.


( b ) MEFs as in a were treated with CCCP ( 30 μM ) for the indicated times and then subjected to immunoblot analysis .

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

(b) MEFs as in a were treated with CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis.


( c ) MEFs stably expressing EGFP - Parkin and FLAG - VAP - A were incubated in the absence or presence of CCCP ( 30 μM ) and lactacystin ( 10 μM ) for 24 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 , to FLAG ( M2 ) and to cytochrome c . The fluorescence of EGFP - Parkin was also monitored directly .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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) MEFs stably expressing EGFP-Parkin and FLAG-VAP-A were incubated in the absence or presence of CCCP (30 μM) and lactacystin (10 μM) for 24 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to FLAG (M2) and to cytochrome c. The fluorescence of EGFP-Parkin was also monitored directly.


( d ) MEFs stably expressing EGFP - Parkin were incubated for 12 h in the absence or presence of CCCP ( 30 μM ) and lactacystin ( 10 μM ) as indicated and then subjected to immunoblot analysis . Scale bar ( a , c ) , 10 μm .

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

(d) MEFs stably expressing EGFP-Parkin were incubated for 12 h in the absence or presence of CCCP (30 μM) and lactacystin (10 μM) as indicated and then subjected to immunoblot analysis. Scale bar (a,c), 10 μm.


( b ) MEFs stably expressing FLAG - Parkin and EGFP - tagged forms of FKBP38 , Omp25 , Omp25 / FKBP38 or FKBP38 / Omp25 were treated with CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG ( rabbit polyclonal ) . The fluorescence of EGFP was also monitored directly . Scale bar , 10 μm .

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

(b) MEFs stably expressing FLAG-Parkin and EGFP-tagged forms of FKBP38, Omp25, Omp25/FKBP38 or FKBP38/Omp25 were treated with CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP was also monitored directly. Scale bar, 10 μm.


( c ) MEFs stably expressing mCherry - tagged Parkin and EGFP - tagged forms of FKBP38 , Omp25 , Omp25 / FKBP38 or FKBP38 / Omp25 were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h and then subjected to immunoblot analysis .

O O O O O O O O O O O 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

(c) MEFs stably expressing mCherry-tagged Parkin and EGFP-tagged forms of FKBP38, Omp25, Omp25/FKBP38 or FKBP38/Omp25 were incubated in the absence or presence of CCCP (30 μM) for 24 h and then subjected to immunoblot analysis.


( a ) MEFs stably expressing FLAG - Parkin and either EGFP - Bcl - 2 or EGFP - Bcl - xL were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h and then subjected to immunoblot analysis .

O O O O O O 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

(a) MEFs stably expressing FLAG-Parkin and either EGFP-Bcl-2 or EGFP-Bcl-xL were incubated in the absence or presence of CCCP (30 μM) for 24 h and then subjected to immunoblot analysis.


( c ) MEFs stably expressing FLAG - Parkin and EGFP - tagged forms of Bcl - 2 , Bcl - xL , Bcl - 2 / xL or Bcl - xL / 2 were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG ( rabbit polyclonal ) . The fluorescence of EGFP was also monitored directly . ( d ) Higher - magnification views of the boxed areas in c . Scale bar ( c , d ) , 10 μm .

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

(c) MEFs stably expressing FLAG-Parkin and EGFP-tagged forms of Bcl-2, Bcl-xL, Bcl-2/xL or Bcl-xL/2 were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP was also monitored directly. (d) Higher-magnification views of the boxed areas in c. Scale bar (c,d), 10 μm.


( a ) MEFs stably expressing EGFP - FKBP38 ( N402K ) and FLAG - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG ( rabbit polyclonal ) . The fluorescence of EGFP - FKBP38 ( N402K ) was also monitored directly . Higher - magnification views of the boxed areas are shown in the right panels of each 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 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

(a) MEFs stably expressing EGFP-FKBP38(N402K) and FLAG-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP-FKBP38(N402K) was also monitored directly. Higher-magnification views of the boxed areas are shown in the right panels of each set.


( b ) MEFs stably expressing EGFP - FKBP38 ( N402K ) , mCherry - FKBP38 and FLAG - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FLAG ( M2 ) . The fluorescence of EGFP - FKBP38 ( N402K ) and mCherry - FKBP38 was also monitored directly .

O O O O 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

(b) MEFs stably expressing EGFP-FKBP38(N402K), mCherry-FKBP38 and FLAG-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FLAG (M2). The fluorescence of EGFP-FKBP38(N402K) and mCherry-FKBP38 was also monitored directly.


( c ) MEFs stably expressing FLAG - Parkin and either EGFP - FKBP38 or EGFP - FKBP38 ( N402K ) were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h and then subjected to immunoblot analysis . ( d ) Densitometric quantitation of EGFP fusion protein abundance in immunoblots similar to those in ( c ) . Data are expressed relative to the corresponding control value ( CCCP 0 h ) and are means ± s . d . , from three independent experiments . * * P0 . 01 ( Student ' s t test ) .

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

(c) MEFs stably expressing FLAG-Parkin and either EGFP-FKBP38 or EGFP-FKBP38(N402K) were incubated in the absence or presence of CCCP (30 μM) for 24 h and then subjected to immunoblot analysis. (d) Densitometric quantitation of EGFP fusion protein abundance in immunoblots similar to those in (c). Data are expressed relative to the corresponding control value (CCCP 0 h) and are means±s.d., from three independent experiments. **P0.01 (Student's t test).


( e ) MEFs stably expressing EGFP - Bcl - 2 ( H235R ) and FLAG - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG ( rabbit polyclonal ) . The fluorescence of EGFP - Bcl - 2 ( H235R ) was also monitored directly . Higher - magnification views of the boxed areas are shown in the right panels of each 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 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

(e) MEFs stably expressing EGFP-Bcl-2(H235R) and FLAG-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP-Bcl-2(H235R) was also monitored directly. Higher-magnification views of the boxed areas are shown in the right panels of each set.