Overexpression of TRIM21 in HEK293T increased EV71 replication by degrading SAMHD1 . HEK293T were transfected with VR1012 or TRIM21 - HA for 24 h then infected with EV71 at a MOI of 0 . 05 . EV71 viral in lysates was detected by RT - qPCR with GAPDH as a control ( n = 3 , mean ± SD , * * P < 0 . 01 , paired t - ) ) .

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Overexpression of TRIM21 in HEK293T cells increased EV71 replication by degrading SAMHD1. HEK293T cells were transfected with VR1012 or TRIM21-HA for 24 h and then infected with EV71 at a MOI of 0.05. EV71 viral RNA in cell lysates was detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P < 0.01, paired t-test)).


TRIM21 itself has no effect on EV71 replication . VR1012 or TRIM21 was transfected into pLKO . 1 or sh - SAMHD1 HEK293T for 24 h infected with EV71 . were harvested subjected to IB .

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TRIM21 itself has no effect on EV71 replication. VR1012 or TRIM21 was transfected into pLKO.1 or sh-SAMHD1 HEK293T cells for 24 h and infected with EV71. Cells were harvested and subjected to IB analysis.


TRIM21 itself has no effect on EV71 replication . VR1012 or TRIM21 was transfected into pLKO . 1 or sh - SAMHD1 HEK293T for 24 h infected with EV71 . Viral titers in supernatants were measured by cytopathic effect method . results represent means ± SD from three independent experiments . Statistical significance was analyzed using Student ' s t ( * P < 0 . 05 ) .

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TRIM21 itself has no effect on EV71 replication. VR1012 or TRIM21 was transfected into pLKO.1 or sh-SAMHD1 HEK293T cells for 24 h and infected with EV71. Viral titers in the supernatants were measured by the cytopathic effect method. The results represent the means ± SD from three independent experiments. Statistical significance was analyzed using Student's t test (*P < 0.05).


mRNA of SAMHD1 TRIM21 in different tissues of neonatal mice were detected by RT - qPCR with GAPDH as a control , expression of SAMHD1 TRIM21 in heart was set as 100 % ( n = 3 , mean ± SD , * P < 0 . 05 , paired t - ) .

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The mRNA levels of SAMHD1 and TRIM21 in different tissues of neonatal mice were detected by RT-qPCR with GAPDH as a control, and the expression of SAMHD1 and TRIM21 in the heart was set as 100% (n=3, mean±SD, * P < 0.05, paired t-test).


of SAMHD1 TRIM21 in different tissues of mice were examined by Near Infrared Spectrum ( NIR ) western blot with tubulin as a control .

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The protein levels of SAMHD1 and TRIM21 in different tissues of mice were examined by Near Infrared Spectrum (NIR) western blot with tubulin as a control.


Immunohistochemical ( ) results of SAMHD1 in various tissues of mice . Bars , 100μm .

Immunohistochemical (IHC) staining results of SAMHD1 in various tissues of mice. Bars, 100μm.


mRNA of EV71 host factors in mock or EV71 infected mice were detected by RT - qPCR with GAPDH as a control . expression of EV71 ( D ) were presented , of corresponding gene in mock infected mice were set as 100 %

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The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT-qPCR with GAPDH as a control. The expression of EV71 (D) were presented, and the levels of corresponding gene in mock infected mice were set as 100%


mRNA of EV71 host factors in mock or EV71 infected mice were detected by RT - qPCR with GAPDH as a control . expression of , IFNα IFNβ ( E ) were presented , of corresponding gene in mock infected mice were set as 100 %

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The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT-qPCR with GAPDH as a control. The expression of , IFNα and IFNβ (E) were presented, and the levels of corresponding gene in mock infected mice were set as 100%


mRNA of EV71 host factors in mock or EV71 infected mice were detected by RT - qPCR with GAPDH as a control . expression of SAMHD1 TRIM21 ( F ) were presented , of corresponding gene in mock infected mice were set as 100 %

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The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT-qPCR with GAPDH as a control. The expression of SAMHD1 and TRIM21 (F) were presented, and the levels of corresponding gene in mock infected mice were set as 100%


IB of THP - 1 treated with scrambled shRNA , TRIM21 - specific shRNA or SAMHD1 - specific shRNA . Tubulin served as loading control . of SAMHD1 TRIM21 in whole in which indicated genes were knocked down by shRNA . Data are normalized to tubulin expressed as fold change over treated with pLKO . 1 without HIV infection ( n = 3 , mean ± SD ) .

IB analysis of THP-1 cells treated with scrambled shRNA, TRIM21-specific shRNA or SAMHD1-specific shRNA. Tubulin served as loading control. Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA. Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO.1 without HIV infection (n=3, mean±SD).


sh - SAMHD1 sh - TRIM21 THP - 1 were treated with PMA for 24 h infected with VSV - G pseudotyped reporter HIV - 1 , then detected 48 h later by flow cytometry ( n = 3 , mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , paired t - ) .

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sh-SAMHD1 and sh-TRIM21 THP-1 cells were treated with PMA for 24 h and infected with the VSV-G pseudotyped reporter HIV-1, then detected 48 h later by flow cytometry analysis (n=3, mean±SD, * P < 0.05, ** P < 0.01, *** P < 0.001, paired t-test).


of SAMHD1 TRIM21 in whole in which indicated genes were knocked down by shRNA . Data are normalized to tubulin expressed as fold change over treated with pLKO . 1 without HIV infection ( n = 3 , mean ± SD ) . IB of MDM from 3 donors transfected with TRIM21 siRNA twice at days 1 3 , respectively , after MDM differentiation .

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Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA. Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO.1 without HIV infection (n=3, mean±SD). IB analysis of MDM cells from 3 donors transfected with TRIM21 siRNA twice at days 1 and 3, respectively, after MDM differentiation.


MDM treated with TRIM21 siRNA were infected with VSV - G - pseudotyped HIV - 1 reporter then detected 48 h later by flow cytometry ( n = 3 , mean ± SD , * * P < 0 . 01 , paired t - ) .

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MDM cells treated with TRIM21 siRNA were infected with VSV-G-pseudotyped HIV-1 reporter and then detected 48 h later by flow cytometry analysis (n=3, mean±SD, ** P < 0.01, paired t-test).


of SAMHD1 TRIM21 in whole in which indicated genes were knocked down by shRNA . Data are normalized to tubulin expressed as fold change over treated with pLKO . 1 without HIV infection ( n = 3 , mean ± SD ) . IB of Jurkat KG - 1 treated with scrambled shRNA or TRIM21 - specific shRNA .

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Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA. Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO.1 without HIV infection (n=3, mean±SD). IB analysis of Jurkat and KG-1 cells treated with scrambled shRNA or TRIM21-specific shRNA.


Jurkat KG - 1 treated with TRIM21 shRNA were infected with HIV - 1 , then detected 48 h later by flow cytometry ( n = 3 , mean ± SD , ns stands for no significance , paired t - ) .

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Jurkat and KG-1 cells treated with TRIM21 shRNA were infected with HIV-1, then detected 48 h later by flow cytometry analysis (n=3, mean±SD, ns stands for no significance, paired t-test).


TRIM21 interacts with SAMHD1 via PRY SPRY domains . effect of TRIM21 on degradation of SAMHD1 . SAMHD1 - flag was cotransfected with VR1012 , TRIM21 WT or indicated mutant into HEK293T for 48 h , were subjected to IB with tubulin as loading control .

TRIM21 interacts with SAMHD1 via PRY and SPRY domains. The effect of TRIM21 on the degradation of SAMHD1. SAMHD1-flag was cotransfected with VR1012, TRIM21 WT or the indicated mutant into HEK293T cells for 48 h, and the cells were subjected to IB with tubulin as loading control.


TRIM21 interacts with SAMHD1 via PRY SPRY domains . SAMHD1 - flag was cotransfected with VR1012 or TRIM21 WT or indicated mutant for 24 h , were then treated with 10 μM MG132 for 12 h before harvest subjected to HA IP IB .

TRIM21 interacts with SAMHD1 via PRY and SPRY domains. SAMHD1-flag was cotransfected with VR1012 or TRIM21 WT or the indicated mutant for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB.


effect of TRIM21 on SAMHD1 WT or its mutants . SAMHD1 - HA or indicated mutant were cotransfected with VR1012 or TRIM21 - flag into HEK293T for 48 h , were subjected to IB with tubulin as a loading control .

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The effect of TRIM21 on SAMHD1 WT or its mutants. SAMHD1-HA or the indicated mutant were cotransfected with VR1012 or TRIM21-flag into HEK293T cells for 48 h, and the cells were subjected to IB with tubulin as a loading control.


SAMHD1 - flag 1 - 547 was cotransfected with VR1012 or TRIM21 - HA for 24 h , were then treated with 10 μM MG132 for 12 h before harvest subjected to HA IP IB .

SAMHD1-flag 1-547 was cotransfected with VR1012 or TRIM21-HA for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB.


SAMHD1 109 - 626 followed with a His tag or TRIM21 - PRYSPRY followed with a GST tag were expressed in Rosetta ( DE3 ) , pull - down were performed with Ni - Sepharose ( up ) GST Sepharose ( down ) , respectively .

SAMHD1 109-626 followed with a His tag or TRIM21-PRYSPRY followed with a GST tag were expressed in Rosetta (DE3), and pull-down assay were performed with Ni-Sepharose (up) and GST Sepharose (down), respectively.


FRET indicates interaction between YFP - SAMHD1 CFP - TRIM21 . A representative image of SAMHD1 - YFP ( yellow ) ECFP - TRIM21 ( cyan ) expressing before after photobleaching acceptor fluorophore , YFP . region chosen for photobleaching is marked ( open box ) , Bars , 10μm . quantization of brightness was analyzed by Image J ( n = 3 , mean ± SD , ns stands for no significance , * P < 0 . 05 , paired t - )

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FRET analysis indicates interaction between YFP-SAMHD1 and CFP-TRIM21. A representative image of SAMHD1-YFP (yellow) and ECFP-TRIM21 (cyan) expressing cells before and after photobleaching the acceptor fluorophore, YFP. The region chosen for photobleaching is marked (white open box), Bars, 10μm. The quantization of fluorescence brightness was analyzed by Image J (n=3, mean±SD, ns stands for no significance, * P < 0.05, paired t-test)


microscale thermophoresis curve for interaction between SAMHD1 TRIM21 . Alexa Fluor ® 647 labelled SAMHD1 at a of 20 nM were incubated with two - fold dilution series of unlabeled TRIM21 ( 3 μM to 9 . 16E - 05 μM ) . curve represents signal recorded from three measurements . normalized thermophoretic signals were plotted against of TRIM21 with mean values ± standard deviation as well as a fitting to a 1 : 1 binding model ( NanoTemper ® software , F Norm = F Hot / F Cold ) . Kd of this interaction was determined to be 270 ± 92nM . data was a representive of three independent experiments using different dilution .

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The microscale thermophoresis curve for the interaction between SAMHD1 and TRIM21. Alexa Fluor®647 labelled SAMHD1 at a concentration of 20 nM were incubated with two-fold dilution series of unlabeled TRIM21 (3 μM to 9.16E-05 μM). The curve represents the signal recorded from three measurements. The normalized fluorescence thermophoretic signals were plotted against the concentration of TRIM21 with mean values ± standard deviation as well as a fitting to a 1:1 binding model (NanoTemper® analysis software, F Norm = F Hot / F Cold). The Kd of this interaction was determined to be 270±92nM. The data was a representive of three independent experiments using different dilution.


effect of TRIM21 on SAMHD1 from various species . HEK293T were transfected with VR1012 or TRIM21 indicated SAMHD1 expression vector , then subjected to IB . Tubulin served as a loading control .

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The effect of TRIM21 on SAMHD1 proteins from various species. HEK293T cells were transfected with VR1012 or TRIM21 and the indicated SAMHD1 expression vector, then subjected to IB analysis. Tubulin served as a loading control.


effect of TRIM21 on hSAMHD1 mutants . HEK293T were transfected with VR1012 or TRIM21 SAMHD1 mutants for 48 h then subjected to IB . Tubulin served as a loading control .

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The effect of TRIM21 on hSAMHD1 mutants. HEK293T cells were transfected with VR1012 or TRIM21 and SAMHD1 mutants for 48 h and then subjected to IB analysis. Tubulin served as a loading control.


SAMHD1 - flag WT or G153S or G183R mutant was cotransfected with VR1012 or TRIM21 - HA for 24 h , were then treated with 10 μM MG132 for 12 h before harvest subjected to HA IP IB . Tubulin served as a loading control .

SAMHD1-flag WT or the G153S or G183R mutant was cotransfected with VR1012 or TRIM21-HA for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB. Tubulin served as a loading control.


SAMHD1 - HA WT or indicated mutants were cotransfected with VR1012 or TRIM21 - flag into HEK293T for 48 h , were subjected to IB with tubulin as a loading control .

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SAMHD1-HA WT or the indicated mutants were cotransfected with VR1012 or TRIM21-flag into HEK293T cells for 48 h, and the cells were subjected to IB with tubulin as a loading control.


SAMHD1 - HA WT VR1012 or TRIM21 were cotransfected with K48 - only K63 - only ubiquitin - flag into HEK293T for 24 h , were then treated with 10 μM MG132 for 12 h before harvest subjected to HA IP IB with tubulin as a loading control .

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SAMHD1-HA WT and VR1012 or TRIM21 were cotransfected with K48-only and K63-only ubiquitin-flag into HEK293T cells for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB with tubulin as a loading control.


K48 - only ubiquitin - flag SAMHD1 - HA WT or mutant K622R were cotransfected with VR1012 or TRIM21 into HEK293T as indicated . were then treated with 10 μM MG132 for 12 h before harvest subjected to HA IP IB with tubulin as a loading control .

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K48-only ubiquitin-flag and SAMHD1-HA WT or mutant K622R were cotransfected with VR1012 or TRIM21 into HEK293T cells as indicated. The cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB with tubulin as a loading control.


VR1012 , SAMHD1 - HA WT or K622R mutant was transfected into HEK293T - shSAMHD1 for 24 h . were then infected with EV71 at a MOI of 0 . 1 harvested at indicated time points . SAMHD1 - HA , TRIM21 EV71 - VP1 were detected by IB with tubulin as a loading control .

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VR1012, SAMHD1-HA WT or the K622R mutant was transfected into HEK293T-shSAMHD1 cells for 24 h. The cells were then infected with EV71 at a MOI of 0.1 and harvested at the indicated time points. SAMHD1-HA, TRIM21 and EV71-VP1 were detected by IB with tubulin as a loading control.


A PTAR1 expression in rat tissues . extracted from various rat tissues ( 15 μg each ) were analyzed by immunoblotting with antibodies against PTAR1 .

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A PTAR1 protein expression in rat tissues. Proteins extracted from various rat tissues (15 μg each) were analyzed by immunoblotting with antibodies against PTAR1.


B Identification of RabGGTβ as a binding of PTAR1 . Anti - FLAG immunoprecipitates from control HeLa S3 or FLAG - PTAR1 - expressing HeLa S3 were analyzed by SDS - PAGE silver . 35 kDa was identified as RabGGTβ by mass spectrometry . asterisk denotes a degradation product of FLAG - PTAR1 .

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B Identification of RabGGTβ as a binding protein of PTAR1. Anti-FLAG immunoprecipitates from control HeLa S3 cells or FLAG-PTAR1-expressing HeLa S3 cells were analyzed by SDS-PAGE and silver staining. The 35 kDa protein was identified as RabGGTβ by mass spectrometry. The asterisk denotes a degradation product of FLAG-PTAR1.


C Co - immunoprecipitation of endogenous PTAR1 RabGGTβ from rat brain thymus cytosol .

C Co-immunoprecipitation of endogenous PTAR1 and RabGGTβ from rat brain and thymus cytosol.


D Fractionation of rat brain cytosol by Mono Q anion exchange column chromatography . Column fractions were analyzed by immunoblotting with antibodies against RabGGTα , PTAR1 , RabGGTβ . RabGGTβ eluted in two peaks ( peak A peak B ) .

D Fractionation of rat brain cytosol by Mono Q anion exchange column chromatography. Column fractions were analyzed by immunoblotting with antibodies against RabGGTα, PTAR1, and RabGGTβ. RabGGTβ eluted in two peaks (peak A and peak B).


E Co - immunodepletion of PTAR1 RabGGTβ by anti - PTAR1 IgG . PTAR1 was immunodepleted from Mono Q peak A fraction using anti - PTAR1 IgG - bound A beads , supernatant was analyzed by immunoblotting with antibodies against PTAR1 RabGGTβ . Normal rabbit IgG anti - PTAR1 IgG preabsorbed with antigen peptide were used as controls .

E Co-immunodepletion of PTAR1 and RabGGTβ by anti-PTAR1 IgG. PTAR1 was immunodepleted from the Mono Q peak A fraction using anti-PTAR1 IgG-bound protein A beads, and the supernatant was analyzed by immunoblotting with antibodies against PTAR1 and RabGGTβ. Normal rabbit IgG and anti-PTAR1 IgG preabsorbed with the antigen peptide were used as controls.


F Fractionation of Mono Q peak A fraction by Superdex 200 gel filtration chromatography . Column fractions were analyzed by immunoblotting with antibodies against PTAR1 RabGGTβ .

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F Fractionation of the Mono Q peak A fraction by Superdex 200 gel filtration chromatography. Column fractions were analyzed by immunoblotting with antibodies against PTAR1 and RabGGTβ.


G Gel filtration chromatography of recombinant PTAR1 - RabGGTβ purified from Sf9 insect on a Superdex 200 column . Inset shows SDS - PAGE Coomassie of purified .

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G Gel filtration chromatography of the recombinant PTAR1-RabGGTβ complex purified from Sf9 insect cells on a Superdex 200 column. Inset shows SDS-PAGE and Coomassie staining analysis of the purified complex.


C Identification of Ykt6 as a biotin - geranylated by GGTase - III . extracted from statin - treated HeLa S3 were applied to GST or GST - GGTase - III affinity columns . Bound were eluted incubated with recombinant GGTase - III BGPP . Reaction products were separated by SDS - PAGE , transferred to a nitrocellulose , probed with horseradish peroxidase ( HRP ) - labeled avidin to detect biotinylated . Mass spectrometry identified 25 kDa as Ykt6 .

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C Identification of Ykt6 as a protein biotin-geranylated by GGTase-III. Cytosolic proteins extracted from statin-treated HeLa S3 cells were applied to GST or GST-GGTase-III affinity columns. Bound proteins were eluted and incubated with recombinant GGTase-III and BGPP. Reaction products were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with horseradish peroxidase (HRP)-labeled avidin to detect biotinylated proteins. Mass spectrometry identified the 25 kDa protein as Ykt6.


D Biotin - geranylation of recombinant Ykt6 by GGTase - III . Bacterially produced recombinant untagged Ykt6 ( left ) was incubated with buffer , GGTase - III , or RabGGTase in absence or presence of BGPP for 30 min at 37ºC . Biotin - geranylated Ykt6 was

D Biotin-geranylation of recombinant Ykt6 by GGTase-III. Bacterially produced recombinant untagged Ykt6 (left) was incubated with buffer, GGTase-III, or RabGGTase in the absence or presence of BGPP for 30 min at 37ºC. Biotin-geranylated Ykt6 was detected


C Farnesylation of recombinant Ykt6 by FTase . Recombinant Ykt6 ( 5 μM ) was incubated with FTase ( 0 or 50 nM ) 3H - FPP ( 1 μM ; ~ 3 , 500 dpm / pmol ) at 37ºC . Reactions were stopped at indicated time points , amount of 3H - farnesyl transferred to Ykt6 was quantified by scintillation counting ( mean ± , n = 3 ) .

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C Farnesylation of recombinant Ykt6 by FTase. Recombinant Ykt6 (5 μM) was incubated with FTase (0 or 50 nM) and 3H-FPP (1 μM; ~3,500 dpm/pmol) at 37ºC. Reactions were stopped at the indicated time points, and the amount of 3H-farnesyl transferred to Ykt6 was quantified by scintillation counting (mean ± SEM, n = 3).


D Geranylgeranylation of recombinant Ykt6 by GGTase - III . Recombinant Ykt6 ( 5 μM ) was incubated with increasing concentrations of GGTase - III ( 0 - 100 nM ) 3H - GGPP ( 1 μM ; ~ 3 , 000 dpm / pmol ) for indicated times , amount of 3H - geranylgeranyl transferred to Ykt6 was quantified ( mean ± , n = 3 ) .

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D Geranylgeranylation of recombinant Ykt6 by GGTase-III. Recombinant Ykt6 (5 μM) was incubated with increasing concentrations of GGTase-III (0-100 nM) and 3H-GGPP (1 μM; ~3,000 dpm/pmol) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3).


E Geranylgeranylation of Cys to Ser mutants of Ykt6 by GGTase - III . WT mutant Ykt6 ( 5 μM each ) were incubated with GGTase - III ( 100 nM ) 3H - GGPP ( 1 μM ) for indicated times , amount of 3H - geranylgeranyl transferred to Ykt6 was quantified ( mean ± , n = 3 ) .

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E Geranylgeranylation of Cys to Ser mutants of Ykt6 by GGTase-III. WT and mutant Ykt6 proteins (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3).


G Mass spectra of prenylated Ykt6 peptides . Recombinant Ykt6 was farnesylated by FTase ( FTase ) , further processed by RCE1 ICMT ( RCE1 / ICMT ) , finally geranylgeranylated by GGTase - III ( GGTase - III ) . After purification to homogeneity , prenylated Ykt6 were digested with chymotrypsin ( left ) or V8 protease ( right ) analyzed by MALDI - TOF mass spectrometry . calculated m / z values for C - terminal peptide ions are shown in parentheses . peaks labeled with an asterisk correspond to 14 - 38 of Ykt6 ( AKVVLLKAAYDVSSFSFFQRSSVQE ; m / z 2806 . 48 ) .

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G Mass spectra of prenylated Ykt6 peptides. Recombinant Ykt6 was farnesylated by FTase (FTase), further processed by RCE1 and ICMT (RCE1/ICMT), and finally geranylgeranylated by GGTase-III (GGTase-III). After purification to homogeneity, the prenylated Ykt6 proteins were digested with chymotrypsin (left) or V8 protease (right) and analyzed by MALDI-TOF mass spectrometry. The calculated m/z values for the C-terminal peptide ions are shown in parentheses. The peaks labeled with an asterisk correspond to amino acids 14-38 of Ykt6 (AKVVLLKAAYDVSSFSFFQRSSVQE; m/z 2806.48).


Effects of C - terminal processing Cys195 farnesylation on Cys194 geranylgeranylation by GGTase - III . Purified recombinant unprenyl Ykt6 , Cys195 - farnesyl Ykt6 , Cys195 - farnesyl Ykt6ΔAIM ( 5 μM each ) were incubated with GGTase - III ( 100 nM ) 3H - GGPP ( 1 μM ) for indicated times , amount of 3H - geranylgeranyl transferred to Ykt6 was quantified . ( mean ± , n = 3 ) .

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Effects of C-terminal processing and Cys195 farnesylation on Cys194 geranylgeranylation by GGTase-III. Purified recombinant unprenyl Ykt6, Cys195-farnesyl Ykt6, and Cys195-farnesyl Ykt6ΔAIM (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified. (mean ± SEM, n = 3).


I Gel filtration chromatography of purified unprenyl Ykt6 , Cys195 - farnesyl Ykt6ΔAIM , Cys194 / 195 - diprenyl Ykt6ΔAIM ( 135 μg each ) on a Superdex 200 column . Dotted lines indicate elution profile of unprenyl Ykt6 for comparison . prenylated Ykt6 were eluted slightly later than unprenylated Ykt6 , suggesting more compact stable folds .

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I Gel filtration chromatography of purified unprenyl Ykt6, Cys195-farnesyl Ykt6ΔAIM, and Cys194/195-diprenyl Ykt6ΔAIM (135 μg each) on a Superdex 200 column. Dotted lines indicate the elution profile of unprenyl Ykt6 for comparison. The prenylated Ykt6 proteins were eluted slightly later than the unprenylated Ykt6, suggesting more compact and stable folds.


E Geranylgeranylation activity of WT GGTase - III indicated mutants ( 100 nM each ) . Cys195 - farnesyl Ykt6ΔAIM ( 1 μM ) 3H - GGPP ( 1 μM ) were used as substrates ( mean ± , n = 3 ) .

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E Geranylgeranylation activity of WT GGTase-III and the indicated mutants (100 nM each). Cys195-farnesyl Ykt6ΔAIM (1 μM) and 3H-GGPP (1 μM) were used as substrates (mean ± SEM, n = 3).


F Kinetic of WT GGTase - III site 1 or site 2 mutant ( 100 nM each ) using increasing concentrations of Cys195 - farnesyl Ykt6ΔAIM ( mean ± , n = 3 for WT Y306A , n = 1 for V48A / V50A ) .

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F Kinetic analysis of WT GGTase-III and the site 1 or site 2 mutant (100 nM each) using increasing concentrations of Cys195-farnesyl Ykt6ΔAIM (mean ± SEM, n = 3 for WT and Y306A, n = 1 for V48A/V50A).


Geranylgeranylation of unprenyl WT Ykt6 site 1 or site 2 mutants by GGTase - III . WT mutant Ykt6 ( 5 μM each ) were incubated with GGTase - III ( 100 nM ) 3H - GGPP ( 1 μM ) , amount of 3H - geranylgeranyl transferred to Ykt6 was quantified ( mean ± , n = 3 ) .

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Geranylgeranylation of unprenyl WT Ykt6 and the site 1 or site 2 mutants by GGTase-III. WT and mutant Ykt6 proteins (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM), and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3).


H Geranylgeranylation activity of WT GGTase - III tunnel mutants ( 100 nM each ) . Cys195 - farnesyl Ykt6ΔAIM ( 1 μM ) 3H - GGPP ( 1 μM ) were used as substrates ( mean ± , n = 3 ) .

H Geranylgeranylation activity of WT GGTase-III and tunnel mutants (100 nM each). Cys195-farnesyl Ykt6ΔAIM (1 μM) and 3H-GGPP (1 μM) were used as substrates (mean ± SEM, n = 3).


I GGPP binding to GGTase - III . WT GGTase - III tunnel mutants ( 1 μM each ) were incubated with 3H - GGPP ( 2 μM ) at 4ºC for 10 min . After desalting , enzyme - bound 3H - GGPP was quantified by scintillation counting ( mean ± , n = 3 ) .

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I GGPP binding to GGTase-III. WT GGTase-III and tunnel mutants (1 μM each) were incubated with 3H-GGPP (2 μM) at 4ºC for 10 min. After desalting, enzyme-bound 3H-GGPP was quantified by scintillation counting (mean ± SEM, n = 3).


A Immunoblot of lysates from WT HAP1 PTAR1 KO HAP1 using anti - Ykt6 antibody . Note slight difference in gel mobility of Ykt6 ( arrows ) .

A Immunoblot of cell lysates from WT HAP1 cells and PTAR1 KO HAP1 cells using anti-Ykt6 antibody. Note the slight difference in the gel mobility of Ykt6 (arrows).


B Prenylation status of rat brain Ykt6 . Recombinant unprenyl Ykt6 , Cys195 - farnesyl Ykt6 , Cys194 / 195 - diprenyl Ykt6 samples ( 1 ng each ) , rat brain cytosol were electrophoresed on a polyacrylamide gel using deoxycholate ( DOC ) - containing buffer analyzed by immunoblotting with anti - Ykt6 antibody ( DOC - PAGE ; upper panel ) . same samples were analyzed by conventional SDS - PAGE immunoblotting with anti - Ykt6 antibody ( SDS - PAGE ; lower panel ) .

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B Prenylation status of rat brain Ykt6. Recombinant unprenyl Ykt6, Cys195-farnesyl Ykt6, Cys194/195-diprenyl Ykt6 samples (1 ng each), and rat brain cytosol were electrophoresed on a polyacrylamide gel using deoxycholate (DOC)-containing buffer and analyzed by immunoblotting with anti-Ykt6 antibody (DOC-PAGE; upper panel). The same samples were analyzed by conventional SDS-PAGE and immunoblotting with anti-Ykt6 antibody (SDS-PAGE; lower panel).


C Prenylation status of Ykt6 in WT PTAR1 KO . Recombinant Ykt6 samples lysates of WT , PTAR1 KO , PTAR1 KO stably expressing PTAR1 ( KO + PTAR1 ) were separated by DOC - PAGE ( upper ) or SDS - PAGE ( lower ) , analyzed by immunoblotting with anti - Ykt6 antibody .

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C Prenylation status of Ykt6 in WT and PTAR1 KO cells. Recombinant Ykt6 samples and cell lysates of WT cells, PTAR1 KO cells, and PTAR1 KO cells stably expressing PTAR1 (KO + PTAR1) were separated by DOC-PAGE (upper) or SDS-PAGE (lower), and analyzed by immunoblotting with anti-Ykt6 antibody.


D In vitro reconstitution of Ykt6 double prenylation . Dialyzed cytosol prepared from PTAR1 KO HAP1 was incubated at 37ºC for indicated times with recombinant GGTase - III ( 100 nM ) or RabGGTase ( 100 nM ) in absence or presence of GGPP ( 10 μM ) . After incubation , reaction products were separated by DOC - PAGE ( upper ) or SDS - PAGE ( lower ) , analyzed by immunoblotting with anti - Ykt6 antibody .

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D In vitro reconstitution of Ykt6 double prenylation. Dialyzed cytosol prepared from PTAR1 KO HAP1 cells was incubated at 37ºC for the indicated times with recombinant GGTase-III (100 nM) or RabGGTase (100 nM) in the absence or presence of GGPP (10 μM). After incubation, reaction products were separated by DOC-PAGE (upper) or SDS-PAGE (lower), and analyzed by immunoblotting with anti-Ykt6 antibody.


A Representative confocal of Golgi apparatus in WT or PTAR1 KO HeLa . were co - immunostained for GM130 ( a cis - Golgi marker ) golgin - 97 ( a trans - Golgi marker ) . were deconvoluted using Huygens software . lower panels show magnified of boxed region of upper panels . Scale bars , 5 μm .

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A Representative confocal images of the Golgi apparatus in WT or PTAR1 KO HeLa cells. Cells were co-immunostained for GM130 (a cis-Golgi marker) and golgin-97 (a trans-Golgi marker). Images were deconvoluted using Huygens software. The lower panels show magnified images of the boxed region of the upper panels. Scale bars, 5 μm.


B Electron of Golgi apparatus in WT or PTAR1 KO HAP1 . Arrows indicate Golgi stacks . Arrowheads indicate examples of unfused vesicles accumulated around swollen Golgi cisternae in PTAR1 KO . Scale bars , 500 nm .

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B Electron micrographs of the Golgi apparatus in WT or PTAR1 KO HAP1 cells. Arrows indicate Golgi stacks. Arrowheads indicate examples of unfused vesicles accumulated around the swollen Golgi cisternae in PTAR1 KO cells. Scale bars, 500 nm.


Defect in intra - Golgi trafficking in PTAR1 KO . ( C ) VSVG - GFP expressing WT PTAR1 KO HeLa were cultured at 40ºC then shifted to 32ºC . VSVG - GFP taken at indicated times after shift are shown . right panel shows of VSVG - GFP in Golgi region after shift ( mean ± , n = 6 ) .

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Defect in intra-Golgi trafficking in PTAR1 KO cells. (C) VSVG-GFP expressing WT and PTAR1 KO HeLa cells were cultured at 40ºC and then shifted to 32ºC. VSVG-GFP fluorescence images taken at the indicated times after the temperature shift are shown. The right panel shows quantification of VSVG-GFP fluorescence in the Golgi region after the temperature shift (mean ± SEM, n = 6).


Defect in intra - Golgi trafficking in PTAR1 KO . ( D ) VSVG - GFP expressing WT HeLa , PTAR1 KO HeLa , PTAR1 KO HeLa stably expressing PTAR1 ( KO + PTAR1 ) were cultured at 40ºC then shifted to 32ºC . At indicated time points , surface were biotinylated using sulfo - NHS - LC - biotin . Biotinylated VSVG - GFP was purified from lysates using avidin agarose analyzed by immunoblotting with anti - GFP antibody . right panel shows of surface biotinylated VSVG - GFP ( means ± , n = 3 ) . Data were analyzed by one - way ANOVA with Dunnett ' s post - hoc . * p < 0 . 05 , * * * p < 0 . 001 .

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Defect in intra-Golgi trafficking in PTAR1 KO cells. (D) VSVG-GFP expressing WT HeLa cells, PTAR1 KO HeLa cells, and PTAR1 KO HeLa cells stably expressing PTAR1 (KO + PTAR1) were cultured at 40ºC and then shifted to 32ºC. At the indicated time points, cell surface proteins were biotinylated using sulfo-NHS-LC-biotin. Biotinylated VSVG-GFP was purified from cell lysates using avidin agarose and analyzed by immunoblotting with anti-GFP antibody. The right panel shows quantification of the cell surface biotinylated VSVG-GFP (means ± SEM, n = 3). Data were analyzed by one-way ANOVA with Dunnett's post-hoc test. *p < 0.05, ***p < 0.001.


E of LAMP1 glycosylation . lysates of WT HeLa , PTAR1 KO HeLa , PTAR1 KO HeLa stably expressing PTAR1 ( KO + PTAR1 ) were analyzed by immunoblotting with anti - LAMP1 antibody ( upper ) . Sialylated LAMP1 was precipitated from lysates using Maackia amurensis leucoagglutinin ( MAL ) agarose analyzed by immunoblotting ( lower ) .

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E Analysis of LAMP1 glycosylation. Cell lysates of WT HeLa cells, PTAR1 KO HeLa cells, and PTAR1 KO HeLa cells stably expressing PTAR1 (KO + PTAR1) were analyzed by immunoblotting with anti-LAMP1 antibody (upper). Sialylated LAMP1 was precipitated from the cell lysates using Maackia amurensis leucoagglutinin (MAL) agarose and analyzed by immunoblotting (lower).


F Defect in Golgi SNARE assembly in PTAR1 KO . Golgi SNARE was immunoprecipitated from NEM - treated WT HAP1 , PTAR1 KO HAP1 , PTAR1 KO HAP1 stably expressing PTAR1 ( KO + PTAR1 ) using control mouse IgG , anti - GS28 IgG , or anti - syntaxin 5 IgG . Immunoprecipitates were analyzed by immunoblotting with antibodies against syntaxin 5 , GS28 , GS15 , Ykt6 . Syntaxin 5 has two isoforms with different translation initiation sites . Inputs were 20 % ( syntaxin 5 , GS28 , GS15 ) 2 % ( Ykt6 ) . data shown are representative of three independent experiments with similar results .

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F Defect in the Golgi SNARE assembly in PTAR1 KO cells. The Golgi SNARE complex was immunoprecipitated from NEM-treated WT HAP1 cells, PTAR1 KO HAP1 cells, and PTAR1 KO HAP1 cells stably expressing PTAR1 (KO + PTAR1) using control mouse IgG, anti-GS28 IgG, or anti-syntaxin 5 IgG. Immunoprecipitates were analyzed by immunoblotting with antibodies against syntaxin 5, GS28, GS15, and Ykt6. Syntaxin 5 has two isoforms with different translation initiation sites. Inputs were 20% (syntaxin 5, GS28, and GS15) and 2% (Ykt6). The data shown are representative of three independent experiments with similar results.


( a ) Morphological . Jurkat T were cultured in presence absence of indicated anticancer drugs for 48 h . Drug - treated were classified as normal , apoptotic or abnormal , with enlarged irregular nuclei , with multipolar mitoses or multinucleated . Cultures treated with etoposide ( 0 . 25 μM ) , cisplatin ( 3 μM ) , taxol ( 4 nM ) nocodazole ( 10 nM ) exhibited high of with abnormal nuclei . None of treated populations showed a significant fraction of apoptosis ( see also Supplementary Fig . S1 ) . Values are means ± s . d . for three independent experiments . Right : representative examples of these morphological categories are shown . Scale bar , 10 μM . Similar abnormalities were also seen after treating NB4 MDA - MA - 231 with etoposide or cisplatin ( Supplementary Fig . S2 ) .

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(a) Morphological analysis. Jurkat T cells were cultured in the presence and absence of the indicated anticancer drugs for 48 h. Drug-treated cells were classified as normal, apoptotic or abnormal, with enlarged and irregular nuclei, with multipolar mitoses or multinucleated. Cultures treated with etoposide (0.25 μM), cisplatin (3 μM), taxol (4 nM) and nocodazole (10 nM) exhibited high levels of cells with abnormal nuclei. None of the treated populations showed a significant fraction of apoptosis (see also Supplementary Fig. S1). Values are means±s.d. for three independent experiments. Right: representative examples of these morphological categories are shown. Scale bar, 10 μM. Similar abnormalities were also seen after treating NB4 and MDA-MA-231 cells with etoposide or cisplatin (Supplementary Fig. S2).


( b ) Immunoblotting . Jurkat T were treated as indicated ATG5 ( monomeric ATG12 - conjugated ) , ATG12 ( conjugated with ATG5 ) , LC3 ( 18 16 kDa ) , Beclin 1 GAPDH detected . Chloroquine ( CQ ) was employed to block lysosomal proteases . Each immunoblot is representative of at least three independent experiments . same assays were performed using NB4 MDA - MA - 231 ( Supplementary Fig . S2 ) . Moreover , as a marker for autophagy onset , increased LC3 - II formation following etoposide or cisplatin treatment was also documented using microscopy ( Supplementary Fig . S2 ) . Full - immunoblots are provided in Supplementary Fig . S8 .

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(b) Immunoblotting. Jurkat T cells were treated as indicated and ATG5 (monomeric and ATG12-conjugated), ATG12 (conjugated with ATG5), LC3 (18 and 16 kDa), Beclin 1 and GAPDH detected. Chloroquine (CQ) was employed to block lysosomal proteases. Each immunoblot is representative of at least three independent experiments. The same assays were performed using NB4 and MDA-MA-231 cells (Supplementary Fig. S2). Moreover, as a marker for autophagy onset, increased LC3-II formation following etoposide or cisplatin treatment was also documented using fluorescence microscopy (Supplementary Fig. S2). Full-length immunoblots are provided in Supplementary Fig. S8.


( a ) Time - lapse microscopy . HeLa H2B - mCherry - α - tubulin - EGFP were cultured in presence absence of etoposide cisplatin , respectively . Treated frequently developed multiple nuclei , including micronuclei ( upper panel ) , demonstrated evidence of multipolar spindles misaligned chromosomes ( lower panel ) . were also much larger than untreated ( see also Supplementary Fig . S3 ) . Scale bar , 10 μm . ( b ) Statistical of data shown above . At least 100 were counted for each drug exposure time according to classification used in Fig . 1 . Values are means ± s . d . ( n = 3 ) .

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(a) Time-lapse microscopy. HeLa H2B-mCherry-α-tubulin-EGFP cells were cultured in the presence and absence of etoposide and cisplatin, respectively. Treated cells frequently developed multiple nuclei, including micronuclei (upper panel), and demonstrated evidence of multipolar spindles and misaligned chromosomes (lower panel). Cells were also much larger than untreated cells (see also Supplementary Fig. S3). Scale bar, 10 μm. (b) Statistical analysis of the data shown above. At least 100 cells were counted for each drug exposure time according to the classification used in Fig. 1. Values are means±s.d. (n=3).


( a ) Immunoblotting . Jurkat T were cultured as indicated for 48 h . Only under conditions of ATG5 induction was evidence of increased autophagic activity obtained . Monomeric ATG5 ( shown under dotted ) was detected after a longer exposure time compared with ATG5 conjugated with ATG12 . Results are representative of three independent experiments .

(a) Immunoblotting. Jurkat T cells were cultured as indicated for 48 h. Only under conditions of ATG5 induction was evidence of increased autophagic activity obtained. Monomeric ATG5 (shown under the dotted line) was detected after a longer exposure time compared with the ATG5 conjugated with ATG12. Results are representative of three independent experiments.


( b ) Morphological . Jurkat T were cultured as in a . Values are means ± s . d . of three independent experiments .

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(b) Morphological analysis. Jurkat T cells were cultured as in a. Values are means±s.d. of three independent experiments.


c ) Morphological . Jurkat T were treated with indicated drugs or transduced with lentivirus ATG5 , ATG5 - K130R , Beclin 1 or control constructs for 48 h . Enlarged multinucleated were seen as a consequence of ATG5 or ATG5 - K130R lentiviral gene transfer or of etoposide treatment . Increased Beclin 1 or rapamycin treatment had no such effect . Representative examples of are shown . Scale bar , 10 μm . Right : statistical of data . At least 100 were counted for each condition . Values are means ± s . d . ( n = 3 ) .

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c) Morphological analysis. Jurkat T cells were treated with the indicated drugs or transduced with lentivirus ATG5, ATG5-K130R, Beclin 1 or control constructs for 48 h. Enlarged and multinucleated cells were seen as a consequence of ATG5 or ATG5-K130R lentiviral gene transfer or of etoposide treatment. Increased Beclin 1 levels or rapamycin treatment had no such effect. Representative examples of morphology are shown. Scale bar, 10 μm. Right: statistical analysis of the data. At least 100 cells were counted for each condition. Values are means±s.d. (n=3).


( d ) Immunoblotting . Enforced expression of ATG5 , Beclin 1 , or ATG5 - K130R following lentiviral gene transfer .

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(d) Immunoblotting. Enforced expression of ATG5, Beclin 1, or ATG5-K130R following lentiviral gene transfer.


( e ) Time - lapse microscopy . HeLa H2B - mCherry - α - tubulin - EGFP were transduced by lentiviral gene transfer as indicated . Only ATG5 - overexpressing demonstrated evidence for mitotic catastrophe ( see also Supplementary Movies ) . As with GFP control , Beclin 1 construct also included GFP , hence in green was very high . Scale bar , 10 μm .

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(e) Time-lapse microscopy. HeLa H2B-mCherry-α-tubulin-EGFP cells were transduced by lentiviral gene transfer as indicated. Only ATG5-overexpressing cells demonstrated evidence for mitotic catastrophe (see also Supplementary Movies). As with the GFP control, the Beclin 1 construct also included GFP, hence the intensity in green was very high. Scale bar, 10 μm.


( a ) cycle . After lentivirus gene transfer , Jurkat T overexpressing ATG5 or GFP were followed for indicated times analysed for content by flow cytometry . Untransduced were left untreated or treated with etoposide for comparison . Values are means ± s . d . ( n = 3 ) . Right : representative flow cytometry diagrams for 48 h time point .

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(a) Cell cycle analysis. After lentivirus gene transfer, Jurkat T cells overexpressing ATG5 or GFP were followed for the indicated times and analysed for DNA content by flow cytometry. Untransduced cells were left untreated or treated with etoposide for comparison. Values are means±s.d. (n=3). Right: representative flow cytometry diagrams for the 48 h time point.


( b ) cycle . Jurkat were cultured as indicated for 48 h . Only under conditions of ATG5 induction , we observed an accumulation of in G2 - or M - phase arrest . Results are representative of three independent experiments .

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(b) Cell cycle analysis. Jurkat cells were cultured as indicated for 48 h. Only under conditions of ATG5 induction, we observed an accumulation of cells in G2- or M-phase arrest. Results are representative of three independent experiments.


( c ) Immunoblotting . Jurkat T overexpressing ATG5 or GFP were cultured for indicated times after transduction analysed . As controls , were treated with etoposide or rapamycin . Although rapamycin , similar to etoposide , induced autophagy , no increase in ATG5 expression no cycle arrest were observed . ATG5 overexpression etoposide treatment resulted in a similar expression pattern for investigated . Each immunoblot is representative of at least three independent experiments .

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(c) Immunoblotting. Jurkat T cells overexpressing ATG5 or GFP were cultured for the indicated times after transduction and analysed. As controls, cells were treated with etoposide or rapamycin. Although rapamycin, similar to etoposide, induced autophagy, no increase in ATG5 expression and no cell cycle arrest were observed. ATG5 overexpression and etoposide treatment resulted in a similar expression pattern for all proteins investigated. Each immunoblot is representative of at least three independent experiments.


( a ) Flow cytometry . Untreated Jurkat T , treated with etoposide continuously ( 0 . 25 μM ) , or transduced with lentivirus ATG5 or GFP were cultured for 48 h , fixed , permeabilized exposed to antiphospho - H2AX ( Ser139 ) antibody , then stained with antimouse - APC - conjugated antibody , analysed using a flow cytometer .

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(a) Flow cytometry. Untreated Jurkat T cells, cells treated with etoposide continuously (0.25 μM), or transduced with lentivirus ATG5 or GFP were cultured for 48 h, fixed, permeabilized and exposed to antiphospho-H2AX (Ser139) antibody, then stained with antimouse-APC-conjugated antibody, and analysed using a flow cytometer.


( b ) Confocal microscopy . Untreated HeLa , treated with etoposide continuously ( 0 . 25 μM ) , or transduced with lentivirus ATG5 were cultured for 48 h . Additional DAPI was used . Scale bar , 10 μm . Right : phospho - H2AX was quantified by measuring mean ( MFI ) in five high - power fields of each condition using IMARIS software . Values are means ± s . d .

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(b) Confocal microscopy. Untreated HeLa cells, cells treated with etoposide continuously (0.25 μM), or transduced with lentivirus ATG5 were cultured for 48 h. Additional DAPI nuclear staining was used. Scale bar, 10 μm. Right: phospho-H2AX was quantified by measuring the mean fluorescence intensity (MFI) in five high-power fields of each condition using IMARIS software. Values are means±s.d.


( c ) Jurkat T were treated as in a analysed by immunoblotting . Besides phospho - H2AX ( Ser139 ) , we also detected phospho - ATM ( Ser1981 ) phospho - ATR ( Ser428 ) , two additional markers of damage response . of bands were quantified with a Licor / Odyssey scanner software ( shown beneath each immunoblot ) . fold change in phosphorylation compared with no treatment ( untreated ) are presented . data in panels are representative of at least three independent experiments . Full - immunoblots are provided in Supplementary Fig . S9 .

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(c) Jurkat T cells were treated as in a and analysed by immunoblotting. Besides phospho-H2AX (Ser139), we also detected phospho-ATM (Ser1981) and phospho- ATR (Ser428), two additional markers of DNA damage response. Intensity of the protein bands were quantified with a Licor/Odyssey scanner and software (shown beneath each immunoblot). The fold change in phosphorylation levels compared with no treatment (untreated) are presented. The data in all panels are representative of at least three independent experiments. Full-length immunoblots are provided in Supplementary Fig. S9.


( a ) Immunoblotting . Jurkat T overexpressing ATG5 or control were cultured in presence absence of 3 - MA for 48 h . 3 - MA blocked formation of LC3 - II p62 degradation , indicating that it indeed inhibited ATG5 - induced autophagy . Results are representative of three independent experiments .

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(a) Immunoblotting. Jurkat T cells overexpressing ATG5 or control cells were cultured in the presence and absence of 3-MA for 48 h. 3-MA blocked the formation of LC3-II and p62 degradation, indicating that it indeed inhibited ATG5-induced autophagy. Results are representative of three independent experiments.


( b ) cycle . Normal Jurkat T overexpressing ATG5 were cultured for 48 h in presence absence of 3 - MA . Induction of G2 - or M - phase arrest was seen as a consequence of ATG5 overexpression independent of treatment with 3 - MA , as normal Jurkat T exhibited no G2 / M arrest following 3 - MA treatment . Representative flow cytometry diagrams are shown ( n = 3 ) . Moreover , induction of autophagy by Beclin 1 overexpression or starvation failed to produce cycle arrest ( Supplementary Fig . S4 ) .

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(b) Cell cycle analysis. Normal Jurkat T cells and cells overexpressing ATG5 were cultured for 48 h in the presence and absence of 3-MA. Induction of G2- or M-phase arrest was seen as a consequence of ATG5 overexpression independent of treatment with 3-MA, as normal Jurkat T cells exhibited no G2/M arrest following 3-MA treatment. Representative flow cytometry diagrams are shown (n=3). Moreover, induction of autophagy by Beclin 1 overexpression or starvation failed to produce cell cycle arrest (Supplementary Fig. S4).


( c ) Morphological . Normal Jurkat T overexpressing ATG5 were cultured for 48 h in presence absence of 3 - MA analysed . Representative examples of are shown ( n = 3 ) ; large multinucleated with abnormal nuclei were seen as a consequence of ATG5 overexpression independent of 3 - MA . Neither induction of autophagy by Beclin 1 overexpression nor starvation resulted in such abnormal ( Supplementary Fig . S4 ) . Scale bar , 10 μm . (

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 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

(c) Morphological analysis. Normal Jurkat T cells and cells overexpressing ATG5 were cultured for 48 h in the presence and absence of 3-MA and analysed. Representative examples of morphology are shown (n=3); large multinucleated cells and cells with abnormal nuclei were seen as a consequence of ATG5 overexpression independent of 3-MA. Neither induction of autophagy by Beclin 1 overexpression nor starvation resulted in such abnormal cells (Supplementary Fig. S4). Scale bar, 10 μm. (


( d ) Viability assays . Jurkat T either overexpressing ATG5 following lentiviral gene transfer or treated with etoposide were cultured in presence absence of indicated pharmacological inhibitors for indicated times to block autophagy caspase activity . values are means ± s . d . ( n = 3 ) .

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(d) Viability assays. Jurkat T cells either overexpressing ATG5 following lentiviral gene transfer or treated with etoposide were cultured in the presence and absence of the indicated pharmacological inhibitors for the indicated times to block autophagy and caspase activity. All values are means±s.d. (n=3).


( a ) Subcellular fractionation immunoblotting . Jurkat T were cultured in presence of etoposide for indicated times . Following etoposide treatment , ATG5 monomer was newly present in both cytoplasm nucleus . exposure of ATG5 monomer ( shown under dotted ) was longer than that shown above for ATG5 conjugated to ATG12 . Full - immunoblots are provided in Supplementary Fig . S10 .

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(a) Subcellular fractionation and immunoblotting. Jurkat T cells were cultured in the presence of etoposide for the indicated times. Following etoposide treatment, the ATG5 monomer was newly present in both cytoplasm and nucleus. The exposure of the ATG5 monomer (shown under the dotted line) was longer than that shown above for the ATG5 conjugated to ATG12. Full-length immunoblots are provided in Supplementary Fig. S10.


b ) A comparison of ATG5 sequences in different species demonstrates presence of a conserved putative NES , similar to that present in Beclin 1 . alignment was performed with Clustal W using data obtained from GenBank . characteristic leucines are indicated in red resulting of ATG5 - ΔNES , following mutation of three leucines within this motif , is shown . ( c )

b) A comparison of ATG5 sequences in different species demonstrates the presence of a conserved putative NES, similar to that present in Beclin 1. The sequence alignment was performed with Clustal W using sequence data obtained from GenBank. The characteristic leucines are indicated in red and the resulting sequence of ATG5-ΔNES, following mutation of three leucines within this motif, is shown. (c)


( c ) Subcellular fractionation immunoblotting of fraction . Jurkat T , either treated with etoposide , or overexpressing ATG5 following lentiviral gene transfer , were cultured in presence absence of leptomycin B for 72 h . Leptomycin B increased relative ATG5 Beclin 1 in nucleus .

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(c) Subcellular fractionation and immunoblotting of the nuclear fraction. Jurkat T cells, either treated with etoposide, or overexpressing ATG5 following lentiviral gene transfer, were cultured in the presence and absence of leptomycin B for 72 h. Leptomycin B increased relative ATG5 and Beclin 1 levels in the nucleus.


( d ) Subcellular fractionation immunoblotting . Jurkat T were transduced to express ATG5 - ΔNES , ATG5 , or GFP . Although increased of ATG5 were observed in both cytoplasm nucleus , ATG5 - ΔNES accumulated following lentiviral gene transfer in cytoplasm only . Full - immunoblots are provided in Supplementary Fig . S11 .

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(d) Subcellular fractionation and immunoblotting. Jurkat T cells were transduced to express ATG5-ΔNES, ATG5, or GFP. Although increased levels of ATG5 were observed in both cytoplasm and nucleus, ATG5-ΔNES accumulated following lentiviral gene transfer in the cytoplasm only. Full-length immunoblots are provided in Supplementary Fig. S11.


( e ) Immunoblotting . ATG5 - ΔNES overexpression induced LC3 - II formation p62 degradation , indicating increased autophagic flux .

(e) Immunoblotting. ATG5-ΔNES overexpression induced LC3-II formation and p62 degradation, indicating increased autophagic flux.


( f ) cycle . Jurkat T overexpressing ATG5 or ATG5 - ΔNES , as well as control were cultured for 48 h . Induction of G2 M phase arrest was seen as a consequence of ATG5 , but not ATG5 - ΔNES overexpression . As an additional control , we transduced with GFP , which , similar to ATG5 - ΔNES - overexpressing , failed to exhibit G2 - M - phase arrest . Representative flow cytometry diagrams are shown ( n = 3 ) .

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(f) Cell cycle analysis. Jurkat T cells overexpressing ATG5 or ATG5-ΔNES, as well as control cells were cultured for 48 h. Induction of G2 and M phase arrest was seen as a consequence of ATG5, but not ATG5-ΔNES overexpression. As an additional control, we transduced cells with GFP, which, similar to ATG5-ΔNES-overexpressing cells, failed to exhibit G2- and M-phase arrest. Representative flow cytometry diagrams are shown (n=3).


( g ) Morphological . Non - transduced Jurkat T overexpressing ATG5 or ATG5 - ΔNES were cultured for 48 h . Large , multinucleated were seen as a consequence of ATG5 , but not ATG5 - ΔNES overexpression . Representative examples are shown ( n = 3 ) . Scale bar , 10 μm .

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(g) Morphological analysis. Non-transduced Jurkat T cells and cells overexpressing ATG5 or ATG5-ΔNES were cultured for 48 h. Large, multinucleated cells were seen as a consequence of ATG5, but not ATG5-ΔNES overexpression. Representative examples are shown (n=3). Scale bar, 10 μm.


( a ) Lysates of Jurkat T overexpressing ATG5 or treated with etoposide ( 48 - h cultures ) were immunoprecipitated with anti - ATG5 or antisurvivin antibodies . A physical interaction between ATG5 survivin , absent in untreated or GFP - expressing control , was detected reciprocally . Full - immunoblots are provided in Supplementary Fig . S12 .

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(a) Lysates of Jurkat T cells overexpressing ATG5 or treated with etoposide (48-h cultures) were immunoprecipitated with anti-ATG5 or antisurvivin antibodies. A physical interaction between ATG5 and survivin, absent in untreated or GFP-expressing control cells, was detected reciprocally. Full-length immunoblots are provided in Supplementary Fig. S12.


( b ) Jurkat T overexpressing ATG5 - ΔNES exhibited no ATG5 / survivin molecular interaction . ( c ) ATG5 - K130R mutant did show coprecipitation of survivin with ATG5 .

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(b) Jurkat T cells overexpressing ATG5-ΔNES exhibited no ATG5/survivin molecular interaction. (c) The ATG5-K130R mutant did show coprecipitation of survivin with ATG5.


( d ) Calpain - mediated , truncated ATG5 ( tATG5 ) , that is , N - terminal part of ATG5 , failed to bind survivin . ATG5 - K130R mutant was control .

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(d) Calpain-mediated, truncated ATG5 (tATG5), that is, the N-terminal part of ATG5, failed to bind survivin. The ATG5-K130R mutant was the control.


( e ) Confocal microscopy . MDA - MA - 231 were etoposide treated or transduced as indicated ( 48 - h cultures ) stained . In untreated , survivin ATG5 were in cytosol with no colocalization . ATG5 - transduced exhibited ATG5 / survivin colocalization in nucleus . ATG5 - ΔNES - transduced showed survivin , but not ATG5 , in nuclei of cycling . Numerical was performed Pearson ' s correlation coefficients are indicated in . Scale bar , 10 μm . Right : Statistical ( of variance ) is presented in which results of ten representative within each group were integrated . Values are means ± s . d .

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(e) Confocal microscopy. MDA-MA-231 cells were etoposide treated or transduced as indicated (48-h cultures) and stained. In untreated cells, survivin and ATG5 were in the cytosol with no colocalization. ATG5-transduced cells exhibited ATG5/survivin colocalization in the nucleus. ATG5-ΔNES-transduced cells showed survivin, but not ATG5, in the nuclei of cycling cells. Numerical analysis was performed and Pearson's correlation coefficients are indicated in the images. Scale bar, 10 μm. Right: Statistical analysis (analysis of variance) is presented in which the results of ten representative cells within each group were integrated. Values are means±s.d.


( f ) physical interaction between Aurora B survivin was analysed reciprocally reduced in expressing high of ATG5 . Neither ATG5 nor HSP90 were detectable in Aurora B - containing immune precipitate , but ATG5 molecularly interacted with survivin . Results representative of three independent experiments .

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(f) The physical interaction between Aurora B and survivin was analysed reciprocally and reduced in cells expressing high levels of nuclear ATG5. Neither ATG5 nor HSP90 were detectable in the Aurora B-containing immune precipitate, but ATG5 molecularly interacted with survivin. Results representative of three independent experiments.


( g ) Confocal microscopy . HeLa treated with etoposide or transduced as indicated ( 48 - h cultures ) were stained with centromere - specific antiserum anti - Aurora B ( top panels ) or antisurvivin antibodies ( lower panels ) . In untreated or ATG5 - ΔNES transduced , both Aurora B survivin were recruited to prometaphase centromeres located at central spindle during anaphase where they no longer colocalized with centromeres . In contrast , etoposide - treated or ATG5 - transduced recruited much less Aurora B to prometaphase centromeres . Such recruitment , however , was observed during anaphase . Compared with in normal mitosis , survivin , although reduced , was recruited to centromeres in prometaphase , but , similar to Aurora B , remained attached to centromeres during anaphase . Such mislocation of Aurora B survivin was associated with severe chromosome alignment segregation defects . Colocalization were prepared using Imaris software . Numerical was performed correlation coefficients are indicated . Scale bar , 2 μm .

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

(g) Confocal microscopy. HeLa cells treated with etoposide or transduced as indicated (48-h cultures) were stained with centromere-specific antiserum and anti-Aurora B (top panels) or antisurvivin antibodies (lower panels). In untreated or ATG5-ΔNES transduced cells, both Aurora B and survivin were recruited to prometaphase centromeres and located at the central spindle during anaphase where they no longer colocalized with centromeres. In contrast, etoposide-treated or ATG5-transduced cells recruited much less Aurora B to prometaphase centromeres. Such recruitment, however, was observed during anaphase. Compared with cells in normal mitosis, survivin, although reduced, was recruited to centromeres in prometaphase, but, similar to Aurora B, remained attached to centromeres during anaphase. Such mislocation of Aurora B and survivin was associated with severe chromosome alignment and segregation defects. Colocalization images were prepared using Imaris software. Numerical analysis was performed and correlation coefficients are indicated. Scale bar, 2 μm.


( h ) Confocal microscopy . Sections of lung ( non - small - lung cancer ) oesophageal carcinoma stained for ATG5 survivin expression . In untreated , ATG5 expression in cancer was slight mainly . Following radio - / or chemotherapy , ATG5 was preferentially expressed in nucleus colocalized with survivin . Overlay present average Pearson ' s correlation coefficient as calculated with Imaris . Results are representative of three independent experiments . Scale bar , 10 μm .

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(h) Confocal microscopy. Sections of lung (non-small-cell lung cancer) and oesophageal carcinoma stained for ATG5 and survivin expression. In untreated patients, ATG5 expression in cancer cells was slight and mainly cytosolic. Following radio- and/or chemotherapy, ATG5 was preferentially expressed in the nucleus and colocalized with survivin. Overlay images present the average Pearson's correlation coefficient as calculated with Imaris. Results are representative of three independent experiments. Scale bar, 10 μm.


A - H Effect of CCCP on localisation of Noc NocNΔ10 . localisation of Noc ‐ mYFP ( DWA206 ) NocNΔ10 ‐ mYFP ( DWA382 ) either with no additions ( NA ) or after CCCP treatment ( 5 min ; 100 μM ) , as indicated . Scale bar , 2 . 5 μm .

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A-H Effect of CCCP on the localisation of Noc and NocNΔ10. Cellular localisation of Noc‐mYFP (DWA206) and NocNΔ10‐mYFP (DWA382) either with no additions (NA) or after CCCP treatment (5 min; 100 μM), as indicated. Scale bar, 2.5 μm.


I - L Effect of Noc overproduction on division . Exponentially growing cultures of DWA119 ( Δnoc , Pspac ( hy ) ‐ noc ) DWA282 ( Δnoc , Pspac ( hy ) ‐ nocNΔ10 ) were examined after growth for 1 h with 1 mM IPTG . membranes were stained with FM5 ‐ 95 DAPI , respectively . Insets show corresponding phase contrast microscopy . Scale bar , 5 μm .

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I-L Effect of Noc overproduction on cell division. Exponentially growing cultures of DWA119 (Δnoc, Pspac(hy)‐noc) and DWA282 (Δnoc, Pspac(hy)‐nocNΔ10) were examined after growth for 1 h with 1 mM IPTG. Cell membranes and DNA were stained with FM5‐95 and DAPI, respectively. Insets show corresponding phase contrast light microscopy images. Scale bar, 5 μm.


M Complementation of noc in a Δnoc ΔminCD background . Strains DWA564 ( Δnoc , ΔminCD , Pxyl ‐ noc ‐ myfp ) 566 ( Δnoc , ΔminCD , Pxyl ‐ nocNΔ10 ‐ myfp ) were streaked on nutrient agar ( NA ) plates in presence of 0 . 5 % w / v xylose incubated at 30 39 ° C , as indicated .

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M Complementation of noc in a Δnoc ΔminCD background. Strains DWA564 (Δnoc, ΔminCD, Pxyl‐noc‐myfp) and 566 (Δnoc, ΔminCD, Pxyl‐nocNΔ10‐myfp) were streaked on nutrient agar (NA) plates in the presence of 0.5% w/v xylose and incubated at 30 and 39°C, as indicated.


N Effect of Noc NocNΔ10 overproduction on sporulation . Strains DWA119 ( Δnoc , Pspac ( hy ) ‐ noc ) DWA282 ( Δnoc , Pspac ( hy ) ‐ nocNΔ10 ) were streaked on NA plates in absence presence of 1 mM IPTG , as indicated , photographed after 48 h at 37 ° C .

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N Effect of Noc and NocNΔ10 overproduction on sporulation. Strains DWA119 (Δnoc, Pspac(hy)‐noc) and DWA282 (Δnoc, Pspac(hy)‐nocNΔ10) were streaked on NA plates in the absence and presence of 1 mM IPTG, as indicated, and photographed after 48 h at 37°C.


O Western blot of fractions ( T , total ; C , ; M , ) of strains DWA119 282 expressing Noc or NocNΔ10 , respectively . were detected using polyclonal antibodies against Noc , DnaA PBP2B . Antibodies were used at a dilution of 1 : 10 , 000 .

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O Western blot analysis of cellular fractions (T, total; C, cytosolic; M, membrane) of strains DWA119 and 282 expressing Noc or NocNΔ10, respectively. Proteins were detected using polyclonal antibodies against Noc, DnaA and PBP2B. Antibodies were used at a dilution of 1:10,000.


C - L Effects of N ‐ terminal substitutions on Noc localisation , in strains : DWA211 ( F5E ) , 318 ( F9E ) , 316 ( K2E ) , 212 ( R7E ) , 322 ( F5A ) , 323 ( F8A ) , 325 ( F9A ) , 206 ( WT ) , 328 ( S4A ) 329 ( S4L ) , as indicated . Insets show corresponding phase contrast microscopy . Scale bar , 5 μm .

C-L Effects of N‐terminal substitutions on Noc localisation, in strains: DWA211 (F5E), 318 (F9E), 316 (K2E), 212 (R7E), 322 (F5A), 323 (F8A), 325 (F9A), 206 (WT), 328 (S4A) and 329 (S4L), as indicated. Insets show the corresponding phase contrast light microscopy images. Scale bar, 5 μm.


B Growth of strains DWA350 ( Δnoc ΔminCD ) DWA307 ( Δnoc ΔminCD , Pxyl ‐ HCVAH ‐ NocNΔ10 ) on nutrient agar plates at 30 48 ° C in absence presence of 0 . 5 % w / v xylose , as indicated .

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B Growth of strains DWA350 (Δnoc ΔminCD) and DWA307 (Δnoc ΔminCD, Pxyl‐HCVAH‐NocNΔ10) on nutrient agar plates at 30 and 48°C in the absence and presence of 0.5% w/v xylose, as indicated.


C - F of strains DWA350 DWA307 following growth in LB at 30 ° C ( C D ) at 42 ° C in LB + 0 . 5 % w / v xylose ( E F ) . Arrowheads indicate minicells . membranes were stained with FM5 - 95 . Insets show corresponding phase contrast microscopy . Scale bar , 5 μm .

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C-F Cell morphology of strains DWA350 and DWA307 following growth in LB at 30°C (C and D) and at 42°C in LB + 0.5% w/v xylose (E and F). Arrowheads indicate minicells. Cell membranes were stained with FM5-95. Insets show the corresponding phase contrast light microscopy images. Scale bar, 5 μm.


G , H localisation of HCVAH ‐ NocNΔ10 ‐ YFP ( G ) in DWA193 ( Δnoc , Pxyl ‐ HCVAH ‐ nocNΔ10 ‐ yfp ) overlay showing DAPI ‐ stained ( H ) . was grown at 30 ° C in CH medium . Inset shows corresponding phase contrast microscopy image . Scale bar , 5 μm .

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G, H Cellular localisation of HCVAH‐NocNΔ10‐YFP (G) in strain DWA193 (Δnoc, Pxyl‐HCVAH‐nocNΔ10‐yfp) and overlay showing DAPI‐stained DNA (H). The strain was grown at 30°C in CH medium. Inset shows the corresponding phase contrast light microscopy image. Scale bar, 5 μm.


A - F Localisation of ParB ‐ box mutants . localisation of ( A ) Noc ‐ mYFP ( DWA206 ) , ( B ) NocQ68R ‐ mYFP ( DWA285 ) , ( C ) NocG86S ‐ mYFP ( DWA286 ) , ( D ) NocR88A ‐ mYFP ( DWA545 ) , ( E ) NocR89A ‐ mYFP ( DWA546 ) ( F ) NocR91A ‐ mYFP ( DWA547 ) . Insets show corresponding phase contrast . Scale bar , 5 μm .

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A-F Localisation of ParB‐box mutants. Cellular localisation of (A) Noc‐mYFP (DWA206), (B) NocQ68R‐mYFP (DWA285), (C) NocG86S‐mYFP (DWA286), (D) NocR88A‐mYFP (DWA545), (E) NocR89A‐mYFP (DWA546) and (F) NocR91A‐mYFP (DWA547). Insets show the corresponding phase contrast images. Scale bar, 5 μm.


G Ability of ParB ‐ box mutants to rescue growth defect of Δnoc ΔminCD . Strains DWA564 ( Pxyl ‐ noc ‐ myfp ) , 590 ( Pxyl ‐ nocQ68R ‐ myfp ) , 568 ( Pxyl ‐ nocG86S ‐ myfp ) , 598 ( Pxyl ‐ nocR88A ‐ myfp ) , 600 ( Pxyl ‐ nocR89A ‐ myfp ) 602 ( Pxyl ‐ nocR91A ‐ myfp ) were streaked on plates containing 0 . 5 % w / v xylose incubated for 18 h at either 30 or 39 ° C , as indicated , before being photographed .

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G Ability of ParB‐box mutants to rescue the growth defect of Δnoc ΔminCD. Strains DWA564 (Pxyl‐noc‐myfp), 590 (Pxyl‐nocQ68R‐myfp), 568 (Pxyl‐nocG86S‐myfp), 598 (Pxyl‐nocR88A‐myfp), 600 (Pxyl‐nocR89A‐myfp) and 602 (Pxyl‐nocR91A‐myfp) were streaked on plates containing 0.5% w/v xylose and incubated for 18 h at either 30 or 39°C, as indicated, before being photographed.


H - O ParB ‐ box mutants are dominant ‐ negative . of strains DWA362 ( ΔminCD , Pspac ( hy ) ‐ noc ) , 363 ( ΔminCD , Pspac ( hy ) ‐ nocNΔ10 ) , 364 ( ΔminCD , Pspac ( hy ) ‐ nocQ68R ) 365 ( ΔminCD , Pspac ( hy ) ‐ nocG86S ) were examined after growth for 2 h at 42 ° C with either no additions ( NA ) ( H - K ) or in presence of 1 mM IPTG ( L - O ) , as indicated . membranes were stained with FM5 ‐ 95 . Insets show corresponding phase contrast . Scale bar , 5 μm .

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H-O ParB‐box mutants are dominant‐negative. Cells of strains DWA362 (ΔminCD, Pspac(hy)‐noc), 363 (ΔminCD, Pspac(hy)‐nocNΔ10), 364 (ΔminCD, Pspac(hy)‐nocQ68R) and 365 (ΔminCD, Pspac(hy)‐nocG86S) were examined after growth for 2 h at 42°C with either no additions (NA) (H-K) or in the presence of 1 mM IPTG (L-O), as indicated. Cell membranes were stained with FM5‐95. Insets show the corresponding phase contrast images. Scale bar, 5 μm.


A - C Representative of TetR ‐ mCherry in lacking ( A ) or containing ( B C ) NBS plasmid pDWA117 . Strains DWA427 ( A ) 429 ( B C ) ( both strains contain Pspac ‐ noc ) were examined after growth for 2 h in absence ( A B ) presence ( C ) of 1 mM IPTG . was stained with DAPI .

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A-C Representative images of TetR‐mCherry in cells lacking (A) or containing (B and C) the NBS plasmid pDWA117. Strains DWA427 (A) and 429 (B and C) (both strains contain Pspac‐noc) were examined after growth for 2 h in the absence (A and B) and presence (C) of 1 mM IPTG. DNA was stained with DAPI.


D - G Representative showing localisation of ( D ) Noc ‐ mYFP ( DWA519 ) , ( E ) NocNΔ10 ‐ mYFP ( DWA522 ) , ( F ) NocQ68R ‐ mYFP ( DWA520 ) ( G ) NocG86S ‐ mYFP ( DWA521 ) , in presence of NBS plasmid pSG4929 .

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D-G Representative images showing the localisation of (D) Noc‐mYFP (DWA519), (E) NocNΔ10‐mYFP (DWA522), (F) NocQ68R‐mYFP (DWA520) and (G) NocG86S‐mYFP (DWA521), in the presence of the NBS plasmid pSG4929.


A , B Effects of Noc overproduction on division nucleoid in E . coli . of DWA261 carrying pDWA37 ( PA1 / 04 / 03 ‐ noc ) were examined after growth in LB with either no additions ( A ) or after induction for 1 h with 1 mM IPTG ( B ) .

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A, B Effects of Noc overproduction on cell division and nucleoid morphology in E. coli. Cells of strain DWA261 carrying pDWA37 (PA1/04/03‐noc) were examined after growth in LB with either no additions (A) or after induction for 1 h with 1 mM IPTG (B).


C - E Effects of overproduction of Noc variants on nucleoid . of strains DWA266 ( PA1 / 04 / 03 ‐ nocNΔ10 ) ( C ) , 270 ( PA1 / 04 / 03 ‐ HCVAH ‐ nocNΔ10 ) ( D ) 267 ( PA1 / 04 / 03 ‐ nocCΔ50 ) ( E ) carrying plasmids for overproduction of indicated mutants ( see cartoons underneath panels ) were grown in LB examined after growth for 1 h in presence of 1 mM IPTG . membranes were stained with FM5 ‐ 95 DAPI , respectively . Scale bar , 5 μm .

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C-E Effects of overproduction of Noc variants on nucleoid morphology. Cells of strains DWA266 (PA1/04/03‐nocNΔ10) (C), 270 (PA1/04/03‐HCVAH‐nocNΔ10) (D) and 267 (PA1/04/03‐nocCΔ50) (E) carrying plasmids for the overproduction of the indicated mutants (see cartoons underneath panels) were grown in LB and examined after growth for 1 h in the presence of 1 mM IPTG. Cell membranes and DNA were stained with FM5‐95 and DAPI, respectively. Scale bar, 5 μm.


A , B Effects of Spo0J Noc30 ‐ Spo0J overproduction on division nucleoid in E . coli . of strains DWA271 ( PA1 / 04 / 03 ‐ spo0J ) ( A ) 272 ( PA1 / 04 / 03 ‐ noc ‐ spo0J ) ( B ) were grown in LB in presence of 1 mM IPTG , to induce expression of either Spo0J ( A ) or Noc ‐ Spo0J hybrid ( B ) , were examined 1 h post ‐ induction . membranes were stained with FM5 - 95 DAPI , respectively . Scale bar , 5 μm .

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A, B Effects of Spo0J and Noc30‐Spo0J overproduction on cell division and nucleoid morphology in E. coli. Cells of strains DWA271 (PA1/04/03‐spo0J) (A) and 272 (PA1/04/03‐noc‐spo0J) (B) were grown in LB in the presence of 1 mM IPTG, to induce the expression of either Spo0J (A) or the Noc‐Spo0J hybrid (B), and were examined 1 h post‐induction. Cell membranes and DNA were stained with FM5-95 and DAPI, respectively. Scale bar, 5 μm.


( A ) Volcano plot shows genes where reads are statistically significant under - or over - expressed in 5 RIRCD affected individuals versus 6 controls resulted from DESeq2 . Decreased genes are represented in red , up - regulated ones are in green while blue dots highlight most outlying genes ( defined by absolute ( log2 ( fold change ) + log10 ( padj ) ) > 10 ) . Genes that were not statistically significant are represented in black . Grey black gene names indicate top 30 % of most dysregulated genes with a highlight on ones involved in metabolism .

(A) Volcano plot shows genes where RNA reads are statistically significant under- or over-expressed in 5 RIRCD affected individuals versus 6 controls resulted from DESeq2 analysis. Decreased genes are represented in red, the up-regulated ones are in green while blue dots highlight most outlying genes (defined by absolute (log2(fold change) + log10(padj))>10).Genes that were not statistically significant are represented in black. Grey and black gene names indicate top 30% of the most dysregulated genes with a highlight on ones involved in metabolism.


( D ) Relevant functional changes in gene expression showing major genes that return to baseline or contribute to recovery in RIRCD . data depicted is between affected recovered biopsies of same ( F6 / 1M ) .

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(D) Relevant functional changes in gene expression showing the major genes that return to baseline or contribute to the recovery in RIRCD patients. The data depicted is between the affected and recovered biopsies of the same patient (F6/1M).


( E ) Comparative proteome profiling of that are significantly under - or over - expressed in RIRCD affected versus control individuals ( p - Anova < 0 . 05 - statistically significant - horizontal ) . which are decreased are represented in red while up - regulated ones are in green while that did not reach statistical significance threshold are marked in black . Vertical lines are delimiting regulated resulted from proteomics ( cut - off values were determined on 2 × standard deviation normal distribution from identified ' s log2 in which bell curve is symmetric around mean , therefore , an average log2 of a which < − 2 . 2 or > 0 . 98 was considered as regulated ) .

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(E) Comparative proteome profiling of proteins that are significantly under- or over-expressed in RIRCD affected versus control individuals (p-Anova <0.05-statistically significant - horizontal line). Proteins which are decreased are represented in red while the up-regulated ones are in green while proteins that did not reach statistical significance threshold are marked in black. Vertical lines are delimiting the regulated proteins resulted from the proteomics analysis (the cut-off values were determined based on the 2× standard deviation and the normal distribution from all identified protein's log2 ratio in which the bell curve is symmetric around the mean, therefore, an average log2 ratio of a protein which <−2.2 or > 0.98 was considered as regulated).


( A ) Western blotting depicts mitochondrial ( NDUFA9 - I , SDHA - II , MTCO1 - IV MTCO2 - IV ) in control derived muscle ( n = 3 ) , derived muscle during symptomatic phase ( n = 3 ) derived muscle after recovery ( n = 1 ) . VCL was used as a loading control while densitometry was done on mitochondrial TOM20 . ( B ) Densitometry of immunoblotting from ( A ) showing an increase in SDHA ( II ) , while NDUFA9 ( I ) probably as a compensatory mechanism MT - CO2 ( IV ) show significant reductions as majority of mitochondrial encoded belong to this two complexes , thus being destabilized by defective mitochondrial mistranslation .

(A) Western blotting analysis depicts mitochondrial proteins (NDUFA9-Complex I, SDHA-Complex II, MTCO1-Complex IV and MTCO2-Complex IV) levels in control derived muscle (n=3), patient derived muscle during the symptomatic phase (n=3) and patient derived muscle after recovery (n=1). VCL was used as a loading control while the densitometry analysis was done based on the mitochondrial protein TOM20. (B) Densitometry analysis of the immunoblotting from (A) showing an increase in SDHA (Complex II), while NDUFA9 (Complex I) probably as a compensatory mechanism and MT-CO2 (Complex IV) show significant reductions as the majority of mitochondrial encoded proteins belong to this two complexes, thus being destabilized by the defective mitochondrial mistranslation.


( C ) Mitochondrial copy numbers were determined in affected ( n = 3 ) recovered individuals ( n = 2 ) compared to control individuals ( n = 4 , 3 months , 3 years , 4 years 7 years ) using qPCR showing a significant increase in recovered muscle .

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(C) Mitochondrial DNA copy numbers were determined in affected (n=3) and recovered individuals (n=2) and compared to control individuals (n=4, 3 months, 3 years, 4 years and 7 years) using qPCR showing a significant increase in recovered muscle.


( D ) Western blotting of involved in - acid sensing integrated stress response : GCN2 , SLC7A11 ATF4 in control derived muscle ( n = 3 ) , derived muscle during symptomatic phase ( n = 3 ) derived muscle after recovery ( n = 1 ) . samples were normalized to VCL which was used as a loading control . ( E ) Densitometry of from ( D ) showing a significant increase of major transducers of ISR such as GCN2 ATF4 . Consistent with ISR activation is also increase in SLC7A1 .

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(D) Western blotting analysis of proteins involved in the amino-acid sensing and integrated stress response: GCN2, SLC7A11 and ATF4 in control derived muscle (n=3), patient derived muscle during the symptomatic phase (n=3) and patient derived muscle after recovery (n=1). All samples were normalized to VCL which was used as a loading control. (E) Densitometry analysis of proteins from (D) showing a significant increase of major transducers of ISR such as GCN2 and ATF4. Consistent with ISR activation is also the increase in SLC7A1.


( F ) Transcriptomic pathways related to ATFs ISR changed in affected compared to controls ( RIRCD n = 5 ; controls n = 6 ) in recovered muscle compared to affected muscle ( n = 1 , affected recovered muscle was collected from same was analysed using 4 technical replicates ) schematic representation of metabolic pathways impacted in muscle ( bottom , right panel ) .

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(F) Transcriptomic pathways related to ATFs and ISR changed in affected patients compared to controls (RIRCD n=5; controls n=6) and in recovered muscle compared to affected muscle (n=1, affected and recovered muscle was collected from the same individual and was analysed using 4 technical replicates) and schematic representation of the metabolic pathways impacted in patient muscle (bottom, right panel).


( A ) n = 4 ( F7 / 7F - healthy carrier , F7 / 2M - RIRCD , F7 / 5F - healthy carrier , F7 / 1M - RIRCD without a clear second mutation ) control fibroblasts ( n = 2 ) were grown for 12 days in media containing 0 . 02 mM cysteine 5 % dialyzed FBS . belonging to mitochondrial complexes ( NDUFA9 - I , SDHA - II , MTCO1 - IV MTCO2 - IV ) were analysed via Western Blotting . VCL was used as a loading control densitometry was done on mitochondrial proteinTOM20 . ( B ) Densitometry of from ( A ) showing no significant changes in OXPHOS upon glutamine / glutamic acid depletion . Control bar represents an average of two control fibroblast lines employed ( F008 F154 ) .

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(A) Patient n= 4 (F7/7F-healthy carrier, F7/2M-RIRCD, F7/5F-healthy carrier, F7/1M-RIRCD without a clear second mutation) and control fibroblasts (n=2) were grown for 12 days in media containing 0.02 mM cysteine and 5% dialyzed FBS. Proteins belonging to mitochondrial complexes (NDUFA9-Complex I, SDHA-Complex II, MTCO1-Complex IV and MTCO2-Complex IV) were analysed via Western Blotting. VCL was used as a loading control and the densitometry analysis was done based on the mitochondrial proteinTOM20. (B) Densitometry analysis of proteins from (A) showing no significant changes in OXPHOS proteins upon glutamine/ glutamic acid depletion. Control bar represents an average of the two control fibroblast lines employed (F008 and F154).


( C ) ( F7 / 7F - healthy carrier , F7 / 2M - RIRCD , F7 / 5F - healthy carrier , F7 / 1M - RIRCD without a clear second mutation ) control fibroblasts were grown for 12 days in media with no added glutamine or glutamic acid 5 % dialyzed FBS . were lysed above mentioned mitochondrial were checked via Immunoblotting . VCL was used as a loading control while densitometry ( D ) was done on mitochondrial proteinTOM20 . Control bar represents an average of two control fibroblast lines employed ( F008 F154 ) . Although we could observe significant changes upon cysteine depletion in conditions , phenotype was exacerbated in digenic fibroblasts compared to controls .

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(C) Patient (F7/7F-healthy carrier, F7/2M-RIRCD, F7/5F-healthy carrier, F7/1M-RIRCD without a clear second mutation) and control fibroblasts were grown for 12 days in media with no added glutamine or glutamic acid and 5% dialyzed FBS. Cells were lysed and the above mentioned mitochondrial proteins were checked via Immunoblotting. VCL was used as a loading control while the densitometry analysis (D) was done based on the mitochondrial proteinTOM20. Control bar represents an average of the two control fibroblast lines employed (F008 and F154). Although we could observe significant changes upon cysteine depletion in all conditions, the phenotype was exacerbated in the digenic fibroblasts compared to the controls.


( A ) Volcano plot of transcriptomic changes where statistically significant genese are delimited by a horizontal . Decreased genes are represented in red while up - regulated ones are in green , while genes that were not statistically significant are represented in black .

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(A) Volcano plot analysis of transcriptomic changes where statistically significant genese are delimited by a horizontal line. Decreased genes are represented in red while the up-regulated ones are in green, while genes that were not statistically significant are represented in black.


( B ) of altered mitochondrial gene expression resulted from our RNAseq showing that affected genes their coding carry a high numbers of glutamic acid glutamine residues , in accordance to mitochondrial translational defect .

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(B) Analysis of altered mitochondrial gene expression resulted from our RNAseq showing that affected genes and their coding proteins carry a high numbers of glutamic acid and glutamine residues, in accordance to the mitochondrial translational defect.


FP titrations show binding of WT α - actinin - 1 _ EF34 to IQ peptides WT ( black ) K1647E ( red ) , of α - actinin - 1 _ EF34 mutant E847K / E851K to IQ peptides WT ( green ) K1647E ( purple ) , of full - α - actinin - 1 to IQ WT ( o ) , lack of IQ binding to α - actinin - 1 _ CH1 - CH2 ( + ) α - actinin - 1 _ EF12 ( x ; see Table 2 for binding parameters standard errors ) . FP titrations showing binding of wild type α - actinin - 1 _ EF34 to IQ peptides WT ( black ) I1654A ( purple ) of ACTN1 _ EF34 mutant F833 to IQ WT ( red ; see Table 2 for binding parameters standard errors ) .

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FP titrations show binding of WT α-actinin-1_EF34 to IQ peptides WT (black) and K1647E (red), of α-actinin-1_EF34 mutant E847K/E851K to IQ peptides WT (green) and K1647E (purple), of full-length α-actinin-1 to IQ WT (o), and lack of IQ binding to α-actinin-1_CH1-CH2 (+) and α-actinin-1_EF12 (x; see Table 2 for binding parameters and standard errors). FP titrations showing binding of wild type α-actinin-1_EF34 to IQ peptides WT (black) and I1654A (purple) and of ACTN1_EF34 mutant F833 to IQ WT (red; see Table 2 for binding parameters and standard errors).


FP titrations showing binding of apoCaM to IQ peptides WT ( black ) K1662E ( red ) of apoCaM mutant E88K to IQ WT ( green ) IQ peptide K1662E ( purple , " charge inversion " ; see Table 4 for binding parameters standard errors ) . FP titrations showing binding of apo - CaM to IQ peptides WT ( black ) , I1654A ( red ) , F1658A ( green ) of apoCaM mutant F90A to IQ WT ( blue ; see Table 4 for binding parameters standard errors ) .

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FP titrations showing binding of apoCaM to IQ peptides WT (black) and K1662E (red) and of apoCaM mutant E88K to IQ WT (green) and IQ peptide K1662E (purple, "charge inversion"; see Table 4 for binding parameters and standard errors). FP titrations showing binding of apo-CaM to IQ peptides WT (black), I1654A (red), and F1658A (green) and of apoCaM mutant F90A to IQ WT (blue; see Table 4 for binding parameters and standard errors).


HEK293 were transfected with α11 . 2 , α2δ - 1 , β2A cultured for 22 - 24h before attached patch recording in 110 mM Ba2 + . Single CaV1 . 2 channel recordings of CaV1 . 2 WT K1647A . Holding potential ( HP ) was - 80 mV potential ( TP ) 0 mV . Shown are 10 consecutive sweeps from representative experiments ( see Fig . EV 4 for more sweeps ) . Dot plots means + for availability ( i . e . , likelihood that a sweep had at least one event ; c ) , NPo ( d ) , Po ( e ) , unitary current amplitude i ( e ) , mean of current of a single channel at any point in time calculated from ensemble averages of each experiment ( g ) ( * * p < 0 . 01 , * * * p < 0 . 001 compared to WT ; one - way ANOVA with Bonferroni post - hoc ( c ) or Welch ANOVA with Tamhane T2 ( d - g ) ; n = 7 to 35 see Table 5 for more details ) .

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HEK293 cells were transfected with α11.2, α2δ-1, and β2A and cultured for 22-24h before cell attached patch recording in 110 mM Ba2+. Single CaV1.2 channel recordings of CaV1.2 WT and K1647A. Holding potential (HP) was -80 mV and test potential (TP) 0 mV. Shown are 10 consecutive sweeps from representative experiments (see Fig. EV 4 for more sweeps). Dot plots and means + SEM for availability (i.e., likelihood that a sweep had at least one event; c), NPo (d), Po (e), unitary current amplitude i (e), and the mean of the current of a single channel at any point in time calculated from the ensemble averages of each experiment (g) (**p<0.01, ***p<0.001 compared to WT; one-way ANOVA with Bonferroni post-hoc test (c) or Welch ANOVA with Tamhane T2 test (d-g); n = 7 to 35 see Table 5 for more details).