( A ) Proportion change of Chinese SL isolates from different phylogenetic clades . Chinese isolates were classified into two groups on their sampling time ( 1993 - 2010 vs 2011 - 2020 ) . of isolates from each clade in each period was compared .

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(A) Proportion change of Chinese SL isolates from different phylogenetic clades. The Chinese isolates were classified into two groups based on their sampling time (1993-2010 vs 2011-2020). The percentage of isolates from each clade in each period was compared.


( A ) resistance to desiccation . Representative isolates that are phylogenetically distinct within Clade - 1 ( n = 2 ) , Clade - 2 ( n = 1 ) , Clade - 4 ( n = 2 ) Clade - 5 ( n = 6 ) were used for desiccation Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( A ) were calculated using unpaired t - ( two - ) ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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(A) Bacterial resistance to desiccation. Representative isolates that are phylogenetically distinct within Clade-1 (n=2), Clade-2 (n=1), Clade-4 (n=2) and Clade-5 (n=6) were used for desiccation assay Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (A) were calculated using unpaired t-test (two-tailed) (*:P<0.05, **:P<0.01, ***:P<0.001).


( B ) survivability under oxidative stress . Representative isolates from C1 ( n = 2 ) , C2 ( n = 1 ) , C4 ( n = 2 ) C5 ( n = 6 ) were treated with hydrogen peroxide . Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( B ) , were calculated using unpaired t - ( two - ) , ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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(B) Bacterial survivability under oxidative stress. Representative isolates from C1 (n=2), C2 (n=1), C4 (n=2) and C5 (n=6) were treated with hydrogen peroxide. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (B), were calculated using unpaired t-test (two-tailed), (*:P<0.05, **:P<0.01, ***:P<0.001).


( C ) Biofilm formation ability . biofilm of representative isolates from C1 ( n = 2 ) , C2 ( n = 1 ) , C4 ( n = 2 ) C5 ( n = 6 ) were formed at 22 ° C for 48 h . Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( C ) were calculated using unpaired t - ( two - ) , ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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(C) Biofilm formation ability. The biofilm of the representative isolates from C1 (n=2), C2 (n=1), C4 (n=2) and C5 (n=6) were formed at 22°C for 48 h. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (C) were calculated using unpaired t-test (two-tailed), (*:P<0.05, **:P<0.01, ***:P<0.001).


( D ) Comparison of survival rates of chick after challenging SL C5 C1 isolates . Comparison was on 6 day - post - infection ( dpi ) . Ten were used for each group . Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( D ) were calculated using Log - rank ( Mantel - Cox , chi - square ) ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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(D) Comparison of survival rates of chick embryo after challenging SL C5 and C1 isolates. Comparison was on 6 day-post-infection (dpi). Ten embryos were used for each group. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. P values for (D) were calculated using Log-rank (Mantel-Cox, chi-square) test (*:P<0.05, **:P<0.01, ***:P<0.001).


( E ) Survival rates of zebrafish after challenging SL C5 C1 isolates . comparison was on 14 dpi . Ten zebrafish were used for each group . Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( E ) were calculated using Log - rank ( Mantel - Cox , chi - square ) ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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(E) Survival rates of zebrafish embryo after challenging SL C5 and C1 isolates. The comparison was on 14 dpi. Ten zebrafish embryos were used for each group. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. P values for (E) were calculated using Log-rank (Mantel-Cox, chi-square) test (*:P<0.05, **:P<0.01, ***:P<0.001).


( F ) loads in distinct organs of mice after challenging SL C5 ( L42 ) C1 ( L1 ) isolates . in liver , spleen , cecum of mice were measured on 3 5 dpi . Ten mice were used per group . Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( F ) were calculated using unpaired t - ( two - ) , ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

(F) Bacterial loads in distinct organs of mice after challenging SL C5 (L42) and C1 (L1) isolates. The bacterial counts in the liver, spleen, and cecum of mice were measured on 3 and 5 dpi. Ten mice were used per group. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (F) were calculated using unpaired t-test (two-tailed), (*:P<0.05, **:P<0.01, ***:P<0.001).


( C ) day 20 ± 2 cerebral organoids derived from controls , EML1 - heKOs stained for neuronal marker MAPT adherens junction marker NCAD , counterstained with DAPI . Ectopic neural rosettes neuronal heterotopia are highlighted with dotted yellow lines . ( D ) of VZ areas with ectopic neural rosettes ( 3 batches , 3 organoids analyzed per batch , significance on Kruskal - Wallis - , P = 0 . 0001 , Dunn ' s post hoc for multiple comparisons performed to define statistical differences between genotypes , single data points are presented colored by batch ) . ( E ) of heterotopic , disorganized or organized cortical areas ( 3 batches , 3 organoids per batch , significance on Kruskal - Wallis - , P = 0 . 0001 for " organized " " heterotopic " ; no significant difference for " disorganized " , Dunn ' s post hoc performed for multiple comparisons to define statistical differences between genotypes ) . Data information : * marks statistical significance in relation to Control 1 , # in relation to Control 2 . P - values : * * * / # # # < 0 . 001 . VZ : ventricular zone ; Data in graphs are represented as means ± SD . Scale bars : 50µm .

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(C) day 20±2 cerebral organoids derived from controls, patients and EML1-heKOs stained for the neuronal marker MAPT and the adherens junction marker NCAD, counterstained with DAPI. Ectopic neural rosettes and neuronal heterotopia are highlighted with dotted yellow lines. (D) Quantification of VZ areas with ectopic neural rosettes (3 batches, 3 organoids analyzed per batch, significance based on Kruskal-Wallis-test, P=0.0001, Dunn's post hoc test for multiple comparisons performed to define statistical differences between genotypes, single data points are presented colored by batch). (E) Quantification of heterotopic, disorganized or organized cortical areas (3 batches, 3 organoids per batch, significance based on Kruskal-Wallis-test, P=0.0001 for "organized" and "heterotopic"; no significant difference for "disorganized", Dunn's post hoc test performed for multiple comparisons to define statistical differences between genotypes). Data information: * marks statistical significance in relation to Control 1, # in relation to Control 2. P-values: ***/###<0.001. VZ: ventricular zone; Data in graphs are represented as means ± SD. Scale bars: 50µm.


( A ) Hybrid organoids generated from controls mixed with control - EGFP + or EML1 - heKO - EGFP + ( day 20 ± 2 ) . Yellow dotted lines indicate VZ areas . ( B ) of control - EGFP + or EML1 - heKO - EGFP + within or at basal side of VZ areas ( 3 batches , 3 organoids each , significance on Mann Whitney , * * * P = 0 . 0001 for " EGFP + at basal side of VZ areas " ) . Data information : Data in graphs are represented as means ± SD . Scale bars : ( A )

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(A) Hybrid organoids generated from controls mixed with control-EGFP+ or EML1-heKO-EGFP+ cells (day 20±2). Yellow dotted lines indicate VZ areas. (B) Quantification of control-EGFP+ or EML1-heKO-EGFP+ cells within or at the basal side of VZ areas (3 batches, 3 organoids each, significance based on Mann Whitney test, ***P= 0.0001 for "EGFP+ cells at the basal side of VZ areas"). Data information: Data in graphs are represented as means ± SD. Scale bars: (A) 50µm


( C ) Representative scheme of cleavage plan orientation . Upper panel : immunostained for pVim ( red ) TPX2 ( green ) , counterstained with DAPI , dotted indicates cleavage plane orientation , yellow dotted VZ - surface . ( D ) of horizontal , vertical oblique plane of divisions in control EML1 - deficient organoids ( day 20 ± 2 ; 3 batches , 3 organoids each ) . Data information : Scale bars :

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

(C) Representative images and scheme of cleavage plan orientation. Upper panel: cells immunostained for pVim (red) and TPX2 (green), counterstained with DAPI, white dotted line indicates cleavage plane orientation, yellow dotted line VZ-surface. (D) Quantification of horizontal, vertical and oblique plane of cell divisions in control and EML1-deficient organoids (day 20±2; 3 batches, 3 organoids each). Data information: Scale bars: 5µm


( F ) Electron microscopy of cilia derived from control or EML1 - deficient cortical progenitors in DMSO - control or following EpothiloneD ( EpoD ) exposure ( blue arrow : basal , yellow arrow : appendages , red arrow : ciliary pockets ) . Data information : Scale bars : 0 , 5µm .

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(F) Electron microscopy of primary cilia derived from control or EML1-deficient cortical progenitors in DMSO-control or following EpothiloneD (EpoD) exposure (blue arrow: basal body, yellow arrow: appendages, red arrow: ciliary pockets). Data information: Scale bars: 0,5µm.


( E ) Control EML1 - heKO - derived cerebral organoid stained for COL1A2 MEIS2 . VZ areas are encircled , squares indicate areas enlarged in F . ( F ) Control EML1 - heKO - derived cerebral organoid stained for COL1A2 , MEIS2 MAPT . ( G ) Representative showing EGFP - control EGFP - EML1 - heKO hybrid cerebral organoid stained for EGFP , FAM107A NCAD . Yellow dotted lines indicate of EGFP + ( 4 independent batches each , 3 organoids per batch , at least 30 ectopic rosettes in total ) . Data information : Scale bars : ( E ) 50µm ; ( F ) 10µm ; ( G ) 50µm , enlarged 10µm .

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

(E) Control and EML1-heKO-derived cerebral organoid stained for COL1A2 and MEIS2. VZ areas are encircled, squares indicate areas enlarged in F. (F) Control and EML1-heKO-derived cerebral organoid stained for COL1A2, MEIS2 and MAPT. (G) Representative images showing EGFP-control and EGFP-EML1-heKO hybrid cerebral organoid stained for EGFP, FAM107A and NCAD. Yellow dotted lines indicate morphology of EGFP+ cells (4 independent batches each, 3 organoids per batch, at least 30 ectopic rosettes in total). Data information: Scale bars: (E) 50µm; (F) 10µm; (G) 50µm, enlarged 10µm.


( D , E ) Immunofluorescence for ( D ) p - VIM YAP1 or ( E ) Pax6 YAP1 in control EML1 - heKO - derived organoids ( day 20 ± 2 , squares indicate areas enlarged in adjacent part of panel respectively ) . ( F ) of basally located pVim + with YAP1 signal per VZ in control EML1 - heKO - derived organoids ( 3 batches , 3 organoids analyzed per batch ) . ( G ) of PAX6 + with YAP1 signal per VZ in control EML1 - heKO - derived organoids ( 3 batches , 3 organoids analyzed per batch ) . Data information : Data in graphs are represented as means ± SD . Significance on Kruskal - Wallis - Dunn ' s post hoc for multiple comparisons were performed to define statistical differences between genotypes . P - values : * * * < 0 . 001 , * * < 0 . 01 , * < 0 . 05 . Scale bars : ( D E ) 50µm , enlarged

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

(D, E) Immunofluorescence staining for (D) p-VIM and YAP1 or (E) Pax6 and YAP1 in control and EML1-heKO-derived organoids (day 20±2, squares indicate areas enlarged in adjacent part of the panel respectively). (F) Quantification of basally located pVim+ cells with nuclear YAP1 signal per VZ area in control and EML1-heKO-derived organoids (3 batches, 3 organoids analyzed per batch). (G) Quantification of PAX6+ cells with nuclear YAP1 signal per VZ area in control and EML1-heKO-derived organoids (3 batches, 3 organoids analyzed per batch). Data information: Data in graphs are represented as means ± SD. Significance based on Kruskal-Wallis-test Dunn's post hoc test for multiple comparisons were performed to define statistical differences between genotypes. P-values: ***<0.001, **<0.01, *<0.05. Scale bars: (D and E) 50µm, enlarged 10µm


( H ) Immunofluorescence for p - Vim in EML1 - heKO control derived organoids in DMSO , Verteporfin or Fluvastatin treated conditions , counterstained with DAPI . Dotted indicates VZ areas . ( I ) of p - VIM + located at basal side of VZ areas in control or EML1 - heKO - derived organoids under DMSO , Verteporfin or Fluvastatin treated condition ( 3 batches , 3 organoids analyzed per batch ) . Data information : Data in graphs are represented as means ± SD . Significance on two - way ANOVA ( on log10 normalized data ) Tukey post hoc for multiple comparisons were performed to define statistical differences between genotypes . P - values : * * * < 0 . 001 , * * < 0 . 01 , * < 0 . 05 . Scale bars : ( H )

O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence staining for p-Vim in EML1-heKO and control derived organoids in DMSO, Verteporfin or Fluvastatin treated conditions, counterstained with DAPI. Dotted line indicates VZ areas. (I) Quantification of p-VIM+ cells located at the basal side of VZ areas in control or EML1-heKO-derived organoids under DMSO, Verteporfin or Fluvastatin treated condition (3 batches, 3 organoids analyzed per batch). Data information: Data in graphs are represented as means ± SD. Significance based on two-way ANOVA (on log10 normalized data) Tukey post hoc test for multiple comparisons were performed to define statistical differences between genotypes. P-values: ***<0.001, **<0.01, *<0.05. Scale bars: (H) 50µm


expression of SAMHD1 is negatively correlated with EV71 viral replication ability . HEK293T RD were infected with EV71 at a MOI of 0 . 1 , then supernatants were harvested at indicated time points . Immunoblotting ( IB ) of EV71 VP1 in , with tubulin as a loading control . EV71 - VP1 in supernatants was detected after ultracentrifugation .

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

The expression of SAMHD1 is negatively correlated with EV71 viral replication ability. HEK293T and RD cells were infected with EV71 at a MOI of 0.1, then the cells and supernatants were harvested at the indicated time points. Immunoblotting (IB) analysis of EV71 VP1 in cells, with tubulin as a loading control. EV71-VP1 protein in the supernatants was detected after ultracentrifugation.


expression of SAMHD1 is negatively correlated with EV71 viral replication ability . HEK293T RD were infected with EV71 at a MOI of 0 . 1 , then supernatants were harvested at indicated time points . EV71 viral in lysates supernatants were detected by RT - qPCR with GAPDH as a control . ( n = 3 , mean ± SD , ns stands for no significance , paired t - )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 expression of SAMHD1 is negatively correlated with EV71 viral replication ability. HEK293T and RD cells were infected with EV71 at a MOI of 0.1, then the cells and supernatants were harvested at the indicated time points. EV71 viral RNA levels in cell lysates and supernatants were detected by RT-qPCR with GAPDH as a control. (n=3, mean±SD, ns stands for no significance, paired t-test)


expression of SAMHD1 is negatively correlated with EV71 viral replication ability . HEK293T RD were infected with EV71 at a MOI of 0 . 1 , then supernatants were harvested at indicated time points . mRNA of host restriction factors were detected by RT - qPCR in infected or uninfected HEK293T or RD , expression of target genes were normalized to GAPDH . ( n = 3 , mean ± SD , ns stands for no significance , paired t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 expression of SAMHD1 is negatively correlated with EV71 viral replication ability. HEK293T and RD cells were infected with EV71 at a MOI of 0.1, then the cells and supernatants were harvested at the indicated time points. The mRNA levels of the host restriction factors were detected by RT-qPCR in infected or uninfected HEK293T or RD cells, and the expression levels of the target genes were normalized to GAPDH. (n=3, mean±SD, ns stands for no significance, paired t-test).


expression of SAMHD1 is negatively correlated with EV71 viral replication ability . HEK293T RD were infected with EV71 at a MOI of 0 . 1 , then supernatants were harvested at indicated time points . IB of SAMHD1 BST - 2 with tubulin as a loading control . densities of bands were analyzed with ImageJ software to calculate values relative to that of tubulin .

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

The expression of SAMHD1 is negatively correlated with EV71 viral replication ability. HEK293T and RD cells were infected with EV71 at a MOI of 0.1, then the cells and supernatants were harvested at the indicated time points. IB analysis of SAMHD1 and BST-2 protein levels with tubulin as a loading control. The densities of bands were analyzed with ImageJ software to calculate the values relative to that of tubulin.


SAMHD1 knockdown enhances EV71 replication . stable lines pLKO . 1 sh - SAMHD1 constructed in HEK293T were infected with EV71 at a MOI of 0 . 1 0 . 05 respectively , supernatants were harvested at indicated time points . IB of EV71 VP1 SAMHD1 in was performed with tubulin as a loading control . EV71 - VP1 in supernatants was detected after ultracentrifugation .

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

SAMHD1 knockdown enhances EV71 replication. The stable cell lines pLKO.1 and sh-SAMHD1 constructed in HEK293T cells were infected with EV71 at a MOI of 0.1 and 0.05 respectively, and cells and supernatants were harvested at the indicated time points. IB analysis of EV71 VP1 and SAMHD1 in cells was performed with tubulin as a loading control. EV71-VP1 protein in the supernatants was detected after ultracentrifugation.


SAMHD1 knockdown enhances EV71 replication . EV71 viral in lysates was detected by RT - qPCR with GAPDH as a control ( n = 3 , mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , paired t - ) .

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

SAMHD1 knockdown enhances EV71 replication. EV71 viral RNA in cell lysates was detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, * P < 0.05, ** P < 0.01, paired t-test).


SAMHD1 knockdown enhances EV71 replication . Viral titers in supernatants were measured by cytopathic effect method .

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

SAMHD1 knockdown enhances EV71 replication. Viral titers in the supernatants were measured by the cytopathic effect method.


SAMHD1 knockdown enhances EV71 replication . stable lines pLKO . 1 sh - SAMHD1 constructed in RD ( H ) were infected with EV71 at a MOI of 0 . 1 0 . 05 respectively , supernatants were harvested at indicated time points . IB of EV71 VP1 SAMHD1 in was performed with tubulin as a loading control . EV71 - VP1 in supernatants was detected after ultracentrifugation .

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

SAMHD1 knockdown enhances EV71 replication. The stable cell lines pLKO.1 and sh-SAMHD1 constructed in RD (H) cells were infected with EV71 at a MOI of 0.1 and 0.05 respectively, and cells and supernatants were harvested at the indicated time points. IB analysis of EV71 VP1 and SAMHD1 in cells was performed with tubulin as a loading control. EV71-VP1 protein in the supernatants was detected after ultracentrifugation.


HEK293T transfected with VR1012 , which is a eukaryotic expression vector , or Vpx - HA were infected with EV71 at a MOI of 0 . 1 , then supernatants were harvested at indicated time points . IB of EV71 - VP1 , SAMHD1 Vpx - HA in lysates was performed with tubulin as a loading control . EV71 - VP1 was detected in supernatants after ultracentrifugation .

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

HEK293T cells transfected with VR1012, which is a eukaryotic expression vector, or Vpx-HA were infected with EV71 at a MOI of 0.1, and then cells and supernatants were harvested at the indicated time points. IB analysis of EV71-VP1, SAMHD1 and Vpx-HA in cell lysates was performed with tubulin as a loading control. EV71-VP1 was detected in the supernatants after ultracentrifugation.


EV71 viral was detected in lysates by RT - qPCR with GAPDH as a control ( n = 3 , mean ± SD , * * P < 0 . 01 , paired t - ) .

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

EV71 viral RNA was detected in cell lysates by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P <0.01, paired t-test).


Viral titres 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 . 01 ) .

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

Viral titres 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 < 0.01).


Stable SAMHD1 - HA - overexpressing lines were constructed in HEK293T RD detected by IB with pLVX as a negative control .

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

Stable SAMHD1-HA-overexpressing cell lines were constructed in HEK293T and RD cells and detected by IB with pLVX as a negative control.


RD SAMHD1 - HA lines were treated with dimethyl sulfoxide ( DMSO ) or 10 μM MG132 for 12 h prior to harvest subjected to IB with tubulin as a loading control .

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RD SAMHD1-HA cell lines were treated with dimethyl sulfoxide (DMSO) or 10 μM MG132 for 12 h prior to harvest and subjected to IB with tubulin as a loading control.


RD SAMHD1 - HA lines were treated as in ( B ) , then HA immunoprecipitation ( IP ) IB were performed . IP elution was analyzed by mass spectrometry .

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RD SAMHD1-HA cell lines were treated as in (B), and then HA immunoprecipitation (IP) and IB were performed. The IP elution was analyzed by mass spectrometry.


TRIM21 induced degradation of SAMHD1 , MG132 rescued TRIM21 - mediated degradation of SAMHD1 . HEK293T were cotransfected with SAMHD1 VR1012 or TRIM21 expression plasmids with or without MG132 treatment for 12 h prior to harvest , then subjected to IB with tubulin as a loading control .

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TRIM21 induced the degradation of SAMHD1, and MG132 rescued the TRIM21-mediated degradation of SAMHD1. HEK293T cells were cotransfected with SAMHD1 and VR1012 or TRIM21 expression plasmids with or without MG132 treatment for 12 h prior to harvest, and then subjected to IB with tubulin as a loading control.


TRIM25 ( E ) induced degradation of SAMHD1 HEK293T were cotransfected with SAMHD1 VR1012 or TRIM25 expression plasmids then subjected to IB with tubulin as a loading control .

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TRIM25 (E) induced the degradation of SAMHD1 HEK293T cells were cotransfected with SAMHD1 and VR1012 or TRIM25 expression plasmids and then subjected to IB with tubulin as a loading control.


Reverse co - IP confirmed interaction between SAMHD1 TRIM21 . SAMHD1 - flag was transfected with TRIM21 - HA into HEK293T , then were treated as in ( B ) subjected to HA IP IB .

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Reverse co-IP confirmed the interaction between SAMHD1 and TRIM21. SAMHD1-flag was transfected with TRIM21-HA into HEK293T cells, and then the cells were treated as in (B) and subjected to HA IP and IB.


Endogenous TRIM21 but not TRIM25 still interacted with SAMHD1 . RD - SAMHD1 - HA treated as in ( B ) were subjected to TRIM21 or TRIM25 IP IB .

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Endogenous TRIM21 but not TRIM25 still interacted with SAMHD1. RD-SAMHD1-HA cells treated as in (B) were subjected to TRIM21 or TRIM25 IP and IB.


mRNA levels of TRIM21 in RD and HEK293T cells.


protein levels of TRIM21 in RD and HEK293T cells.


TRIM21 ( J ) but not TRIM25 ( K ) colocalized with SAMHD1 . were taken under a Zeiss LZM710 confocal microscope , Bars , 10μm .

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TRIM21 (J) but not TRIM25 (K) colocalized with SAMHD1. Images were taken under a Zeiss LZM710 confocal microscope, Bars, 10μm.


TRIM21 Knockout in RD released SAMHD1 inhibition on EV71 replication . Negative control or RD - CRISPR - Cas9 - TRIM21 RD were infected with EV71 at a MOI of 0 . 05 for indicated time harvested for SAMHD1 , TRIM21 EV71 - VP1 detection by IB . Tubulin served as a loading control .

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TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication. Negative control or RD-CRISPR-Cas9-TRIM21 RD cells were infected with EV71 at a MOI of 0.05 for the indicated time and harvested for SAMHD1, TRIM21 and EV71-VP1 detection by IB. Tubulin served as a loading control.


TRIM21 Knockout in RD released SAMHD1 inhibition on EV71 replication . EV71 in ( B ) were detected by RT - qPCR with GAPDH as a control ( n = 3 , mean ± SD , * * P < 0 . 01 , paired t - ) .

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TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication. EV71 RNA levels in (B) were detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P < 0.01, paired t-test).


TRIM21 Knockout in RD released SAMHD1 inhibition on EV71 replication . Viral titres in supernatant were measured by cytopathic effect method . results represent means ± SD from three independent experiments . Statistical significance was analyzed using Student ' s t ( * * P < 0 . 01 ) .

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TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication. Viral titres in the supernatant 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 < 0.01).


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 . were harvested at indicated time points subjected to IB with tubulin as a loading control .

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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. Cells were harvested at the indicated time points and subjected to IB with tubulin as a loading control.


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 .

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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 ) .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 ) .

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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 .

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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

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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 ) .

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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 .

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(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 . (

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(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 )

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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 .

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