Figure 1 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.


Figure 2 ( 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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 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 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.


Figure 3 ( 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 .

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Figure 3(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.


Figure 4 ( 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 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 )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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 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


Figure 1The 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 . 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 ) 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 ) . 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 . 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 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 the supernatants were measured by the cytopathic effect method . 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 . 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 the supernatants were measured by the cytopathic effect method . 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 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1The 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.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)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).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. 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 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 the supernatants were measured by the cytopathic effect method.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.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 the supernatants were measured by the cytopathic effect method.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 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 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).


Figure 2Stable 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 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 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 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 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 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 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 . Images were taken under a Zeiss LZM710 confocal microscope , Bars , 10μm .

O B-PANEL_START O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O B-PANEL_START O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Stable 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 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 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 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 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 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 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. Images were taken under a Zeiss LZM710 confocal microscope, Bars, 10μm.


Figure 3TRIM21 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 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 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 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 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 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 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 ) .

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Figure 3TRIM21 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 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 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 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 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 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 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).


Figure 4The 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 ) . 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 ( IHC ) staining results of SAMHD1 in various tissues of mice . Bars , 100μm . 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 % 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 % 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 %

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Figure 4The 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).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 (IHC) staining results of SAMHD1 in various tissues of mice. Bars, 100μm.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%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%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%


Figure 5IB 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 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 ) . 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 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 ) . 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 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 ) .

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Figure 5IB 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 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).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 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).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 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).


Figure 6TRIM21 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 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 . 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 , 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 ) , and pull - down assay were performed with Ni - Sepharose ( up ) and GST Sepharose ( down ) , respectively . 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 ) 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 .

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Figure 6TRIM21 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 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.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, 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), and pull-down assay were performed with Ni-Sepharose (up) and GST Sepharose (down), respectively.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)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.


Figure 7The 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 . 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 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 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 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 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 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 .

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Figure 7The 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.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 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 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 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 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 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.


Figure 1A 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 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 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 , and RabGGTβ . RabGGTβ eluted in two peaks ( peak A and peak B ) . 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 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 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 .

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Figure 1A 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 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 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, and RabGGTβ. RabGGTβ eluted in two peaks (peak A and peak B).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 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 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.


Figure 2C 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 the absence or presence of BGPP for 30 min at 37ºC . Biotin - geranylated Ykt6 was

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Figure 2C 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 the absence or presence of BGPP for 30 min at 37ºC. Biotin-geranylated Ykt6 was detected


Figure 3C 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 ) 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 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 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 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 , 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 .

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Figure 3C 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) 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 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 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 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, 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.


Figure 4E 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 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 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 ) .

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Figure 4E 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 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 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).


Figure 5H 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 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 ) .

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Figure 5H 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 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).


Figure 6A 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 ) , 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 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 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 .

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Figure 6A 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), 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 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 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.


Figure 7A 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 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 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 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 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 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 .

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Figure 7A 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 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 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 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 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 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.


f1 ( 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 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 .

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f1(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 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.


f2 ( 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 ) .

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f2(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).


f3 ( 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 analysis . Jurkat T cells were cultured as in a . Values are means ± s . d . of three independent experiments . 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 . ( 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f3(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 analysis. Jurkat T cells were cultured as in a. Values are means±s.d. of three independent experiments.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.(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.


f4 ( 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 ) 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f4(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) 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 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.


f5 ( 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f5(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 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 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.


f6 ( 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 ) 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 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f6(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) 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 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 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).


f7 ( 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 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 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 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 and p62 degradation , indicating increased autophagic flux . ( 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f7(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 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 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 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 and p62 degradation, indicating increased autophagic flux.(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 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.