DU145 were transfected for 48h with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . ( B ) Immunoblots of p - YAP ( ser127 ) , YAP UBTD1 ( left ) of YAP p - YAP / YAP ( right ) in control or UBTD1 - depleted DU145 at low high density . Immunoblot of UBTD1 shows level of siRNA - mediated depletion . Actin was used as a loading controls . , immunoblots for loading control ( actin ) UBTD1 knock - down efficiency ( UBTD1 ) are duplicated because they belong to same experiment shown in both panels .

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DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (B) Immunoblots of p-YAP (ser127), YAP and UBTD1 (left) and quantification of YAP and p-YAP/YAP levels (right) in control or UBTD1-depleted DU145 at low and high cell density. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading controls. , immunoblots for the loading control (actin) and UBTD1 knock-down efficiency (UBTD1) are duplicated because they belong to the same experiment shown in both panels.


DU145 were transfected for 48h with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . ( C ) Immunoblots of Hippo signaling pathway using phosphorylated ( p - ser909LATS p - thr183MST1 / p - thr180MST2 ) total LATS , MST at low high DU145 density . Immunoblot of UBTD1 shows level of siRNA - mediated depletion . Actin Rho - GDI were used as loading controls . , immunoblots for loading control ( actin ) UBTD1 knock - down efficiency ( UBTD1 ) are duplicated because they belong to same experiment shown in both panels .

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DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (C) Immunoblots of the Hippo signaling pathway using phosphorylated (p-ser909LATS and p-thr183MST1/p-thr180MST2) and total LATS, MST at low and high DU145 cells density. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin and Rho-GDI were used as loading controls. , immunoblots for the loading control (actin) and UBTD1 knock-down efficiency (UBTD1) are duplicated because they belong to the same experiment shown in both panels.


DU145 were transfected for 48h with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . Immunoblots ( up ) ( down ) of YAP in confluent ( D ) DU145 UBTD1 depleted treated or not with cycloheximide ( CHX ) for 2h . Immunoblot of UBTD1 shows level of siRNA - mediated depletion . Actin was used as a loading control .

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DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) of YAP levels in confluent (D) DU145 UBTD1 depleted cells treated or not with cycloheximide (CHX) for 2h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control.


A549 were transfected for 48h with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . Immunoblots ( up ) ( down ) of YAP in confluent ( E ) A549 UBTD1 depleted treated or not with cycloheximide ( CHX ) for 2h . Immunoblot of UBTD1 shows level of siRNA - mediated depletion . Actin was used as a loading control .

A549 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) of YAP levels in confluent (E) A549 UBTD1 depleted cells treated or not with cycloheximide (CHX) for 2h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control.


DU145 or A549 were transfected for 48h with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . Immunoblots ( up ) ( down ) showing YAP in UBTD1 depleted confluent DU145 A549 treated or not with MG132 for 6h . Immunoblot of UBTD1 shows level of siRNA - mediated depletion . Actin was used as a loading control .

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DU145 cells or A549 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) showing YAP levels in UBTD1 depleted confluent DU145 A549 cells treated or not with MG132 for 6h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control.


( A ) Co - immunoprecipitation in DU145 A549 between endogenous YAP UBTD1 . YAP was used as bait . IgG isotype was used as a negative control .

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(A) Co-immunoprecipitation in DU145 and A549 cells between endogenous YAP and UBTD1. YAP was used as bait. The IgG isotype was used as a negative control.


( B ) Co - immunoprecipitation in DU145 A549 between endogenous YAP UBTD1 . UBTD1 was used as bait . IgG isotype was used as a negative control .

(B) Co-immunoprecipitation in DU145 and A549 cells between endogenous YAP and UBTD1. UBTD1 was used as bait. The IgG isotype was used as a negative control.


( C ) Proximal ligation monitoring ( left ) ( right ) of UBTD1 associated with YAP of DU145 A549 transfected with indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool ) . Nuclei were stained with DAPI ( Blue ) on MERGE image .

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(C) Proximal ligation assay monitoring (left) and quantification (right) of UBTD1 associated with YAP of DU145 and A549 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.


( D ) Co - immunoprecipitation in confluent DU145 between endogenous β - TrCP , Ubch5c UBTD1 . UBTD1 was used as bait . IgG isotype was used as a negative control .

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(D) Co-immunoprecipitation in confluent DU145 cells between endogenous β-TrCP, Ubch5c and UBTD1. UBTD1 was used as bait. IgG isotype was used as a negative control.


( E ) Co - immunoprecipitation in confluent DU145 between endogenous β - TrCP , Ubch5c , YAP β - catenin . DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool or siUBTD1single ) . β - TrCP was used as bait . IgG isotype was used as a negative control . Immunoblot of UBTD1 shows level of siRNA - mediated depletion .

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(E) Co-immunoprecipitation in confluent DU145 cells between endogenous β-TrCP, Ubch5c, YAP and β-catenin. DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single). β-TrCP was used as bait. IgG isotype was used as a negative control. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion.


Proximal ligation monitoring ( F ) of β - TrCP associated with Ubch5 , YAP or β - catenin in DU145 transfected with indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool ) . Nuclei were stained with DAPI ( Blue ) on MERGE image .

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Proximal ligation assay monitoring (F) of β-TrCP associated with Ubch5, YAP or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.


( G ) of β - TrCP associated with Ubch5 , YAP or β - catenin in DU145 transfected with indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool ) .

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quantification (G) of β-TrCP associated with Ubch5, YAP or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool).


( H ) Immunoblots show YAP ubiquitylation in HEK in different experimental conditions . were transfected , as indicated , with expression vectors of histidine - tagged ubiquitin ( His - Ub ) together with control siRNA or UBTD1 siRNA . His - Ub crosslinked forms of YAP were purified ( IP : His ) immunoblot of YAP showed YAP ubiquitylation . immunoblot of YAP ( lower panel ) was performed in parallel to verify amounts of YAP engaged in His - Ub purifications . immunoblot of UBTD1 shows level of siRNA depletion .

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(H) Immunoblots show YAP ubiquitylation in HEK cells in different experimental conditions. Cells were transfected, as indicated, with expression vectors of histidine-tagged ubiquitin (His-Ub) together with control siRNA or UBTD1 siRNA. His-Ub crosslinked forms of YAP were purified (IP: His) and the immunoblot of YAP showed YAP ubiquitylation. The immunoblot of YAP (lower panel) was performed in parallel to verify the amounts of YAP protein engaged in the His-Ub purifications. The immunoblot of UBTD1 shows the level of siRNA depletion.


DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . ( A ) Representative wide - field immunofluorescence ( left ) ( right ) showing endogenous YAP translocation in DU145 after UBTD1 depletion . Nuclei were stained with DAPI ( Blue ) on MERGE image .

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DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (A) Representative wide-field immunofluorescence images (left) and quantification (right) showing endogenous YAP nuclear translocation in DU145 cells after UBTD1 depletion. Nuclei were stained with DAPI (Blue) on the MERGE image.


DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . ( B ) of CTGF1 , CYR61 BIRC5 mRNA .

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DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (B) Quantification of CTGF1, CYR61 and BIRC5 mRNA levels.


( C ) Immunoblots of YAP in DU145 depleted ( siUBTD1single2 ) or not ( siCTRL ) for UBTD1 transfected or not with GFP - UBTD1 ( + ) . Immunoblot of UBTD1 shows level of siRNA - mediated depletion . Immunoblot of GFP shows level of UBTD1 transfection . CHC was used as a loading control .

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(C) Immunoblots of YAP in DU145 cells depleted (siUBTD1single2) or not (siCTRL) for UBTD1 and transfected or not with GFP-UBTD1 (+). Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Immunoblot of GFP shows the level of UBTD1 transfection. CHC was used as a loading control.


( D ) Representative confocal immunofluorescence ( left ) ( right ) showing endogenous YAP translocation in DU145 depleted ( siUBTD1single2 ) or not ( siCTRL ) for UBTD1 transfected or not with GFP - UBTD1 . Nuclei were stained with DAPI ( Blue ) on MERGE image .

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(D) Representative confocal immunofluorescence images (left) and quantification (right) showing endogenous YAP nuclear translocation in DU145 cells depleted (siUBTD1single2) or not (siCTRL) for UBTD1 and transfected or not with GFP-UBTD1. Nuclei were stained with DAPI (Blue) on the MERGE image.


( E ) Immunoblots of p - YAP YAP in DU145 control ( siCTRL ) or depleted for LATS1 LATS2 ( siLATS1 + siLATS2 ) / or UBTD1 ( siUBTD1pool ) . Immunoblot of p - YAP reflects activity of Hippo pathway . Immunoblot of UBTD1 LATS show siRNA knock - down efficiency . Actin was used as a loading control .

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(E) Immunoblots of p-YAP and YAP in DU145 control cells (siCTRL) or depleted for LATS1 and LATS2 (siLATS1+siLATS2) and/or UBTD1 (siUBTD1pool). Immunoblot of p-YAP reflects the activity of the Hippo pathway. Immunoblot of UBTD1 and LATS show siRNA knock-down efficiency. Actin was used as a loading control.


Representative confocal immunofluorescence ( F ) showing endogenous YAP translocation in DU145 control ( siCTRL ) or depleted for LATS1 LATS2 ( siLATS1 + siLATS2 ) / or UBTD1 ( siUBTD1pool ) . Nuclei were stained with DAPI ( Blue ) on MERGE image .

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Representative confocal immunofluorescence images (F) showing endogenous YAP nuclear translocation in DU145 control cells (siCTRL) or depleted for LATS1 and LATS2 (siLATS1+siLATS2) and/or UBTD1 (siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.


( G ) showing endogenous YAP translocation in DU145 control ( siCTRL ) or depleted for LATS1 LATS2 ( siLATS1 + siLATS2 ) / or UBTD1 ( siUBTD1pool ) .

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quantification (G) showing endogenous YAP nuclear translocation in DU145 control cells (siCTRL) or depleted for LATS1 and LATS2 (siLATS1+siLATS2) and/or UBTD1 (siUBTD1pool).


( H ) Immunoblots of YAP in DU145 control ( siCTRL ) or depleted for ROCK1 ( siROCK1 ) or ROCK2 ( siROCK2 ) / or UBTD1 ( siUBTD1pool ) . Immunoblots of UBTD1 , ROCK1 ROCK2 show level of siRNA knock - down efficiency . Actin was used as a loading control .

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(H) Immunoblots of YAP in DU145 control (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool). Immunoblots of UBTD1, ROCK1 and ROCK2 show the level of siRNA knock-down efficiency. Actin was used as a loading control.


Representative confocal immunofluorescence ( I ) showing endogenous YAP localization in DU145 control ( siCTRL ) or depleted for ROCK1 ( siROCK1 ) or ROCK2 ( siROCK2 ) / or UBTD1 ( siUBTD1pool ) . Nuclei were stained with DAPI ( Blue ) on MERGE image .

Representative confocal immunofluorescence images (I) showing endogenous YAP localization in DU145 control cells (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.


( J ) showing endogenous YAP localization in DU145 control ( siCTRL ) or depleted for ROCK1 ( siROCK1 ) or ROCK2 ( siROCK2 ) / or UBTD1 ( siUBTD1pool ) .

quantification (J) showing endogenous YAP localization in DU145 control cells (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool).


( K ) of CTGF1 , CYR61 , UBTD1 BIRC5 mRNA in DU145 control ( siCTRL ) or depleted for ROCK1 ( siROCK1 ) or ROCK2 ( siROCK2 ) / or UBTD1 ( siUBTD1pool ) .

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(K) Quantification of CTGF1, CYR61, UBTD1 and BIRC5 mRNA in DU145 control cells (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool).


DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool or siUBTD1single1 ; UBTD1 + YAP + TAZ , siUBTD1single1 + siYAPsingle1 + siTAZsingle1 or siUBTD1single1 + siYAPsingle2 + siTAZsingle2 ) . ( A ) of EMT marker genes ( vimentin , slug , snail , fibronectin , α - SMA , E - cadherin ) UBTD1 .

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single1; UBTD1 +YAP+TAZ, siUBTD1single1+siYAPsingle1+ siTAZsingle1 or siUBTD1single1+siYAPsingle2+ siTAZsingle2). (A) Quantification of EMT marker genes (vimentin, slug, snail, fibronectin, α-SMA, E-cadherin) and UBTD1.


( B ) Immunoblots of EMT markers ( vimentin , E - cadherin , slug ) . Immunoblot of UBTD1 shows siRNA knock - down efficiency . Hsp90 tubulin were used as loading controls .

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(B) Immunoblots of EMT markers (vimentin, E-cadherin, slug). Immunoblot of UBTD1 shows siRNA knock-down efficiency. Hsp90 and tubulin were used as loading controls.


DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool or siUBTD1single1 ; UBTD1 + YAP + TAZ , siUBTD1single1 + siYAPsingle1 + siTAZsingle1 or siUBTD1single1 + siYAPsingle2 + siTAZsingle2 ) . ( C ) of DU145 migration in Transwell chamber inserts .

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single1; UBTD1 +YAP+TAZ, siUBTD1single1+siYAPsingle1+ siTAZsingle1 or siUBTD1single1+siYAPsingle2+ siTAZsingle2). (C) Quantification of DU145 cell migration in Transwell chamber inserts.


DU145 were transfected with indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool or siUBTD1single1 ; UBTD1 + YAP + TAZ , siUBTD1single1 + siYAPsingle1 + siTAZsingle1 or siUBTD1single1 + siYAPsingle2 + siTAZsingle2 ) . ( D ) of DU145 invasion in Transwell chamber inserts .

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool or siUBTD1single1; UBTD1 +YAP+TAZ, siUBTD1single1+siYAPsingle1+ siTAZsingle1 or siUBTD1single1+siYAPsingle2+ siTAZsingle2). (D) Quantification of DU145 cell invasion in Transwell chamber inserts.


( E ) Invasive properties of human prostate tumoroids depleted for UBTD1 .

(E) Invasive properties of human prostate tumoroids depleted for UBTD1.


( F ) of UBTD1 mRNA in prostate ( n = 70 ) adjacent normal tissues ( n = 47 ) .

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(F) Quantification of UBTD1 mRNA levels in prostate tumor (n=70) and adjacent normal tissues (n=47).


( G ) Representative UBTD1 immunohistochemistry on sections of human prostate normal tissues ( n = 8 ) .

(G) Representative UBTD1 immunohistochemistry staining on sections of human prostate tumor and normal tissues (n=8).


( H ) Representative UBTD1 immunohistochemistry on sections of human lung normal tissues ( n = 6 ) .

(H) Representative UBTD1 immunohistochemistry staining on sections of human lung tumor and normal tissues (n=6).


( I ) of UBTD1 mRNA in grade 3 prostate adenocarcinoma ( 56 ) are correlated with overall survival ( OS ) . OS was calculated from subgroups with mRNA that were less or greater than first quartile value .

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(I) The levels of UBTD1 mRNA in grade 3 prostate adenocarcinoma patients (56 patients) are correlated with overall survival (OS). OS was calculated from patient subgroups with mRNA levels that were less or greater than the first quartile value.


( J ) of UBTD1 mRNA in lung adenocarcinoma ( 522 ) are correlated with OS .

(J) The levels of UBTD1 mRNA in lung adenocarcinoma patients (522 patients) are correlated with OS.


( C ) Confocal of formaldehyde fixed GFP + S . flexneri following 20 min incubation with 2 mM GTP 10 µM Alexa - Fluor647 - labeled . associated with hGBP1 mutants after 20 min were quantified . Mean frequencies ± of combined data from two independent experiments are shown . Significance was determined by one - way ANOVA with Tukey ' s multiple comparison . * * * , P ≤ 0 . 001 . Scale bars equal 5 µm . Flow diagram depicts effects of K51A , R48A , H74A hGBP1 mutations on nucleotide binding hydrolysis .

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(C) Confocal images of formaldehyde fixed GFP+ S. flexneri following 20 min incubation with 2 mM GTP and 10 µM Alexa-Fluor647-labeled protein. Bacteria associated with hGBP1 mutants after 20 min were quantified. Mean frequencies ± SEM of combined data from two independent experiments are shown. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ***, P ≤ 0.001. Scale bars equal 5 µm. Flow diagram depicts effects of K51A, R48A, and H74A hGBP1 mutations on nucleotide binding and hydrolysis.


( A ) Translocation of ectopically expressed mCherry - hGBP1 to GFP + S . flexneri ΔipaH9 . 8 in HeLa hGBP1 - KO was monitored by time - lapse microscopy . time frames of Movie EV1 starting at 55 min post infection ( mpi ) are shown .

(A) Translocation of ectopically expressed mCherry-hGBP1 to cytosolic GFP+ S. flexneri ΔipaH9.8 in HeLa hGBP1-KO cells was monitored by time-lapse microscopy. Individual time frames of Movie EV1 starting at 55 min post infection (mpi) are shown.


( B ) Confocal time - lapse microscopy was used to image 10 µM Alexa - Fluor647 - hGBP1F supplemented with 2 mM GTP in presence or absence of formaldehyde fixed GFP + S . flexneri . time frames of Movie EV2 depict hGBP1F . Merged of hGBP1F S . flexneri are shown for 60 min time points .

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(B) Confocal time-lapse microscopy was used to image 10 µM Alexa-Fluor647-hGBP1F supplemented with 2 mM GTP in the presence or absence of formaldehyde fixed GFP+ S. flexneri. Individual time frames of Movie EV2 depict hGBP1F fluorescence intensity. Merged images of hGBP1F and S. flexneri fluorescence are shown for the 60 min time points.


( C ) were taken at 45 min after addition of 10 µM Alexa - Fluor647 - hGBP1F to formaldehyde fixed GFP + S . flexneri in presence of indicated nucleotides ( GTP , natural substrate ; GppNHp , non - hydrolysable GTP analog ; GTPγS , slowly hydrolysable GTP analog ; GDP · AlFX , GTP transition state analog ) . hGBP1 - associated after 45 min were quantified . Combined data from two independent experiments are shown as mean ± . Significance was determined by one - way ANOVA with Tukey ' s multiple comparison . * * * * , P ≤ 0 . 0001 .

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(C) Images were taken at 45 min after addition of 10 µM Alexa-Fluor647-hGBP1F to formaldehyde fixed GFP+ S. flexneri in the presence of indicated nucleotides (GTP, natural substrate; GppNHp, non-hydrolysable GTP analog; GTPγS, slowly hydrolysable GTP analog; GDP·AlFX, GTP transition state analog). hGBP1-associated bacteria after 45 min were quantified. Combined data from two independent experiments are shown as mean ± SEM. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.


( A ) Polymerization of 10 µM hGBP1F hGBP1FR584 - 586A in presence of 2 mM GTP was monitored over time by absorption spectroscopy at 350 nm . Absorbance signals were superimposed with nucleotide composition of same solution analyzed at defined time points , revealing characteristic first phase of slow polymer nucleation second phase of fast polymer growth cooperative hydrolysis .

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(A) Polymerization of 10 µM hGBP1F and hGBP1FR584-586A in the presence of 2 mM GTP was monitored over time by absorption spectroscopy at 350 nm. Absorbance signals were superimposed with nucleotide composition of the same solution analyzed at defined time points, revealing the characteristic first phase of slow polymer nucleation and the second phase of fast polymer growth and cooperative hydrolysis.


( B ) Scanning - electron of live S . flexneri incubated with no or with 5 µM of either hGBP1F , hGBP1FR584 - 586A , or hGBP1FR48A ( non - polymerizing mutant ) in presence of 2 mM GTP for 4 min . Arrowheads point to unattached hGBP1 polymers , arrows point to hGBP1 polymers attached to , asterisks mark polymeric structures that appear to fuse with surfaces .

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(B) Scanning-electron micrographs of live S. flexneri incubated with no protein or with 5 µM of either hGBP1F, hGBP1FR584-586A, or hGBP1FR48A (non-polymerizing mutant) in the presence of 2 mM GTP for 4 min. Arrowheads point to unattached hGBP1 polymers, arrows point to hGBP1 polymers attached to bacteria, and asterisks mark polymeric structures that appear to fuse with bacterial surfaces.


( C ) Confocal time - lapse microscopy frames of formaldehyde fixed GFP + S . flexneri following admixture of 10 µM Alexa - Fluor647 - labeled hGBP1F or hGBP1FR584 - 586A 2 mM GTP . Binding of hGBP1F polymers to at 5 min enclosure of with hGBP1F coats after 60 min were quantified . Mean frequencies ± of combined data from at least three independent experiments are shown . Significance was determined by by unpaired t - , two - . * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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

(C) Confocal time-lapse microscopy frames of formaldehyde fixed GFP+ S. flexneri following admixture of 10 µM Alexa-Fluor647-labeled hGBP1F or hGBP1FR584-586A and 2 mM GTP. Binding of hGBP1F polymers to bacteria at 5 min and enclosure of bacteria with hGBP1F protein coats after 60 min were quantified. Mean frequencies ± SEM of combined data from at least three independent experiments are shown. Significance was determined by by unpaired t-tests, two-tailed. ***, P ≤ 0.001; ****, P ≤ 0.0001.


( A ) Representative confocal taken at 60 min after admixture of 2 mM GTP 10 µM Alexa - Fluor647 - or Alexa - Fluor488 - hGBP1F to formaldehyde fixed gram - negative gram - positive expressing GFP ( S . enterica Typhimurium [ ST ] , L . monocytogenes [ Lm ] ) , or RFP ( S . flexneri [ Sf ] , uropathogenic E . coli [ UPEC ] ) , or dsRed ( L . pneumophila [ Lp ] , S . aureus [ Sa ] ) .

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

(A) Representative confocal images taken at 60 min after admixture of 2 mM GTP and 10 µM Alexa-Fluor647- or Alexa-Fluor488-hGBP1F to formaldehyde fixed gram-negative and gram-positive bacteria expressing GFP (S. enterica Typhimurium [ST], L. monocytogenes [Lm]), or RFP (S. flexneri [Sf], uropathogenic E. coli [UPEC]), or dsRed (L. pneumophila [Lp], S. aureus [Sa]).


( B ) ( Left panel ) Confocal time - lapse microscopy frames of Alexa - Fluor488 - LPS - O55 : B5 after addition of 5 µM Alexa - Fluor647 - hGBP1F supplemented with 2 mM GTP . ( Right panel ) Confocal taken of 5 µM Alexa - Fluor488 - LPS - O55 : B5 20 min after addition of 5 µM Alexa - Fluor647 - hGBP1F or - hGBP1FR48A supplemented with 2 mM GTP . Graphs depict average aggregate of Alexa - Fluor488 - LPS - O55 : B5 supplemented with 2 mM GTP 5 µM of indicated hGBP1 variant . Mean ± of combined data from three independent experiments . Significance was determined by one - way ANOVA with Tukey ' s multiple comparison . . n . s . , not significant ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (Left panel) Confocal time-lapse microscopy frames of Alexa-Fluor488-LPS-O55:B5 after addition of 5 µM Alexa-Fluor647-hGBP1F supplemented with 2 mM GTP. (Right panel) Confocal images taken of 5 µM Alexa-Fluor488-LPS-O55:B5 20 min after addition of 5 µM Alexa-Fluor647-hGBP1F or -hGBP1FR48A supplemented with 2 mM GTP. Graphs depict average aggregate area of Alexa-Fluor488-LPS-O55:B5 supplemented with 2 mM GTP and 5 µM of the indicated hGBP1 variant. Mean area ± SEM of combined data from three independent experiments. Significance was determined by one-way ANOVA with Tukey's multiple comparison test.. n.s., not significant; ***, P ≤ 0.001; ****, P ≤ 0.0001.


( C ) Polymerization of 5 µM hGBP1F induced by addition of 2 mM GTP in presence absence of 5 µM LPS - O55 : B5 was monitored by absorption spectroscopy at 350 nm . absorbance signal was superimposed with nucleotide composition of same solution analyzed at defined time points . Maximal hydrolysis rates ( turnover numbers ) were determined for hGBP1F variants in presence absence of 5 µM LPS - O55 : B5 . Graphs show mean turnover numbers ± of combined data from at least three independent experiments . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison . n . s . , not significant ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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

(C) Polymerization of 5 µM hGBP1F induced by addition of 2 mM GTP in the presence and absence of 5 µM LPS-O55:B5 was monitored by absorption spectroscopy at 350 nm. The absorbance signal was superimposed with nucleotide composition of the same solution analyzed at defined time points. Maximal hydrolysis rates (turnover numbers) were determined for hGBP1F variants in the presence and absence of 5 µM LPS-O55:B5. Graphs show mean turnover numbers ± SEM of combined data from at least three independent experiments. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ***, P ≤ 0.001; ****, P ≤ 0.0001.


( D ) Formaldehyde fixed GFP + S . flexneri supplemented with varying LPS - O55 : B5 concentrations were mixed with 5 µM hGBP1F 2 mM GTP . After 60 min hGBP1F - enclosed S . flexneri were quantified . Combined data from three independent experiments are shown as mean ± . Significance was determined by one - way ANOVA with Tukey ' s multiple comparison . * * * * , P ≤ 0 . 0001 .

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

(D) Formaldehyde fixed GFP+ S. flexneri supplemented with varying LPS-O55:B5 concentrations were mixed with 5 µM hGBP1F and 2 mM GTP. After 60 min hGBP1F-enclosed S. flexneri were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.


( E ) Confocal of formaldehyde fixed GFP - , RFP - , or dsRed - expressing gram - negative 60 min after addition of 5 µM Alexa - Fluor647 - or Alexa - Fluor488 - hGBP1F 2 mM GTP in presence absence of 100 µM LPS - O55 : B5 . After 60 min hGBP1F - enclosed gram - negative were quantified . Combined data from three independent experiments are shown as mean ± . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison . * * * * , P ≤ 0 . 0001 .

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

(E) Confocal images of formaldehyde fixed GFP-, RFP-, or dsRed-expressing gram-negative bacteria 60 min after addition of 5 µM Alexa-Fluor647- or Alexa-Fluor488-hGBP1F and 2 mM GTP in the presence and absence of 100 µM LPS-O55:B5. After 60 min hGBP1F-enclosed gram-negative bacteria were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.


( B ) Time - lapse microscopy of fixed GFP + co - isogenic S . flexneri wild type rfaL mutant after adding 10 µM Alexa - Fluor647 - hGBP1F 2 mM GTP . bound by hGBP1F at 5 min hGBP1F - enclosed after 60 min were quantified . Combined data from three independent experiments are shown as mean ± . Significance was determined by unpaired t - , two - . n . s . , not significant ; * * * * , P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Time-lapse microscopy of fixed GFP+ co-isogenic S. flexneri wild type and rfaL mutant after adding 10 µM Alexa-Fluor647-hGBP1F and 2 mM GTP. Bacteria bound by hGBP1F at 5 min and hGBP1F-enclosed bacteria after 60 min were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by unpaired t-tests, two-tailed. n.s., not significant; ****, P ≤ 0.0001.


( C ) Scanning - electron of live wild type rfaL mutant strains incubated with no or with 5 µM of hGBP1F in presence of 2 mM GTP for 4 min . Arrowheads point to unattached hGBP1 polymers , arrows point to hGBP1 polymers attached to , asterisks mark polymeric structures that appear to fuse with

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

(C) Scanning-electron micrographs of live wild type and rfaL mutant strains incubated with no protein or with 5 µM of hGBP1F in the presence of 2 mM GTP for 4 min. Arrowheads point to unattached hGBP1 polymers, arrows point to hGBP1 polymers attached to bacteria, and asterisks mark polymeric structures that appear to fuse with bacterial surfaces


( E ) Confocal of formaldehyde fixed , co - isogenic GFP + wild type mutant S . flexneri strains harboring rfb regions of E . coli serotypes O8 O25 after 60 min of incubation time in presence of 10 µM Alexa - Fluor647 - hGBP1F 2 mM GTP .

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(E) Confocal images of formaldehyde fixed, co-isogenic GFP+ wild type and mutant S. flexneri strains harboring rfb regions of E. coli serotypes O8 and O25 after 60 min of incubation time in the presence of 10 µM Alexa-Fluor647-hGBP1F and 2 mM GTP.


( A ) Following incubation for 1 . 5 h with buffer in absence or presence of 10 µM hGBP1F 5 mM GTP , live were treated with ( 2 . 5 µg / ml PMB or 5 mg / ml lysozyme ) for 30 min or left untreated . number of viable was subsequently determined by colony forming unit ( CFU ) . Graphs show mean CFUs ± of combined data from at least three independent experiments . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison . n . s . , not significant ; * * * * , P ≤ 0 . 0001 .

O O O O O O O O O O O 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 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

(A) Following incubation for 1.5 h with buffer in the absence or presence of 10 µM hGBP1F and 5 mM GTP, live bacteria were treated with antibiotics (2.5 µg/ml PMB or 5 mg/ml lysozyme) for 30 min or left untreated. The number of viable bacteria was subsequently determined by colony forming unit (CFU) counts. Graphs show mean CFUs ± SEM of combined data from at least three independent experiments. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ****, P ≤ 0.0001.


( B ) Formaldehyde fixed RFP + UPEC were mixed with 10 µM Alexa - Fluor488 - hGBP1F or - hGBP1FR584 - 586A supplemented with 2 mM GTP . Following 60 min of incubation hGBP1F - enclosed UPEC were quantified . Graphs depict mean ± of combined data from three independent experiments . Significance was determined by unpaired t - , two - . * * * * , P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Formaldehyde fixed RFP+ UPEC were mixed with 10 µM Alexa-Fluor488-hGBP1F or -hGBP1FR584-586A supplemented with 2 mM GTP. Following 60 min of incubation hGBP1F-enclosed UPEC were quantified. Graphs depict mean ± SEM of combined data from three independent experiments. Significance was determined by unpaired t-test, two-tailed. ****, P ≤ 0.0001.


( C ) Formaldehyde fixed RFP + UPEC were mixed with 10 µM Alexa - Fluor488 - hGBP1F or - hGBP1FR584 - 586A 2 mM GTP . Samples were fixed after 60 min incubation stained with an antibody against inner / lipid A fragment of E . coli LPS ( anti - LPS ) . frequency of anti - LPS stained UPEC was quantified . Graphs show mean ± of combined data from three independent experiments . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison . n . s . , not significant ; * * * * , P ≤ 0 . 0001 .

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

(C) Formaldehyde fixed RFP+ UPEC were mixed with 10 µM Alexa-Fluor488-hGBP1F or -hGBP1FR584-586A and 2 mM GTP. Samples were fixed after 60 min incubation and stained with an antibody against the inner core/ lipid A fragment of E. coli LPS (anti-LPS). The frequency of anti-LPS stained UPEC was quantified. Graphs show mean ± SEM of combined data from three independent experiments. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ****, P ≤ 0.0001.


( D ) IFNγ - primed wild type hGBP1 - KO HeLa stably expressing inactive mutant YFP - Caspase - 4C258S were infected with either cytosol - entering S . enterica Typhimurium mutant ΔsifA ( MOI = 25 ) or with S . flexneri ΔospC3ΔipaH9 . 8 ( MOI = 6 ) , a mutant lacking Caspase - 4 antagonist OspC3 as well as hGBP1 antagonist IpaH9 . 8 . were fixed at 4 hpi ( ST ΔsifA ) or at 2 hpi ( Sf ΔospC3ΔipaH9 . 8 ) stained for endogenous hGBP1 . YFP - Caspase - 4C258S associated were quantified mean ± of combined data from three independent experiments are shown . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison . * * * * , P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IFNγ-primed wild type and hGBP1-KO HeLa cells stably expressing inactive mutant YFP-Caspase-4C258S were infected with either cytosol-entering S. enterica Typhimurium mutant ΔsifA (MOI = 25) or with S. flexneri ΔospC3ΔipaH9.8 (MOI = 6), a mutant strain lacking Caspase-4 antagonist OspC3 as well as hGBP1 antagonist IpaH9.8. Cells were fixed at 4 hpi (ST ΔsifA) or at 2 hpi (Sf ΔospC3ΔipaH9.8) and stained for endogenous hGBP1. YFP-Caspase-4C258S associated bacteria were quantified and mean ± SEM of combined data from three independent experiments are shown. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.


( B were infected with poly - D - lysine treated GFP + S . flexneri strains at an MOI of 6 . were stained for indicated Z - stacks were recorded using confocal microscopy . Actin tails were classified as tails when ≥ 2 . 5 µm . graphs show mean values ± of combined data from three independent experiments . scale bars are 5 µm . Arrows point to associated with indicated , arrowheads point to lacking indicated , asterisks mark lacking unipolar IcsA localization . ( B ) Unprimed HeLa were infected with wild type or rfaL S . flexneri . Total unipolar localization of IcsA as well as actin tail formation were quantified at 1 hpi . Significance was determined by unpaired two - t - . n . s . , not significant ; * , P ≤ 0 . 05 ; * * , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Cells were infected with poly-D-lysine treated GFP+ S. flexneri strains at an MOI of 6. Cells were stained for indicated proteins and Z-stacks were recorded using confocal fluorescence microscopy. Actin tails were classified as tails when ≥ 2.5 µm. All graphs show mean values ± SEM of combined data from three independent experiments. All scale bars are 5 µm. Arrows point to bacteria associated with indicated proteins, arrowheads point to bacteria lacking indicated proteins, and asterisks mark bacteria lacking unipolar IcsA localization. (B) Unprimed HeLa cells were infected with wild type or rfaL S. flexneri. Total and unipolar localization of IcsA as well as the actin tail formation were quantified at 1 hpi. Significance was determined by unpaired two-tailed t-tests. n.s., not significant; *, P ≤ 0.05; **, P ≤ 0.01.


were infected with poly - D - lysine treated GFP + S . flexneri strains at an MOI of 6 . were stained for indicated Z - stacks were recorded using confocal microscopy . Actin tails were classified as tails when ≥ 2 . 5 µm . graphs show mean values ± of combined data from three independent experiments . scale bars are 5 µm . Arrows point to associated with indicated , arrowheads point to lacking indicated , asterisks mark lacking unipolar IcsA localization . ( C - mCherry - hGBP1 was expressed in HeLa hGBP1 - KO infected with S . flexneri ΔipaH9 . 8 stained for IcsA , N - WASP Significance was determined by unpaired t - , two - . n . s . , not significant ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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

Cells were infected with poly-D-lysine treated GFP+ S. flexneri strains at an MOI of 6. Cells were stained for indicated proteins and Z-stacks were recorded using confocal fluorescence microscopy. Actin tails were classified as tails when ≥ 2.5 µm. All graphs show mean values ± SEM of combined data from three independent experiments. All scale bars are 5 µm. Arrows point to bacteria associated with indicated proteins, arrowheads point to bacteria lacking indicated proteins, and asterisks mark bacteria lacking unipolar IcsA localization. (C- mCherry-hGBP1 was expressed in HeLa hGBP1-KO cells infected with S. flexneri ΔipaH9.8 and stained for IcsA, N-WASP Significance was determined by unpaired t-tests, two-tailed. n.s., not significant; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.


were infected with poly - D - lysine treated GFP + S . flexneri strains at an MOI of 6 . were stained for indicated Z - stacks were recorded using confocal microscopy . Actin tails were classified as tails when ≥ 2 . 5 µm . graphs show mean values ± of combined data from three independent experiments . scale bars are 5 µm . Arrows point to associated with indicated , arrowheads point to lacking indicated , asterisks mark lacking unipolar IcsA localization . D ) mCherry - hGBP1 was expressed in HeLa hGBP1 - KO infected with S . flexneri ΔipaH9 . 8 stained for Arp2 , F - actin at 2 . 5 hpi . Significance was determined by unpaired t - , two - . n . s . , not significant ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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

Cells were infected with poly-D-lysine treated GFP+ S. flexneri strains at an MOI of 6. Cells were stained for indicated proteins and Z-stacks were recorded using confocal fluorescence microscopy. Actin tails were classified as tails when ≥ 2.5 µm. All graphs show mean values ± SEM of combined data from three independent experiments. All scale bars are 5 µm. Arrows point to bacteria associated with indicated proteins, arrowheads point to bacteria lacking indicated proteins, and asterisks mark bacteria lacking unipolar IcsA localization. D) mCherry-hGBP1 was expressed in HeLa hGBP1-KO cells infected with S. flexneri ΔipaH9.8 and stained for Arp2, and F-actin at 2.5 hpi. Significance was determined by unpaired t-tests, two-tailed. n.s., not significant; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.


were infected with poly - D - lysine treated GFP + S . flexneri strains at an MOI of 6 . were stained for indicated Z - stacks were recorded using confocal microscopy . Actin tails were classified as tails when ≥ 2 . 5 µm . graphs show mean values ± of combined data from three independent experiments . scale bars are 5 µm . Arrows point to associated with indicated , arrowheads point to lacking indicated , asterisks mark lacking unipolar IcsA localization . ( E ) IFNγ - primed unprimed wild type hGBP1 - KO HeLa were infected with S . flexneri ΔipaH9 . 8 subcellular localization of IcsA , N - WASP , Arp2 , F - actin was assessed quantified at 2 . 5 hpi . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 .

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

Cells were infected with poly-D-lysine treated GFP+ S. flexneri strains at an MOI of 6. Cells were stained for indicated proteins and Z-stacks were recorded using confocal fluorescence microscopy. Actin tails were classified as tails when ≥ 2.5 µm. All graphs show mean values ± SEM of combined data from three independent experiments. All scale bars are 5 µm. Arrows point to bacteria associated with indicated proteins, arrowheads point to bacteria lacking indicated proteins, and asterisks mark bacteria lacking unipolar IcsA localization. (E) IFNγ-primed and unprimed wild type and hGBP1-KO HeLa cells were infected with S. flexneri ΔipaH9.8 and subcellular localization of IcsA, N-WASP, Arp2, and F-actin was assessed and quantified at 2.5 hpi. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.


( B ) Starvation - induced autophagosomes ( AP ) autolysosomes ( AL ) in fat ( functional analogue of liver ) of a fruit fly larva . Note that APs contain intact cytoplasm , whereas contents of ALs show various stages of degradation .

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(B) Starvation-induced autophagosomes (AP) and autolysosomes (AL) in the fat body (the functional analogue of the liver) of a fruit fly larva. Note that APs contain intact cytoplasm, whereas the contents of ALs show various stages of degradation.


( C ) Liver of starved mice carrying a fluorescently tagged LC3 transgene , labeling cup - shaped ring - shaped structures that correspond to IMs autophagosomes , respectively . courtesy of Ryan Scott ( B ) Dr . Noboru Mizushima ( C ) .

(C) Liver cells of starved mice carrying a fluorescently tagged LC3 transgene, labeling cup-shaped and ring-shaped structures that correspond to IMs and autophagosomes, respectively. Images courtesy of Ryan Scott (B) and Dr. Noboru Mizushima (C).


( A ) Northern blot experiments show expression of RsaI during growth phase in HG001 in MHB medium with or without addition of 1 , 5 g / L of D - glucose . Glucose was added either at beginning of growth ( + glucose , left panel ) or after 3h of growth ( right panel ) .

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

(A) Northern blot experiments show the expression of RsaI during growth phase in the HG001 strain in MHB medium with or without the addition of 1,5 g/L of D-glucose. Glucose was added either at the beginning of growth (+ glucose, left panel) or after 3h of growth (right panel).


( B ) Northern blot experiment shows expression of RsaI during growth phase in HG001 , ∆ ccpA mutant , ∆ codY mutant , in MHB medium with ( + ) or without ( - ) addition of 1 , 5 g / L of D - glucose .

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

(B) Northern blot experiment shows the expression of RsaI during growth phase in the HG001, ∆ccpA mutant strain, and ∆codY mutant strain, in MHB medium with (+) or without (-) the addition of 1,5 g/L of D-glucose.


( C ) Northern blot experiment shows expression of RsaI in HG001 ∆ ccpA mutant strains . Total was prepared after 2 , 3 , 4 , 5 6h of culture in BHI medium at 37 ° C .

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(C) Northern blot experiment shows the expression of RsaI in the HG001 and ∆ccpA mutant strains. Total RNA was prepared after 2, 3, 4, 5 and 6h of culture in BHI medium at 37°C.


( D ) Northern blot of RsaI in HG001 grown in MHB medium with or without addition of 1 g / L of glucose , fructose or xylose . For experiments , loading controls were done using expression of 5S rRNA ( 5S ) as revealed after hybridization of membranes with a specific probe . However for these controls , we used aliquots of same preparations but migration of samples was performed in parallel to experiments on a separate agarose gel because RsaI 5S rRNA have very similar sizes .

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(D) Northern blot analysis of RsaI in the HG001 strain grown in MHB medium with or without the addition of 1 g/L of glucose, fructose or xylose. For all the experiments, loading controls were done using the expression of 5S rRNA (5S) as revealed after hybridization of the membranes with a specific probe. However for these controls, we used aliquots of the same RNA preparations but the migration of the samples was performed in parallel to the experiments on a separate agarose gel because RsaI and 5S rRNA have very similar sizes.


( B ) Gel retardation assays show formation of between RsaI icaR , glcU _ 2 fn3K mRNAs . 5 ' end - labeled wild - type RsaI ( RsaI ) , RsaI mutant 3 ( RsaI mut3 , deletion of two G - track motifs ) , RsaI mutant 4 ( RsaI mut4 , deletion of C / U rich unpaired interhelical region ) were incubated with increasing concentrations of mRNAs : lane 1 , 0 nM ; lane 2 , 25 nM ; lane 3 , 50 nM ; lane 4 , 100 nM lane 5 , 200 nM . Below gels , predicted interactions between RsaI its targets are shown . Translation start codons are in green SD is for Shine Dalgarno . Graphs represented % of formed between either RsaI or its two mutant forms ( RsaI mut3 RsaI mut4 ) target mRNA ( icaR , glcU _ 2 , fn3K ) as function of mRNA concentrations .

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(B) Gel retardation assays show the formation of the complex between RsaI and icaR, glcU_2 and fn3K mRNAs. The 5' end-labeled wild-type RsaI (RsaI), RsaI mutant 3 (RsaI mut3, deletion of the two G-track motifs), and RsaI mutant 4 (RsaI mut4, deletion of the C/U rich unpaired interhelical region) were incubated with increasing concentrations of mRNAs: lane 1, 0 nM; lane 2, 25 nM; lane 3, 50 nM; lane 4, 100 nM and lane 5, 200 nM. Below the gels, the predicted interactions between RsaI and its targets are shown. Translation start codons are in green and SD is for Shine Dalgarno sequence. Graphs represented the % of complex formed between either RsaI or its two mutant forms (RsaI mut3 and RsaI mut4) and the target mRNA (icaR, glcU_2, fn3K) as the function of mRNA concentrations.


( C ) Gel retardation assays show formation of between RsaI RsaG . 5 ' end - labeled wild - type RsaI ( RsaI ) , RsaI mutant 2 ( RsaI mut2 ) , RsaI mutant 4 ( RsaI mut4 ) , RsaI mutant 1 ( RsaI mut1 ) were incubated with increasing concentrations of RsaG given in nM on top of autoradiographies .

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(C) Gel retardation assays show the formation of the complex between RsaI and RsaG. The 5' end-labeled wild-type RsaI (RsaI), RsaI mutant 2 (RsaI mut2), RsaI mutant 4 (RsaI mut4), and RsaI mutant 1 (RsaI mut1) were incubated with increasing concentrations of RsaG given in nM on the top of the autoradiographies.


( A ) Toe - print assays showing effect of RsaI on formation of ribosomal initiation of glcU _ 2 fn3K mRNAs , respectively . Lane 1 : incubation control of mRNA alone ; lane 2 : incubation control of mRNA with 30S subunits ; lane 3 : incubation control of mRNA with RsaI ; lane 4 : formation of ribosomal initiation containing mRNA , 30S initiator tRNAfMet ( tRNAi ) ; lanes 5 to 9 : formation of initiation in presence of increasing concentrations of RsaI , respectively : 50 nM ( lane 5 ) , 100 nM ( lane 6 ) , 150 nM ( lane 7 ) , 300 nM ( lane 8 ) , 400 nM ( lane 9 ) . Lanes T , A , C , G : sequencing ladders . Shine Dalgarno ( SD ) , start site of translation ( START ) toe - printing signals ( + 16 ) are indicated . At bottom of gels are shown predicted interactions between RsaI its targets . Translation start codons are in green , Shine Dalgarno ( SD ) is underlined , arrowheads depict toe - printing signals .

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

(A) Toe-print assays showing the effect of RsaI on the formation of the ribosomal initiation complex of glcU_2 and fn3K mRNAs, respectively. Lane 1 : incubation control of mRNA alone; lane 2 : incubation control of mRNA with 30S subunits; lane 3: incubation control of mRNA with RsaI; lane 4 : formation of the ribosomal initiation complex containing mRNA, 30S and the initiator tRNAfMet (tRNAi); lanes 5 to 9 : formation of the initiation complex in the presence of increasing concentrations of RsaI, respectively : 50 nM (lane 5), 100 nM (lane 6), 150 nM (lane 7), 300 nM (lane 8), and 400 nM (lane 9). Lanes T, A, C, G: sequencing ladders. The Shine and Dalgarno (SD) sequence, the start site of translation (START) and the toe-printing signals (+16) are indicated. At the bottom of the gels are shown the predicted interactions between RsaI and its targets. Translation start codons are in green, and the Shine and Dalgarno (SD) sequence is underlined, the arrowheads depict the toe-printing signals.


( B ) β - galactosidase activity ( Miller Units ) has been measured from various fusions expressed from a plasmid which also carries rsaI gene under its own promoter : PrpoB : : glcU : : lacZ : : rsaI , PrpoB : : fn3K : : lacZ : : rsaI , PrpoB : : treB : : lacZ : : rsaI , PrpoB : : HG001 _ 1242 : : lacZ : : rsaI , PrpoB : : HG001 _ 2520 : : lacZ : : rsaI expressed in mutant HG001 - ∆ rsaI . same constructs were made in absence of rsaI gene . For fn3K - lacZ fusion , we also used an additional construct PrpoB : : fn3K : : lacZ : : rsaImut4 expressing both fusion FN3K - LacZ RsaI mut4 . β - galactosidase activity was normalized for density results represented mean of four independent experiments . error bars are standard deviations statistical significance was determined using Student ' s t - . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 , * * * * p < 0 . 0001 , ns is for not significant .

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(B) The β-galactosidase activity (Miller Units) has been measured from various fusions expressed from a plasmid which also carries rsaI gene under its own promoter: PrpoB::glcU::lacZ::rsaI, PrpoB::fn3K::lacZ::rsaI, PrpoB::treB::lacZ::rsaI, PrpoB::HG001_1242::lacZ::rsaI, and PrpoB::HG001_2520::lacZ::rsaI expressed in the mutant strain HG001-∆rsaI. The same constructs were made in the absence of rsaI gene. For the fn3K-lacZ fusion, we also used an additional construct PrpoB::fn3K::lacZ::rsaImut4 expressing both the fusion FN3K-LacZ protein and RsaI mut4. The β-galactosidase activity was normalized for bacterial density and the results represented the mean of four independent experiments. The error bars are standard deviations and the statistical significance was determined using the Student's t-test. * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001, ns is for not significant.


( B ) Gel retardation assays show formation of between RsaI icaR full , icaR SUBST , icaR - 5 ' UTR , icaR - 3 ' UTR . 5 ' end - labeled of RsaI was incubated with increasing concentrations of various mRNAs . UTR is for untranslated region , SUBST stands for substitution of UCCCCUG by AGGGGAC . This mutation , which is located in 3 ' UTR of icaR , significantly destabilizes long - range interaction , enhances icaR translation . Lane C represents binding between radiolabelled RsaI full - icaR mRNA ( 50 nM ) . Due to rather short migration of gel , full - mRNA is still observed in pocket .

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(B) Gel retardation assays show the formation of the complex between RsaI and icaR full length, icaR SUBST, icaR-5'UTR, and icaR-3'UTR. The 5' end-labeled of RsaI was incubated with increasing concentrations of the various mRNAs. UTR is for untranslated region, SUBST stands for the substitution of UCCCCUG sequence by AGGGGAC. This mutation, which is located in the 3'UTR of icaR, significantly destabilizes the long-range interaction, and enhances icaR translation. Lane C represents binding between radiolabelled RsaI and full-length icaR mRNA (50 nM). Due to the rather short migration of the gel, the full-length mRNA is still observed in the pocket.


( C ) In vivo effect of RsaI on PIA - PNAG synthesis in S . aureus wild - type ( WT ) 132 , ∆ rsaI mutant , carrying a deletion of icaR 3 ' UTR ( ∆ 3 ' UTR ) . This last mutant has been transformed with pES plasmids expressing rsaI or rsaI mut5 . PIA - PNAG exopolysaccharide biosynthesis was quantified using dot - blot assays . Serial dilutions ( 1 / 5 ) of samples were spotted onto nitrocellulose membranes PIA - PNAG production was detected with specific anti - PIA - PNAG antibodies . RsaI was detected in same samples by Northern blot using a probe directed against RsaI . Ethidium bromide of rRNA was used as loading controls of same gel .

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

(C) In vivo effect of RsaI on PIA-PNAG synthesis in the S. aureus wild-type (WT) 132 strain, the ∆rsaI mutant, and the strain carrying a deletion of icaR 3'UTR (∆3'UTR). This last mutant strain has been transformed with the pES plasmids expressing rsaI or rsaI mut5. The PIA-PNAG exopolysaccharide biosynthesis was quantified using dot-blot assays. Serial dilutions (1/5) of the samples were spotted onto nitrocellulose membranes and PIA-PNAG production was detected with specific anti-PIA-PNAG antibodies. RsaI was detected in the same samples by Northern blot using a probe directed against RsaI. Ethidium bromide staining of rRNA was used as loading controls of the same gel.


( A ) Ternary formation between RsaI , various mRNAs ( glcU _ 2 , HG001 _ 00942 HG001 _ 1242 ) , RsaG . 5 ' - end labeled RsaI was incubated with increasing concentrations of target mRNA alone or in presence of 50 nM of RsaG . various complexes are notified on sides of autoradiographies .

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(A) Ternary complex formation between RsaI, various mRNAs (glcU_2, HG001_00942 and HG001_1242), and RsaG. The 5'-end labeled RsaI was incubated with increasing concentrations of the target mRNA alone or in the presence of 50 nM of RsaG. The various complexes are notified on the sides of the autoradiographies.


( B ) RsaG does not form stable complexes with glcU _ 2 HG001 _ 01242 . Binding assays were done in presence of 5 ' end - labeled RsaG either cold RsaI ( 300 nM ) or increasing concentrations of glcU _ 2 HG001 _ 01242 .

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(B) RsaG does not form stable complexes with glcU_2 and HG001_01242. Binding assays were done in the presence of 5' end-labeled RsaG and either cold RsaI (300 nM) or increasing concentrations of glcU_2 and HG001_01242.


( C ) Measurements of half - lives of RsaI RsaG in HG001 - ∆ rsaG or HG001 - ∆ rsaI mutant strains using Northern blot experiments . were treated with rifampicin at 4h of growth total RNAs were extracted after 2 , 4 , 8 , 15 , 30 60 min at 37 ° C in BHI . 5S rRNA was probed to quantify yield of RNAs in each lane using same samples , which were however run on two different gels . Calculated half - lives are shown beneath autoradiographies are average of 2 experiments . data were normalized to 5S rRNA .

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(C) Measurements of the half-lives of RsaI and RsaG in HG001-∆rsaG or HG001-∆rsaI mutant strains using Northern blot experiments. The cells were treated with rifampicin at 4h of growth and total RNAs were extracted after 2, 4, 8, 15, 30 and 60 min at 37°C in BHI. 5S rRNA was probed to quantify the yield of RNAs in each lane using the same samples, which were however run on two different gels. Calculated half-lives are shown beneath the autoradiographies and are the average of 2 experiments. The data were normalized to 5S rRNA.


B Hops − / − mouse genotyping expression . Genotyping by PCR using genomic showed a 242bp band for wild type ( Hops + / + ) allele a 331bp band for mutant ( Hops − / − ) allele . NTC is Null Template Control ( left panel ) . Genotyping by real time PCR confirmed absence of Hops mRNA in Hops − / − mice ( middle panel ) . extracts from Hops + / + Hops − / − MEFs were analysed by Western Blot using anti - HOPS antibody anti - α - tubulin antibody as loading control ( right panel ) . Data information : in B data are presented as mean ± SD .

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B Hops−/− mouse genotyping and protein expression. Genotyping by PCR using genomic DNA showed a 242bp band for wild type (Hops+/+) allele and a 331bp band for mutant (Hops−/−) allele. NTC is Null Template Control (left panel). Genotyping by real time PCR confirmed the absence of Hops mRNA in Hops−/− mice (middle panel). Protein extracts from Hops+/+ and Hops−/− MEFs were analysed by Western Blot using anti-HOPS antibody and anti-α-tubulin antibody as loading control (right panel). Data information: in B data are presented as mean ±SD.


C Offspring distribution . Different HOPS genotypes at weaning ( approximately postnatal day 21 ) from crosses between homozygous knockout mice heterozygous ( n = 1525 pups ) . Data information : In C χ2 was performed to determine significant differences between expected observed ratios of Hops − / − to Hops + / − mice . P < 0 . 001 , by two Student ' s t - .

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C Offspring distribution. Different HOPS genotypes at weaning age (approximately postnatal day 21) from crosses between homozygous knockout mice males and heterozygous females (n=1525 pups). Data information: In C the χ2 test was performed to determine significant differences between the expected and observed ratios of Hops−/− to Hops+/− mice. P < 0.001, by two tailed Student's t-test.


A Reduced caspase - 3 activation in Hops − / − mice upon apoptotic stimulus . Hops + / + Hops − / − mice were treated with etoposide for indicated times thymus , spleen , testis liver were harvested Tissue - Tek ® O . C . T . embedded . Sections were immunostained with anti - caspase - 3 antibody followed by appropriate secondary antibody labelling ( green ) . graphs display relative positive number . Scale bars , 10µm . Data information : experiments were performed three times in three different mice per condition a representative example is shown . In A data are presented as mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 , by two Student ' s t - .

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A Reduced caspase-3 activation in Hops−/− mice upon apoptotic stimulus. Hops+/+ and Hops−/− mice were treated with etoposide for the indicated times and thymus, spleen, testis and liver were harvested and Tissue-Tek® O.C.T. embedded. Sections were immunostained with anti-cleaved caspase-3 antibody followed by appropriate secondary antibody labelling (green). The graphs display the relative positive cells number. Scale bars, 10µm. Data information: all the experiments were performed three times in three different mice per condition and a representative example is shown. In A data are presented as mean ±SD. ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.


B HOPS affects p53 increase after stress in vivo . Whole - homogenates from organs , were immediately prepared immunoblotted with antibodies to detect p53 GAPDH . p53 were semi - quantified using GAPDH as loading control relative p53 at time 0 were assumed as 1 . Data information : experiments were performed three times in three different mice per condition a representative example is shown . In B data are presented as mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 , by two Student ' s t - .

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B HOPS affects p53 increase after stress in vivo. Whole-cell homogenates from organs, were immediately prepared and immunoblotted with antibodies to detect p53 and GAPDH. The p53 levels were semi-quantified using GAPDH as loading control and relative p53 levels at time 0 were assumed as 1. Data information: all the experiments were performed three times in three different mice per condition and a representative example is shown. In B data are presented as mean ±SD. ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.


A Thymocytes from Hops + / + Hops − / − mice were treated with dexamethasone ( Dex ) apoptotic were measured by flow cytometry . flow cytometry plots are shown on left data represented as a histogram on right . Data information : experiments were performed three times in from three different mice per condition . In A data are presented as mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by two Student ' s t - .

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A Thymocytes from Hops+/+ and Hops−/− mice were treated with dexamethasone (Dex) and the apoptotic cells were measured by flow cytometry. The flow cytometry analysis plots are shown on the left and the data represented as a histogram on the right. Data information: the experiments were performed three times in primary cells from three different mice per condition. In A data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.


B HOPS was pivotal for apoptosis induction . Hops + / + Hops − / − MEFs were subjected to etoposide ( Eto ) apoptosis was analysed by flow cytometry ( left ) showed as a graph ( right ) . Data information : experiments were performed three times in from three different mice per condition . experiments in MEFs were performed three times . Representative panels are shown . In data are presented as mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by two Student ' s t - .

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B HOPS was pivotal for apoptosis induction. Hops+/+ and Hops−/− MEFs were subjected to etoposide (Eto) and apoptosis was analysed by flow cytometry (left) and showed as a graph (right). Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown. In data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.


C HOPS overexpression in MEFs promotes apoptosis . Hops + / + Hops − / − MEFs were transiently transfected with an empty vector ( pEGFP - N1 ) or with a Hops encoding vector ( pEGFP - N1 - Hops ) . apoptotic were measured by flow cytometry ( left ) on GFP positive negative populations showed as a graph ( right ) . Data information : experiments were performed three times in from three different mice per condition . experiments in MEFs were performed three times . Representative panels are shown . In C data are presented as mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by two Student ' s t - .

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C HOPS overexpression in MEFs promotes apoptosis. Hops+/+ and Hops−/− MEFs were transiently transfected with an empty vector (pEGFP-N1) or with a Hops encoding vector (pEGFP-N1-Hops). The apoptotic cells were measured by flow cytometry (left) on the GFP positive and negative populations and showed as a graph (right). Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown. In C data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.


D HOPS affects p53 following stress in vitro . Hops + / + Hops − / − MEFs were treated with etoposide harvested at indicated time points . p53 were semi - quantified using α - tubulin as loading control relative p53 at 0 µM dosage were assumed as 1 . Data information : experiments were performed three times in from three different mice per condition . experiments in MEFs were performed three times . Representative panels are shown .

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D HOPS affects p53 levels following stress in vitro. Hops+/+ and Hops−/− MEFs were treated with etoposide and harvested at the indicated time points. The p53 protein levels were semi-quantified using α-tubulin as loading control and relative p53 levels at 0 µM dosage were assumed as 1. Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown.


A HOPS loss p53 half - life . Hops + / + Hops − / − MEFs untransfected or transfected with HOPS , were analysed by immunoblotting following cycloheximide treatment for indicated time ( left panel ) . p53 were semi - quantified using GAPDH as loading control relative p53 at time 0 were assumed as 100 % ( right panel ) . Data information : experiments in MEFs were performed three times . Representative panels are shown . In A data are presented as mean ± SD .

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A HOPS loss and p53 half-life. Hops+/+ and Hops−/− MEFs untransfected or transfected with HOPS, were analysed by immunoblotting following cycloheximide treatment for the indicated time (left panel). The p53 protein levels were semi-quantified using GAPDH as loading control and relative p53 levels at time 0 were assumed as 100% (right panel). Data information: all the experiments in MEFs were performed three times. Representative panels are shown. In A data are presented as mean ±SD.


B p19Arf - independent HOPS control of p53 half - life . p19Arf − / − MEFs untransfected ( CTRL ) , transfected with empty vector ( pSGV ) or HOPS were treated with cycloheximide for indicated times subjected to immunoblot with anti - p53 , anti - HOPS anti - GAPDH antibodies ( left panel ) . p53 were semi - quantified as above ( right panel ) . Data information : experiments in MEFs were performed three times . Representative panels are shown . In B data are presented as mean ± SD .

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B p19Arf-independent HOPS control of p53 half-life. p19Arf−/− MEFs untransfected (CTRL), transfected with empty vector (pSGV) or HOPS were treated with cycloheximide for the indicated times and subjected to immunoblot analysis with anti-p53, anti-HOPS and anti-GAPDH antibodies (left panel). The p53 protein levels were semi-quantified as above (right panel). Data information: all the experiments in MEFs were performed three times. Representative panels are shown. In B data are presented as mean ±SD.


C HOPS p53 ubiquitination . p53 , HDM2 , His - tagged ubiquitin were co - expressed in H1299 with HOPS or HOPS - G176A or HOPS - K126A mutants treated with MG132 . p53 ubiquitination rate was evaluated by elution using Ni2 + - NTA His - bind resin followed by immunoblotting with anti - p53 indicated antibodies .

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C HOPS and p53 ubiquitination. p53, HDM2, His-tagged ubiquitin were co-expressed in H1299 cells with HOPS or HOPS-G176A or HOPS-K126A mutants and treated with MG132. p53 ubiquitination rate was evaluated by elution using Ni2+-NTA His-bind resin followed by immunoblotting with anti-p53 and the indicated antibodies.


D HOPS depletion HOPS - G176A mutant affect p53 ubiquitination . Hops + / + Hops − / − MEFs untransfected Hops − / − MEFs transfected with HOPS or HOPS - G176A mutant were co - expressed with p53 , HDM2 His - tagged ubiquitin . were treated with MG132 ubiquitinated p53 fraction was recovered as above . Immunodetection of ubiquitinated p53 was performed using anti - p53 antibody samples were tested with indicated antibodies . Data information : experiments in MEFs were performed three times . Representative panels are shown .

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D HOPS depletion and HOPS-G176A mutant affect p53 ubiquitination. Hops+/+ and Hops−/− MEFs untransfected and Hops−/− MEFs transfected with HOPS or HOPS-G176A mutant were co-expressed with p53, HDM2 and His-tagged ubiquitin. Cells were treated with MG132 and the ubiquitinated p53 fraction was recovered as above. Immunodetection of ubiquitinated p53 was performed using anti-p53 antibody and the samples were tested with the indicated antibodies. Data information: all the experiments in MEFs were performed three times. Representative panels are shown.


E HOPS - G176A mutant ability to stabilize p53 . Hops − / − MEFs were transfected with HOPS - G176A , subjected to cycloheximide treatment for indicated times analysed by immunoblotting using indicated antibodies . Data information : experiments in MEFs were performed three times . Representative panels are shown .

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E HOPS-G176A mutant ability to stabilize p53. Hops−/− MEFs were transfected with HOPS-G176A, subjected to cycloheximide treatment for the indicated times and analysed by immunoblotting using the indicated antibodies. Data information: all the experiments in MEFs were performed three times. Representative panels are shown.


A binding in RKO . PLA showed HOPS / p53 interaction with or without etoposide treatment at indicated time . Red spots showed a single interaction . counterstaining was performed with DAPI . α - Tubulin - FITC antibody was used to identify edge . Background control was performed according to manufacturer ' s instruction . Scale bars , 10µm . graph represents binding increase . Non treated binding was assumed as 1 . Data information : experiments were performed three times , representative examples are shown . In A data are presented as mean ± SD .

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A Protein binding in RKO cells. PLA showed HOPS/p53 interaction with or without etoposide treatment at the indicated time. Red spots showed a single interaction. DNA counterstaining was performed with DAPI. α-Tubulin-FITC antibody was used to identify the cellular edge. Background control was performed according to the manufacturer's instruction. Scale bars, 10µm. The graph represents the binding increase. Non treated cells binding was assumed as 1. Data information: all the experiments were performed three times, and representative examples are shown. In A data are presented as mean ±SD.


B Biochemical of - distribution . RKO were treated with etoposide , collected after 2 4 hours processed to obtain fractions . Aliquots relative to same number per fraction were immunoblotted using anti - HOPS anti - p53 antibodies . GAPDH Lamin B1 are purity controls of fraction respectively .

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B Biochemical analysis of nuclear-cytoplasmic protein distribution. RKO cells were treated with etoposide, collected after 2 and 4 hours and processed to obtain nuclear and cytoplasmic fractions. Aliquots relative to the same cell number per fraction were immunoblotted using anti-HOPS and anti-p53 antibodies. GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively.


C HOPS p53 binding in cytoplasm following apoptosis induction . RKO untreated or treated with etoposide as above were used to isolate fractions . WCL fractions were immunoprecipitated with anti - HOPS antibody subsequently immunoblotted using anti - p53 antibody .

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C HOPS and p53 binding in cytoplasm following apoptosis induction. RKO cells untreated or treated with etoposide as above were used to isolate cytoplasmic fractions. WCL and cytoplasmic fractions were immunoprecipitated with anti-HOPS antibody and subsequently immunoblotted using anti-p53 antibody.


A HOPS localization at mitochondria . RKO were stained by MitoTracker ™ ( red ) , immediately fixed stained to visualize HOPS ( green ) . Nuclei were DAPI stained ( blue ) . Samples were analysed with microscope representative were shown . Scale bars , 10µm .

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A HOPS localization at mitochondria. RKO cells were stained by MitoTracker™ (red), immediately fixed and stained to visualize HOPS (green). Nuclei were DAPI stained (blue). Samples were analysed with fluorescent microscope and representative images were shown. Scale bars, 10µm.


B RKO were lysed processed for mitochondria isolation . WCL , cytoplasm mitochondria were analysed using anti - HOPS antibody . GAPDH , Lamin B1 Tom20 are purity controls of mitochondrial fraction .

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B RKO cells were lysed and processed for mitochondria isolation. WCL, cytoplasm and mitochondria were analysed using anti-HOPS antibody. GAPDH, Lamin B1 and Tom20 are purity controls of mitochondrial fraction.


C HOPS p53 translocation to mitochondria . RKO were treated with etoposide as indicated processed for mitochondrial extraction subsequent immunoblotting for p53 , HOPS reported extraction purity controls .

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C HOPS and p53 translocation to mitochondria. RKO cells were treated with etoposide as indicated and processed for mitochondrial extraction and subsequent immunoblotting for p53, HOPS and the reported extraction purity controls.


D HOPS / p53 binding at mitochondria . RKO were treated with etoposide subjected to mitochondrial purification , subsequent immunoprecipitation using anti - HOPS antibody . Samples were immunoblotted to detect p53 , HOPS reported controls .

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D HOPS/p53 binding at mitochondria. RKO cells were treated with etoposide and subjected to mitochondrial purification, and subsequent immunoprecipitation using anti-HOPS antibody. Samples were immunoblotted to detect p53, HOPS and the reported controls.


E HOPS p53 mitochondrial translocation in vivo . Hops + / + Hops − / − mice were treated with etoposide thymus , spleen testis were harvested . Organs were homogenized mitochondrial fraction were isolated analysed by immunoblotting versus p53 . GAPDH Tom20 are purity controls . p53 mitochondrial were semi - quantified using Tom20 as loading control p53 at time 0 were assumed as 1 . Data information : assays involving were performed three times in three different mice per condition . In E data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 , by two Student ' s t - .

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E HOPS and p53 mitochondrial translocation in vivo. Hops+/+ and Hops−/− mice were treated with etoposide and thymus, spleen and testis were harvested. Organs were homogenized and mitochondrial fraction were isolated and analysed by immunoblotting versus p53. GAPDH and Tom20 are purity controls. p53 mitochondrial protein levels were semi-quantified using Tom20 as loading control and p53 levels at time 0 were assumed as 1. Data information: assays involving animals were performed three times in three different mice per condition. In E data are presented as mean ±SD. * P < 0.05; *** P < 0.001, by two tailed Student's t-test.


F p53 - dependent mitochondrial potential ( Δψm ) induction in MEFs . Hops + / + Hops − / − MEFs were treated with etoposide then incubated with JC - 1 vital dye . were collected by microscopy representative are shown ( left panel ) . Scale bars , 10µm . was analysed by TECAN plate reader plotted in graph ( right panel ) . Data information : assays involving were performed three times in three different mice per condition . Experiments on mitochondria from MEFs were performed five times . In F data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 , by two Student ' s t - .

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F p53-dependent mitochondrial membrane potential (Δψm) induction in MEFs. Hops+/+ and Hops−/− MEFs were treated with etoposide and then incubated with JC-1 vital dye. The images were collected by fluorescent microscopy and representative images are shown (left panel). Scale bars, 10µm. Fluorescence was analysed by TECAN plate reader and plotted in the graph (right panel). Data information: assays involving animals were performed three times in three different mice per condition. Experiments on mitochondria from MEFs were performed five times. In F data are presented as mean ±SD. * P < 0.05; *** P < 0.001, by two tailed Student's t-test.


G p53 MOMP induction in HOPS deficiency . Mitochondria from Hops + / + Hops − / − MEFs , were purified incubated with p53 recombinant . Cytochrome C release from mitochondria as metabolic marker was assayed by immunoblotting . Samples were tested to detect p53 as control Tom20 as mitochondria structural marker . Data information : assays involving were performed three times in three different mice per condition . Experiments on mitochondria from MEFs were performed five times .

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G p53 MOMP induction in HOPS deficiency. Mitochondria from Hops+/+ and Hops−/− MEFs, were purified and incubated with p53 recombinant protein. Cytochrome C release from mitochondria as metabolic marker was assayed by immunoblotting. Samples were tested to detect p53 as control and Tom20 as mitochondria structural marker. Data information: assays involving animals were performed three times in three different mice per condition. Experiments on mitochondria from MEFs were performed five times.


A HOPS expression p53 accumulation . HOPS was overexpressed in RKO were fixed stained with antibody against p53 ( green ) . Nuclei were stained with DAPI ( blue ) . Samples were analysed with microscope merged are shown ( right panel ) . Scale bars , 10µm . were scored for p53 localization . results are shown as of versus p53 localization in untransfected HOPS transfected ( left panel ) . Data information : experiments detailed above were performed three times , representative panels are shown . In A data are presented as mean ± SD .

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A HOPS expression and p53 nuclear accumulation. HOPS was overexpressed in RKO and cells were fixed and stained with antibody against p53 (green). Nuclei were stained with DAPI (blue). Samples were analysed with fluorescent microscope and merged images are shown (right panel). Scale bars, 10µm. Cells were scored for p53 localization. The results are shown as percentage of nuclear versus cytoplasmic p53 localization in untransfected and HOPS transfected cells (left panel). Data information: all the experiments detailed above were performed three times, and representative panels are shown. In A data are presented as mean ±SD.


B Biochemical of HOPS intracellular localization . RKO were transfected or not with HOPS fractionated into component . Aliquots relative to same number per fraction were immunoblotted using anti - p53 anti - HOPS antibodies . GAPDH Lamin B1 are purity controls of fraction respectively . Data information : experiments detailed above were performed three times , representative panels are shown .

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B Biochemical analysis of HOPS intracellular localization. RKO cells were transfected or not with HOPS and fractionated into nuclear and cytoplasmic component. Aliquots relative to the same cell number per fraction were immunoblotted using anti-p53 and anti-HOPS antibodies. GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively. Data information: all the experiments detailed above were performed three times, and representative panels are shown.


HOPS p53 binding to importin α . Untransfected or HOPS transfected RKO were treated with etoposide each sample was evaluated by immunoprecipitation with anti - p53 analysed by immunoblotting using anti - importin α antibody . Arrowhead indicates importin α specific signal . Data information : experiments detailed above were performed three times , representative panels are shown .

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HOPS and p53 binding to importin α. Untransfected or HOPS transfected RKO cells were treated with etoposide and each sample was evaluated by immunoprecipitation with anti-p53 and analysed by immunoblotting using anti-importin α antibody. Arrowhead indicates the importin α specific signal. Data information: all the experiments detailed above were performed three times, and representative panels are shown.


D PCAF - mediated p53 acetylation . p53 PCAF Flag - tagged were co - expressed in H1299 with or without HOPS treated with trichostatin A nicotinamide . p53 acetylation were evaluated by immunoprecipitation with anti - p53 antibody followed by immunoblotting with anti - acetylated - Lysine ( AcK ) , anti - p53 , anti - Flag , anti - HOPS anti - α - tubulin antibodies . Data information : experiments detailed above were performed three times , representative panels are shown .

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D PCAF-mediated p53 acetylation. p53 and PCAF Flag-tagged were co-expressed in H1299 cells with or without HOPS and treated with trichostatin A and nicotinamide. p53 acetylation levels were evaluated by immunoprecipitation with anti-p53 antibody followed by immunoblotting with anti-acetylated-Lysine (AcK), anti-p53, anti-Flag, anti-HOPS and anti-α-tubulin antibodies. Data information: all the experiments detailed above were performed three times, and representative panels are shown.


( A ) Wild type ( left middle panels ) p53 - / - ( right panel ) HCT116 were transfected with control ( mixture of control DS scrambledNeg , siLuci , siGFP ) or GRWD1 - targeting ( mixture of siGRWD1 - 3 4 ) siRNAs for 36 h , treated with 5 nM actinomycin D , harvested at indicated times . Whole extracts were analyzed by immunoblotting with indicated antibodies . Coomassie Brilliant Blue ( CBB ) serves as a loading control .

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(A) Wild type (left and middle panels) and p53-/- (right panel) HCT116 cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4) siRNAs for 36 h, treated with 5 nM actinomycin D, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies. Coomassie Brilliant Blue (CBB) staining serves as a loading control.


( B ) Control U2OS infected with control retroviruses with empty vector U2OS overexpressing siRNA - resistant HA - GRWD1 were treated analyzed as above .

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(B) Control U2OS cells infected with control retroviruses with the empty vector and U2OS cells overexpressing the siRNA-resistant HA-GRWD1 were treated and analyzed as above.


( C ) HCT116 were transfected with control ( mixture of control DS scrambledNeg , siLuci , siGFP ) or GRWD1 - targeting ( mixture of siGRWD1 - 3 4 ) siRNAs for 24 h , treated with 50 µg / ml cycloheximide , harvested at indicated times . Whole extracts were analyzed by immunoblotting with indicated antibodies .

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(C) HCT116 cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4) siRNAs for 24 h, treated with 50 µg/ml cycloheximide, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies.


( D ) HFF2 / T stably overexpressing HA - GRWD1 were established by retroviral infection . were cultured in presence or absence of actinomycin D ( 5 nM ) for 12h then treated with or without MG132 ( 20uM ) for 6 h as indicated . Whole extracts were analyzed as above .

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(D) HFF2/T cells stably overexpressing HA-GRWD1 were established by retroviral infection. Cells were cultured in the presence or absence of actinomycin D (5 nM) for 12h and then treated with or without MG132 (20uM) for 6 h as indicated. Whole cell extracts were analyzed as above.


( E ) Control HA - GRWD1 - overexpressing HFF2 / T were first treated with actinomycin D ( 5nM ) for 12 h , then further treated with 50 ug / ml cycloheximide , harvested at indicated times . Whole extracts were analyzed by immunoblotting with indicated antibodies . means SDs from two independent experiments are shown .

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(E) Control and HA-GRWD1-overexpressing HFF2/T cells were first treated with actinomycin D (5nM) for 12 h, then further treated with 50 ug/ml cycloheximide, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies. The means and SDs from two independent experiments are shown.


( A ) U2OS were transfected with control ( mixture of control DS scrambledNeg , siLuci , siGFP ) or GRWD1 - targeting ( mixture of siGRWD1 - 3 4 or siGRWD1 - 1 ) siRNAs for 24 h . Whole extracts were then analyzed by immunoblotting with indicated antibodies .

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(A) U2OS cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4 or siGRWD1-1) siRNAs for 24 h. Whole cell extracts were then analyzed by immunoblotting with the indicated antibodies.


B ) U2OS treated as above were stained with propidium iodide subjected to flow cytometry .

B) U2OS cells treated as above were stained with propidium iodide and subjected to flow cytometry.


( C ) HCT116 treated with actinomycinD ( 5nM ) or vehicle ( DMSO ) for 12h were first extracted with Triton X - 100 to remove nucleoplasmic , double - immunostained with anti - GRWD1 ( green ) anti - fibrillarin ( red ) antibodies as a marker for nucleoli , counterstained with DAPI . Scale bar , 20 um .

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(C) HCT116 cells treated with actinomycinD (5nM) or vehicle (DMSO) for 12h were first extracted with Triton X-100 to remove nucleoplasmic proteins, double-immunostained with anti-GRWD1 (green) and anti-fibrillarin (red) antibodies as a marker for nucleoli, and counterstained with DAPI. Scale bar, 20 um.


( D ) HCT116 were transfected with control ( mixture of control siLuci siGFP ) or GRWD1 - targeting ( mixture of siGRWD1 - 3 4 or siGRWD1 - 1 ) siRNAs for 24 h then immunostained as above . Scale bar , 20 um .

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(D) HCT116 cells were transfected with control (mixture of control siLuci and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4 or siGRWD1-1) siRNAs for 24 h and then immunostained as above. Scale bar, 20 um.