Total p53 , p53 acetylated at K382 and K370 as well as GAPDH were measured by Western Blot at indicated time points in the context of different p53 dynamics : pulsing p53 ( 10 Gy IR ) , transient p53 ( 10 Gy IR + BML - 277 , central lanes ) and sustained p53 ( 10 Gy IR + Nutlin - 3 , right lanes ) . See Figure 3 and Methods section for details . The relative change in p53 acetylation at K370 ( light green ) and K382 ( dark green ) was quantified from Western Blot and normalized to the abundance 3 h post IR . Means and propagated standard errors from 3 independent experiments are indicated . Acetylation increased over time in the context of sustained p53 . See also Appendix Fig S12 .

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Total p53, p53 acetylated at K382 and K370 as well as GAPDH were measured by Western Blot at indicated time points in the context of different p53 dynamics: pulsing p53 (10 Gy IR), transient p53 (10 Gy IR + BML-277, central lanes) and sustained p53 (10 Gy IR + Nutlin-3, right lanes). See Figure 3 and Methods section for details. The relative change in p53 acetylation at K370 (light green) and K382 (dark green) was quantified from Western Blot and normalized to the abundance 3 h post IR. Means and propagated standard errors from 3 independent experiments are indicated. Acetylation increased over time in the context of sustained p53. See also Appendix Fig S12.


The p53 - K370 methylase Smyd2 was down - regulated in a clonal stable A549 cell line expressing a corresponding shRNA . Transcript levels were measured in wild type and knockdown cells by qRT - PCR . Mean levels and standard deviation from technical triplicates are indicated .

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The p53-K370 methylase Smyd2 was down-regulated in a clonal stable A549 cell line expressing a corresponding shRNA. Transcript levels were measured in wild type and knockdown cells by qRT-PCR. Mean levels and standard deviation from technical triplicates are indicated.


Promoter activity of CDKN1A ( E ) and MDM2 ( F ) were quantified in Smyd2 knockdown cells before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) . Left panel : The percentage of cells with active TSS , subdivided into populations with strong ( > 75 % of TSS , solid colors ) and weak ( < 75 % of TSS , shaded colors ) activity , is shown as stacked bar graphs , the mean fraction of active promoters is indicated above each bar . Right panel : Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates ( PDF , see Data Visualization section ) . We measured a higher fraction of active promoters upon damage compared to A549 wild type cells ( Fig 3 ) , while transcription rates remained unchanged .

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Promoter activity of CDKN1A (E) and MDM2 (F) were quantified in Smyd2 knockdown cells before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). We measured a higher fraction of active promoters upon damage compared to A549 wild type cells (Fig 3), while transcription rates remained unchanged.


The p53 - K382 methylase Set8 was down - regulated in a clonal stable A549 cell line expressing a corresponding shRNA . Transcript levels were measured in wild type and knockdown cells by qRT - PCR . Mean levels and standard deviation from technical triplicates are indicated .

The p53-K382 methylase Set8 was down-regulated in a clonal stable A549 cell line expressing a corresponding shRNA. Transcript levels were measured in wild type and knockdown cells by qRT-PCR. Mean levels and standard deviation from technical triplicates are indicated.


Promoter activity of CDKN1A ( E ) and MDM2 ( F ) were quantified in Set8 knock - down cells before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) . Left panel : The percentage of cells with active TSS , subdivided into populations with strong ( > 75 % of TSS , solid colors ) and weak ( < 75 % of TSS , shaded colors ) activity , is shown as stacked bar graphs , the mean fraction of active promoters is indicated above each bar . Right panel : Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates ( PDF , see Data Visualization section ) . We measured a higher fraction of active promoters upon damage compared to A549 wild type cells ( Figure 3 ) , while transcription rates remained unchanged .

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

Promoter activity of CDKN1A (E) and MDM2 (F) were quantified in Set8 knock-down cells before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). We measured a higher fraction of active promoters upon damage compared to A549 wild type cells (Figure 3), while transcription rates remained unchanged.


( A and B ) Endogenous FYCO1 binds to GST - LC3B . GST - LC3B bound to glutathione - Sepharose beads was incubated with HeLa cell lysate , and copurified proteins were identified by Coomassie blue staining and mass spectrometry ( A ) or immunoblotting with anti - FYCO1 antibody ( B ) .

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(A and B) Endogenous FYCO1 binds to GST-LC3B. GST-LC3B bound to glutathione-Sepharose beads was incubated with HeLa cell lysate, and copurified proteins were identified by Coomassie blue staining and mass spectrometry (A) or immunoblotting with anti-FYCO1 antibody (B).


( C ) GST - LC3B but not GST - p62 or GST alone binds to FYCO1 in a GST pull - down assay . GST , GST - LC3B , or GST - p62 bound to glutathione - Sepharose beads was incubated with [ 35S ] methionine - labeled myc - FYCO1 . Formed protein complexes were isolated and visualized by autoradiography ( top ) or Coomassie blue staining ( bottom ) .

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(C) GST-LC3B but not GST-p62 or GST alone binds to FYCO1 in a GST pull-down assay. GST, GST-LC3B, or GST-p62 bound to glutathione-Sepharose beads was incubated with [35S]methionine-labeled myc-FYCO1. Formed protein complexes were isolated and visualized by autoradiography (top) or Coomassie blue staining (bottom).


( D ) GFP - LC3B immunoprecipitates endogenous FYCO1 from HeLa cell lysate . HeLa cells expressing GFP or GFP - LC3B were lysed and processed as in B . IP , immunoprecipitation ; MM , molecular mass ; WB , Western blot .

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(D) GFP-LC3B immunoprecipitates endogenous FYCO1 from HeLa cell lysate. HeLa cells expressing GFP or GFP-LC3B were lysed and processed as in B. IP, immunoprecipitation; MM, molecular mass; WB, Western blot.


( B ) The region of FYCO1 between aa 1 , 276 and 1 , 294 is essential and sufficient for the interaction with LC3B . GST or GST - LC3B were incubated with [ S35 ] methionine - labeled deletion mutants of FYCO1 and processed as in Fig . 1 C .

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(B) The region of FYCO1 between aa 1,276 and 1,294 is essential and sufficient for the interaction with LC3B. GST or GST-LC3B were incubated with [S35]methionine-labeled deletion mutants of FYCO1 and processed as in Fig. 1 C.


( C ) LIR is essential for colocalization of FYCO1 with LC3B in HeLa cells . HeLa cells expressing the indicated constructs were imaged by confocal microscopy . Insets show an enlarged field of interest .

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(C) LIR is essential for colocalization of FYCO1 with LC3B in HeLa cells. HeLa cells expressing the indicated constructs were imaged by confocal microscopy. Insets show an enlarged field of interest.


( D ) Both N - and C - terminal domains of LC3B are needed for efficient interaction with FYCO1 . GST , GST - LC3B , or its deletion mutants were incubated with [ S35 ] methionine - labeled myc - FYCO1 and processed as in Fig . 1 C . ARG , autoradiography ; CB , Coomassie blue .

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(D) Both N- and C-terminal domains of LC3B are needed for efficient interaction with FYCO1. GST, GST-LC3B, or its deletion mutants were incubated with [S35]methionine-labeled myc-FYCO1 and processed as in Fig. 1 C. ARG, autoradiography; CB, Coomassie blue.


( E ) The interaction between FYCO1 and LC3B is direct . GST or GST - LC3B was incubated with recombinant MBP - FYCO1863 - 1 , 478 . Protein complexes were isolated and visualized by immunoblotting with anti - GST or anti - MBP antibodies . WB , Western blot . Bars : ( C ) 10 µm ; ( C , insets ) 1 µm .

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(E) The interaction between FYCO1 and LC3B is direct. GST or GST-LC3B was incubated with recombinant MBP-FYCO1863-1,478. Protein complexes were isolated and visualized by immunoblotting with anti-GST or anti-MBP antibodies. WB, Western blot. Bars: (C) 10 µm; (C, insets) 1 µm.


( A ) Endogenous FYCO1 localizes to the cytosolic punctuated structures . HeLa cells cultured in complete growth medium ( left ) or in HBSS for 3 h ( right ) were fixed and stained with anti - FYCO1 antibody ( green ) and Draq5 ( blue ) .

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(A) Endogenous FYCO1 localizes to the cytosolic punctuated structures. HeLa cells cultured in complete growth medium (left) or in HBSS for 3 h (right) were fixed and stained with anti-FYCO1 antibody (green) and Draq5 (blue).


( B ) Overexpressed mCherry - FYCO1 localizes to the cytosolic punctuated structures and cytosolic vesicles in stably transfected HeLa cells .

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(B) Overexpressed mCherry-FYCO1 localizes to the cytosolic punctuated structures and cytosolic vesicles in stably transfected HeLa cells.


( C ) FYCO1 partially colocalizes with LC3B , LAMP1 , ATG5 , ATG16L , p62 , and HuntingtinQ64 and does not colocalize with EEA1 . HeLa cells or mouse embryonic fibroblasts ( for ATG16L staining ) were transfected with the indicated vectors or stained with the indicated antibodies and imaged 24 h after transfection . For p62 staining , HeLa cells were incubated with 0 . 2 µM BafA1 for 12 h before fixation .

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(C) FYCO1 partially colocalizes with LC3B, LAMP1, ATG5, ATG16L, p62, and HuntingtinQ64 and does not colocalize with EEA1. HeLa cells or mouse embryonic fibroblasts (for ATG16L staining) were transfected with the indicated vectors or stained with the indicated antibodies and imaged 24 h after transfection. For p62 staining, HeLa cells were incubated with 0.2 µM BafA1 for 12 h before fixation.


( D ) FYCO1 colocalizes with LC3B on the rim but not inside of the vesical structures . HeLa cells cotransfected with GFP - FYCO1 , mCherry - LC3B , and myc - p62 were imaged by confocal microscopy 48 h after transfection . ( E and F ) FYCO1 - decorated structures are heterogenous in their luminal content .

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(D) FYCO1 colocalizes with LC3B on the rim but not inside of the vesical structures. HeLa cells cotransfected with GFP-FYCO1, mCherry-LC3B, and myc-p62 were imaged by confocal microscopy 48 h after transfection. (E and F) FYCO1-decorated structures are heterogenous in their luminal content.


( E ) HeLa cells transfected with CFP - FYCO1 and mCherry - YFP - p62 were imaged 48 h after transfection .

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(E) HeLa cells transfected with CFP-FYCO1 and mCherry-YFP-p62 were imaged 48 h after transfection.


( F ) HeLa cells transfected with GFP - FYCO1 were labeled with LysoTracker red for 60 min and Alexa Fluor 647 - dextran ( 10 , 000 D ) for 4 h in normal growth medium to stain late endocytic compartments ( left ) . Relative number of FYCO1 structures ± SD colocalized with LysoTracker red or Alexa Fluor 647 - dextran ( 10 , 000 D ; right ) .

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(F) HeLa cells transfected with GFP-FYCO1 were labeled with LysoTracker red for 60 min and Alexa Fluor 647-dextran (10,000 D) for 4 h in normal growth medium to stain late endocytic compartments (left). Relative number of FYCO1 structures ± SD colocalized with LysoTracker red or Alexa Fluor 647-dextran (10,000 D; right).


( A ) HeLa cells transfected with the indicated constructs were imaged by confocal microscopy 24 h after transfection . Insets show an enlarged field of interest .

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(A) HeLa cells transfected with the indicated constructs were imaged by confocal microscopy 24 h after transfection. Insets show an enlarged field of interest.


( B - D ) Immunoelectron micrographs of HeLa cells transfected with GFP - FYCO1 and stained with anti - GFP polyclonal antibody ( 10 - nm gold particle ) .

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(B-D) Immunoelectron micrographs of HeLa cells transfected with GFP-FYCO1 and stained with anti-GFP polyclonal antibody (10-nm gold particle).


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( B ) The FYVE domain of FYCO1 is essential but not sufficient for its recruitment to the cytosolic vesicles . HeLa cells were imaged 24 h after transfection with the indicated constructs .

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(B) The FYVE domain of FYCO1 is essential but not sufficient for its recruitment to the cytosolic vesicles. HeLa cells were imaged 24 h after transfection with the indicated constructs.


( C ) FYCO1 can self - interact in a CC - dependent manner . Full - length GFP - FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of myc - tagged FYCO1 in rabbit reticulocyte lysate . S35 - labeled FYCO1 complexes were immunoprecipitated with anti - myc antibody , separated by SDS - PAGE , and visualized by autoradiography . IP , immunoprecipitation .

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(C) FYCO1 can self-interact in a CC-dependent manner. Full-length GFP-FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of myc-tagged FYCO1 in rabbit reticulocyte lysate. S35-labeled FYCO1 complexes were immunoprecipitated with anti-myc antibody, separated by SDS-PAGE, and visualized by autoradiography. IP, immunoprecipitation.


( D ) Dimers but not a monomer of the FYVE domain from FYCO1 can bind PI3P in protein - lipid overlay assay . PIP Strips were incubated with 1 - µg / ml solutions of MBP - FYVEFYCO1 , MBP - 2xFYVEFYCO1 , or MBP - FYCO1863 - 1 , 233 for 1 h , and bound proteins were detected by immunostaining with anti - MBP antibody . LPA , lysophosphatic acid ; LPC , lysophosphocholine ; PI , phosphatidylinositol ; PE , phosphatidylethanolamine ; PC , phosphatidylcholine ; S1P , sphingosine - 1 - phosphate ; PA , phosphatidic acid ; PS , phosphatidylserine .

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(D) Dimers but not a monomer of the FYVE domain from FYCO1 can bind PI3P in protein-lipid overlay assay. PIP Strips were incubated with 1-µg/ml solutions of MBP-FYVEFYCO1, MBP-2xFYVEFYCO1, or MBP-FYCO1863-1,233 for 1 h, and bound proteins were detected by immunostaining with anti-MBP antibody. LPA, lysophosphatic acid; LPC, lysophosphocholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine.


( E ) The construct containing a tandem fusion of two FYVE domains from FYCO1 ( 2xFYVEFYCO1 ) localizes to the nuclear envelope and ER in HeLa cells . HeLa cells were imaged 24 h after transfection with the indicated constructs . ( A and E ) Insets show an enlarged field of interest .

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(E) The construct containing a tandem fusion of two FYVE domains from FYCO1 (2xFYVEFYCO1) localizes to the nuclear envelope and ER in HeLa cells. HeLa cells were imaged 24 h after transfection with the indicated constructs. (A and E) Insets show an enlarged field of interest.


( F ) 2xFYVEFYCO1 does not colocalize with 2xFYVEHrs ( left ) and PXp40phox ( right ) . HeLa cells were imaged 24 h after transfection with the indicated constructs . Bars , 10 µm .

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(F) 2xFYVEFYCO1 does not colocalize with 2xFYVEHrs (left) and PXp40phox (right). HeLa cells were imaged 24 h after transfection with the indicated constructs. Bars, 10 µm.


( A ) FYCO1990 - 1 , 233 is the smallest FYVE domain - containing deletion mutant of FYCO1 consistently recruited to cytosolic vesicles . HeLa cells transiently transfected with FYCO1990 - 1 , 233 were imaged 24 h after transfection .

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(A) FYCO1990-1,233 is the smallest FYVE domain-containing deletion mutant of FYCO1 consistently recruited to cytosolic vesicles. HeLa cells transiently transfected with FYCO1990-1,233 were imaged 24 h after transfection.


( B ) FYCO1990 - 1 , 233 is the smallest FYVE domain - containing deletion mutant of FYCO1 that can efficiently interact with the full - length FYCO1 . Full - length myc - FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of GFP - tagged FYCO1 in rabbitreticulocyte lysate . S35 - labeled FYCO1 complexes were immunoprecipitated with anti - SDS - PAGE antibody , separated by SDS - PAGE , and visualized by autoradiography . IP , immunoprecipitation .

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(B) FYCO1990-1,233 is the smallest FYVE domain-containing deletion mutant of FYCO1 that can efficiently interact with the full-length FYCO1. Full-length myc-FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of GFP-tagged FYCO1 in rabbitreticulocyte lysate. S35-labeled FYCO1 complexes were immunoprecipitated with anti-SDS-PAGE antibody, separated by SDS-PAGE, and visualized by autoradiography. IP, immunoprecipitation.


( C ) FYCO1990 - 1 , 233 strongly colocalizes with Rab7 , LysoTracker red , and Alexa Fluor 647 - dextran ( 10 , 000 D ) but only weakly with PXp40phox . HeLa cells transfected with the indicated constructs or labeled with LysoTracker red for 60 min and Alexa Fluor 647 - dextran ( 10 , 000 D ) for 4 h were imaged 24 h after transfection .

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(C) FYCO1990-1,233 strongly colocalizes with Rab7, LysoTracker red, and Alexa Fluor 647-dextran (10,000 D) but only weakly with PXp40phox. HeLa cells transfected with the indicated constructs or labeled with LysoTracker red for 60 min and Alexa Fluor 647-dextran (10,000 D) for 4 h were imaged 24 h after transfection.


( D ) FYCO1 construct containing a CC region followed by a tandem repeat of two FYVE domains from FYCO1 ( FYCO1990 - 1 , 233 + 1 , 156 - 1 , 233 ) localizes to the ER and perinuclear vesicles and only weakly colocalizes with PXp40phox . HeLa cells were imaged 24 h after transfection with the indicated constructs . ( A , C , and D ) Insets show an enlarged field of interest . Bars , 10 µm .

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(D) FYCO1 construct containing a CC region followed by a tandem repeat of two FYVE domains from FYCO1 (FYCO1990-1,233+1,156-1,233) localizes to the ER and perinuclear vesicles and only weakly colocalizes with PXp40phox. HeLa cells were imaged 24 h after transfection with the indicated constructs. (A, C, and D) Insets show an enlarged field of interest. Bars, 10 µm.


( A ) Rab7Q67L recruits FYCO11 , 038 - 1 , 233 and FYCO11 , 091 - 1 , 233 but not FYCO11 , 156 - 1 , 233 to the cytosolic vesicles . HeLa cells transfected with GFP - FYCO11 , 038 - 1 , 233 ( top ) , GFP - FYCO11 , 091 - 1 , 233 ( bottom ) , or GFP - FYCO11 , 156 - 1 , 233 ( middle right ) with or without mCherry - Rab7Q67L were imaged 24 h after transfection . The graph shows the relative number of cells ± SD with GFP - FYCO11 , 038 - 1 , 233 recruited to intracellular membranes without and with cotransfected mCherry - Rab7Q67L .

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(A) Rab7Q67L recruits FYCO11,038-1,233 and FYCO11,091-1,233 but not FYCO11,156-1,233 to the cytosolic vesicles. HeLa cells transfected with GFP-FYCO11,038-1,233 (top), GFP-FYCO11,091-1,233 (bottom), or GFP-FYCO11,156-1,233 (middle right) with or without mCherry-Rab7Q67L were imaged 24 h after transfection. The graph shows the relative number of cells ± SD with GFP-FYCO11,038-1,233 recruited to intracellular membranes without and with cotransfected mCherry-Rab7Q67L.


( B ) Rab7Q67L recruits the 2xFYVEFYCO1 construct containing a short CC region in front of the first FYVE domain ( FYCO11 , 091 - 1 , 233 + 1 , 156 - 1 , 233 ) but not a 2xFYVE alone to the perinuclear vesicles . HeLa cells transfected with GFP - FYCO11 , 091 - 1 , 233 + 1 , 156 - 1 , 233 or GFP - FYCO11 , 156 - 1 , 233 + 1 , 156 - 1 , 233 with or without mCherry - Rab7Q67L were imaged 24 h after transfection .

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(B) Rab7Q67L recruits the 2xFYVEFYCO1 construct containing a short CC region in front of the first FYVE domain (FYCO11,091-1,233+1,156-1,233) but not a 2xFYVE alone to the perinuclear vesicles. HeLa cells transfected with GFP-FYCO11,091-1,233+1,156-1,233 or GFP-FYCO11,156-1,233+1,156-1,233 with or without mCherry-Rab7Q67L were imaged 24 h after transfection.


( C ) Rab7 coimmunoprecipitates with FYCO1 from HEK293 cell lysate . HEK293 cells stably transfected with 3xFlag - FYCO1 were cotransfected with GTP - locked mutants of Rab5 , - 7 , - 11 , and - 24 and lysed 24 h after transfection , and protein complexes were immunoprecipitated with anti - Flag M2 agarose affinity gel . Bound proteins were eluted with Flag peptide , resolved by SDS - PAGE , and visualized by immunoblotting with anti - GFP and anti - Flag antibody . IP , immunoprecipitation ; WB , Western blot .

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(C) Rab7 coimmunoprecipitates with FYCO1 from HEK293 cell lysate. HEK293 cells stably transfected with 3xFlag-FYCO1 were cotransfected with GTP-locked mutants of Rab5, -7, -11, and -24 and lysed 24 h after transfection, and protein complexes were immunoprecipitated with anti-Flag M2 agarose affinity gel. Bound proteins were eluted with Flag peptide, resolved by SDS-PAGE, and visualized by immunoblotting with anti-GFP and anti-Flag antibody. IP, immunoprecipitation; WB, Western blot.


( D ) Leucine - 1151 and tryptophan - 1152 are important for the recruitment of FYCO1 to Rab7 - containing vesicles . HeLa cells transfected with GFP - FYCO1990 - 1 , 233L1151A / W1152A with or without mCherry - Rab7Q67L were imaged 24 h after transfection . ( B and D ) Insets show an enlarged field of interest .

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(D) Leucine-1151 and tryptophan-1152 are important for the recruitment of FYCO1 to Rab7-containing vesicles. HeLa cells transfected with GFP-FYCO1990-1,233L1151A/W1152A with or without mCherry-Rab7Q67L were imaged 24 h after transfection. (B and D) Insets show an enlarged field of interest.


( E ) RILP but not Rab7 or ORP1L competes with FYCO1 for recruitment to intracellular vesicles . ( right ) HeLa cells cotransfected with GFP - RILP together with mCherry - FYCO1 and stained with Draq5 were imaged 24 h after transfection . ( left ) Relative number of HeLa cells cotransfected with mCherry - FYCO1 and the indicated constructs , in which FYCO1 is recruited to the intracellular vesicles . Error bars represent SD . Bars , 10 µm .

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(E) RILP but not Rab7 or ORP1L competes with FYCO1 for recruitment to intracellular vesicles. (right) HeLa cells cotransfected with GFP-RILP together with mCherry-FYCO1 and stained with Draq5 were imaged 24 h after transfection. (left) Relative number of HeLa cells cotransfected with mCherry-FYCO1 and the indicated constructs, in which FYCO1 is recruited to the intracellular vesicles. Error bars represent SD. Bars, 10 µm.


( A ) Overexpressed FYCO1 is enriched in the cell periphery , close to the MT plus end . HeLa cells were imaged 48 h after transfection with mCherry - FYCO1 and GFP - tubulin .

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(A) Overexpressed FYCO1 is enriched in the cell periphery, close to the MT plus end. HeLa cells were imaged 48 h after transfection with mCherry-FYCO1 and GFP-tubulin.


( B ) Peripheral enrichment of FYCO1 is dependent on intact MTs . HeLa cells were transfected with GFP - FYCO1 and cultured in normal medium ( left ) or treated with 5 µM colcemid ( middle ) or 5 µM latrunculin A ( right ) for 2 h .

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(B) Peripheral enrichment of FYCO1 is dependent on intact MTs. HeLa cells were transfected with GFP-FYCO1 and cultured in normal medium (left) or treated with 5 µM colcemid (middle) or 5 µM latrunculin A (right) for 2 h.


( C ) FYCO1 - decorated vesicles move along the MT tracks . HeLa cells transfected with mCherry - FYCO1 and GFP - tubulin were live imaged by confocal microscopy 48 h after transfection . Movements of FYCO1 - decorated structures were tracked with ImageJ MTrackJ plug - in ( National Institutes of Health ) and presented as 8 - min - long tracks in the overlay image .

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(C) FYCO1-decorated vesicles move along the MT tracks. HeLa cells transfected with mCherry-FYCO1 and GFP-tubulin were live imaged by confocal microscopy 48 h after transfection. Movements of FYCO1-decorated structures were tracked with ImageJ MTrackJ plug-in (National Institutes of Health) and presented as 8-min-long tracks in the overlay image.


( D ) Full - length FYCO1 but not the FYCO1 lacking aa 555 - 1 , 136 can redistribute Rab7 and ORP1L to the cell periphery . HeLa cells were transfected with the indicated constructs and stained with DRAQ5 .

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(D) Full-length FYCO1 but not the FYCO1 lacking aa 555-1,136 can redistribute Rab7 and ORP1L to the cell periphery. HeLa cells were transfected with the indicated constructs and stained with DRAQ5.


( E ) The region of FYCO1 between aa 675 and 771 is essential for the MT plus end translocation of FYCO1 - decorated structures . HeLa cells were imaged 24 h after transfection with the indicated constructs .

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(E) The region of FYCO1 between aa 675 and 771 is essential for the MT plus end translocation of FYCO1-decorated structures. HeLa cells were imaged 24 h after transfection with the indicated constructs.


( A ) Western blot ( WB ) of cell lysates from HeLa cells stably expressing GFP - LC3 48 h after transient transfection with siRNA against FYCO1 ( siRNAs : A , Applied Biosystems ; D , Thermo Fisher Scientific ) . MM , molecular mass .

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(A) Western blot (WB) of cell lysates from HeLa cells stably expressing GFP-LC3 48 h after transient transfection with siRNA against FYCO1 (siRNAs: A, Applied Biosystems; D, Thermo Fisher Scientific). MM, molecular mass.


( B ) Representative images of HeLa cells with or without the perinuclear clusters of GFP - LC3 - positive vesicles ( right ) and quantification of the number of cells with this phenotype 48 h after transient transfection with siRNAs against FYCO1 ( left ) . Error bars represent SDs based on three independent experiments .

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(B) Representative images of HeLa cells with or without the perinuclear clusters of GFP-LC3-positive vesicles (right) and quantification of the number of cells with this phenotype 48 h after transient transfection with siRNAs against FYCO1 (left). Error bars represent SDs based on three independent experiments.


( C , D , and E ) The perinuclear clusters of GFP - LC3 - positive vesicles colocalize with mCherry - Rab7 but not with Golgi protein marker 58K or LysoTracker red . GFP - LC3 HeLa cells were transiently transfected with siRNA against FYCO1 ( Thermo Fisher Scientific ) 48 h before imaging or cotransfected with mCherry - Rab7 . Cells were either fixed and stained with anti - Golgi 58K antibody or incubated with 50 nM LysoTracker red for 1 h . Insets show an enlarged field of interest . Bars , 10 µm .

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(C, D, and E) The perinuclear clusters of GFP-LC3-positive vesicles colocalize with mCherry-Rab7 but not with Golgi protein marker 58K or LysoTracker red. GFP-LC3 HeLa cells were transiently transfected with siRNA against FYCO1 (Thermo Fisher Scientific) 48 h before imaging or cotransfected with mCherry-Rab7. Cells were either fixed and stained with anti-Golgi 58K antibody or incubated with 50 nM LysoTracker red for 1 h. Insets show an enlarged field of interest. Bars, 10 µm.


D , E Dot plots showing the expression level of transcripts coding for established markers of intrathymic T cell differentiation ( D ) , and for molecules involved in V ( D ) J recombination and TCR signaling ( E ) . In D and E , dot color represents the scaled average expression of the specified gene across the various clusters , and dot size indicates the percentage of cells expressing the specified gene .

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D, E Dot plots showing the expression level of transcripts coding for established markers of intrathymic T cell differentiation (D), and for molecules involved in V(D)J recombination and TCR signaling (E). In D and E, dot color represents the scaled average expression of the specified gene across the various clusters, and dot size indicates the percentage of cells expressing the specified gene.


C UMAP plots colored according to the expression level of the specified genes .

C UMAP plots colored according to the expression level of the specified genes.


D Dot plot showing the expression level of genes belonging to the γδ TCR - induced signature across the cell subsets corresponding to Dataset 2 . Based on the gene set that was specifically upregulated during the DPsmall → DPCD69 + / SP transition ( see Fig EV3B ) , it allowed to subdivide the γδ TCR - induced signature into a ' γδ TCR only ' signature and a ' αβ and γδ TCR shared '

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D Dot plot showing the expression level of genes belonging to the γδ TCR-induced signature across the cell subsets corresponding to Dataset 2. Based on the gene set that was specifically upregulated during the DPsmall → DPCD69+/SP transition (see Fig EV3B), it allowed to subdivide the γδ TCR-induced signature into a 'γδ TCR only' signature and a 'αβ and γδ TCR shared' signature


A , B UMAP plots of Dataset 3 which contains five WT ( DN3a , DN3b γδ + , DN4 γδ + , DN3b γδ - and DN4 γδ - ) and two Lat - / - ( DN3 γδ - and DN3 γδint ) scRNAseq datasets . The UMAP plot is colored according to sorted samples ( A ) , and to expression levels of the specified genes ( B ) .

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A, B UMAP plots of Dataset 3 which contains five WT (DN3a, DN3b γδ+, DN4 γδ+, DN3b γδ- and DN4 γδ-) and two Lat-/- (DN3 γδ- and DN3 γδint) scRNAseq datasets. The UMAP plot is colored according to sorted samples (A), and to expression levels of the specified genes (B).


A , B , C AUCell scores for the γδ TCR - induced signature ( A ) , γδ TCR only signature ( B ) and αβ and γδ TCR shared signature ( C ) are displayed on UMAP ( left ) and violin ( right ) plots of the 7 sorted subsets corresponding to Dataset 3 . As expected , AUCell scores for the 3 signatures were very low for DN3a cells , low for WT DN3b γδ - and WT DN4 γδ - cells , and high for WT DN3b γδ + and WT DN4 γδ + cells .

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A,B,C AUCell scores for the γδ TCR-induced signature (A), γδ TCR only signature (B) and αβ and γδ TCR shared signature (C) are displayed on UMAP (left) and violin (right) plots of the 7 sorted subsets corresponding to Dataset 3. As expected, AUCell scores for the 3 signatures were very low for DN3a cells, low for WT DN3b γδ- and WT DN4 γδ- cells, and high for WT DN3b γδ+ and WT DN4 γδ+ cells.


B Bar plot showing the percentage of cells in the G0 / G1 ( red bars ) , S ( blue bars ) and G2 / M ( green bars ) phases of the cell cycle among each of the 7 sorted cell subsets of Dataset 3 .

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B Bar plot showing the percentage of cells in the G0/G1 (red bars), S (blue bars) and G2/M (green bars) phases of the cell cycle among each of the 7 sorted cell subsets of Dataset 3.


( C ) High - performance thin - layer chromatography ( HPTLC ) profile of HeLa cells pulsed for 2 hours with [ 3H ] - sphingosine and chased for 24 hours . The peaks corresponding to each SL species is indicated and numbers represent each SL species as percentage of total SL .

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(C) High-performance thin-layer chromatography (HPTLC) profile of HeLa cells pulsed for 2 hours with [3H]-sphingosine and chased for 24 hours. The peaks corresponding to each SL species is indicated and numbers represent each SL species as percentage of total SL.


( A ) HeLa cells were treated with control or indicated siRNA for 72 hours and SL biosynthesis measured by [ 3H ] - sphingosine pulse - chase assay . GSL levels are expressed as fold changes with respect to control . CERT and FAPP2 knockdowns ( blue bars ) were used as controls .

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(A) HeLa cells were treated with control or indicated siRNA for 72 hours and SL biosynthesis measured by [3H] - sphingosine pulse-chase assay. GSL levels are expressed as fold changes with respect to control. CERT and FAPP2 knockdowns (blue bars) were used as controls.


( D ) Control and GRASP55KO cells were processed for Cy3 - conjugated Shiga Toxin ( ShTxB ) and Alexa488 - conjugated Cholera Toxin ( ChTxB ) staining followed by flow cytometry analysis . Mean fluorescence intensity was measured and represented .

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(D) Control and GRASP55KO cells were processed for Cy3-conjugated Shiga Toxin (ShTxB) and Alexa488-conjugated Cholera Toxin (ChTxB) staining followed by flow cytometry analysis. Mean fluorescence intensity was measured and represented.


( C ) Control and GRASP55 KO cells were transfected for 16 hours with GCS - HA or LCS - HA and processed for cryoimmunolabeling with anti - HA antibody ( 10 - nm gold particles ) and anti - GM130 antibody ( 15 - nm gold particles ) in case of GCS - HA and anti - HA antibody ( 15 - nm gold particles ) and anti - GM130 antibody ( 10 - nm gold particles ) in case of LCS - HA . Representative images of the distribution of GCS - HA and LCS - HA are shown . Scale bar 200nm . Red asterisk marks C4 cisterna and blue circles indicate GM130 labelling .

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(C) Control and GRASP55 KO cells were transfected for 16 hours with GCS-HA or LCS-HA and processed for cryoimmunolabeling with anti-HA antibody (10-nm gold particles) and anti-GM130 antibody (15-nm gold particles) in case of GCS-HA and anti-HA antibody (15-nm gold particles) and anti-GM130 antibody (10-nm gold particles) in case of LCS-HA. Representative images of the distribution of GCS-HA and LCS-HA are shown. Scale bar 200nm. Red asterisk marks C4 cisterna and blue circles indicate GM130 labelling.


( A ) HeLa cells co - transfected with indicated HA tagged enzymes ( GCS , LCS , GM3S and Gb3S ) and GRASP55 - GFP were lysed , immunoprecipitated with anti - GFP antibody or control IgG and were analysed by western blotting for interaction by immunoblotting ( IB ) with anti - HA antibody . I represents 5 % on the input lysate , U 5 % of the unbound fraction and IP the immunoprecipitate . Red asterisks indicate IgG bands and arrow heads indicates the expected position of HA - tagged

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(A) HeLa cells co-transfected with indicated HA tagged enzymes (GCS, LCS, GM3S and Gb3S) and GRASP55-GFP were lysed, immunoprecipitated with anti-GFP antibody or control IgG and were analysed by western blotting for interaction by immunoblotting (IB) with anti-HA antibody. I represents 5% on the input lysate, U 5% of the unbound fraction and IP the immunoprecipitate. Red asterisks indicate IgG bands and arrow heads indicates the expected position of HA-tagged enzymes


( E ) Chemically synthesized biotinylated peptides corresponding to cytosolic portions of GCS ( WT and Δ3C ) and indicated purified GST - tagged GRASP domain or SPR region of GRASP55 were incubated together and their interaction was monitored by pulling down the biotinylated peptides with avidin beads followed by western blotting with an anti - GST tag antibody .

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(E) Chemically synthesized biotinylated peptides corresponding to cytosolic portions of GCS (WT and Δ3C) and indicated purified GST-tagged GRASP domain or SPR region of GRASP55 were incubated together and their interaction was monitored by pulling down the biotinylated peptides with avidin beads followed by western blotting with an anti-GST tag antibody.


( A - C ) Control and GRASP55 KO ( # 2 ) cells were transfected for 16 hours with the indicated HA - tagged enzymes and processed for cryo - immunolabeling with anti - HA antibody ( 10 - nm gold particles ) and anti - GM130 antibody ( 15 - nm gold particles ) . Representative images of the distribution of HA - tagged enzymes are shown . Red circles indicate GM130 labelling . Arrow heads represent the peri - Golgi vesicles . Scale bar 150 nm . Quantification of the distribution of enzymes in vesicles represented as normalized linear

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(A-C) Control and GRASP55 KO (#2) cells were transfected for 16 hours with the indicated HA-tagged enzymes and processed for cryo-immunolabeling with anti-HA antibody (10-nm gold particles) and anti-GM130 antibody (15-nm gold particles). Representative images of the distribution of HA-tagged enzymes are shown. Red circles indicate GM130 labelling. Arrow heads represent the peri-Golgi vesicles. Scale bar 150 nm. Quantification of the distribution of enzymes in vesicles represented as normalized linear density


( F ) HeLa cells co - transfected with HA - tagged GCS and GRASP55 - GFP were incubated for 16 hours and were processed for cryoimmunolabeling with anti - HA antibody ( 10 - nm gold particles ) and anti - GFP antibody ( 15 - nm gold particles ) . Representative images of the distribution of GCS and GRASP55 - GFP are shown . Red arrowhead indicates the clathrin - coated vesicle that marks the TGN area and black arrowheads indicate the presence of GCS - HA in TGN . Scale bar 150 nm . Distribution of GCS across the Golgi stack and TGN was quantified and represented as fraction of gold particles .

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(F) HeLa cells co-transfected with HA-tagged GCS and GRASP55-GFP were incubated for 16 hours and were processed for cryoimmunolabeling with anti-HA antibody (10-nm gold particles) and anti-GFP antibody (15-nm gold particles). Representative images of the distribution of GCS and GRASP55-GFP are shown. Red arrowhead indicates the clathrin-coated vesicle that marks the TGN area and black arrowheads indicate the presence of GCS-HA in TGN. Scale bar 150 nm. Distribution of GCS across the Golgi stack and TGN was quantified and represented as fraction of gold particles.


( A Control and GRASP55 KO clones were pre - treated with DMSO or BFA ( 5μg / ml ) for 30 min and SL output monitored by [ 3H ] - sphingosine pulse - chase assay ( 8 h chase ) . Total GSL levels were quantified and expressed as fold changes with respect to control ( A ) .

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(A Control and GRASP55 KO clones were pre-treated with DMSO or BFA (5μg/ml) for 30 min and SL output monitored by [3H] - sphingosine pulse-chase assay (8 h chase). Total GSL levels were quantified and expressed as fold changes with respect to control (A).


B ) Control and GRASP55 KO clones were pre - treated with DMSO or BFA ( 5μg / ml ) for 30 min and SL output monitored by [ 3H ] - sphingosine pulse - chase assay ( 8 h chase ) . The GM / Gb ratio was calculated and represented ( B ) .

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B) Control and GRASP55 KO clones were pre-treated with DMSO or BFA (5μg/ml) for 30 min and SL output monitored by [3H] - sphingosine pulse-chase assay (8 h chase). The GM/Gb ratio was calculated and represented (B).


( F ) CERT was silenced using siRNAs for 72 hours in control and GRASP55 KO ( # 2 ) clone before subjecting to [ 3H ] - sphingosine pulse chase assay . GSLs were quantified and represented as fold change with respect to control .

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(F) CERT was silenced using siRNAs for 72 hours in control and GRASP55 KO (#2) clone before subjecting to [3H] -sphingosine pulse chase assay. GSLs were quantified and represented as fold change with respect to control.


A Sphere formation of GB2 cells transfected with an shRNA targeting SIRT2 was analyzed by an In Cell Analyzer 2000 . Primary spheres were re - plated to evaluate secondary sphere formation . Bars indicate mean ± s . d . of 10 wells .

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A Sphere formation of GB2 cells transfected with an shRNA targeting SIRT2 was analyzed by an In Cell Analyzer 2000. Primary spheres were re-plated to evaluate secondary sphere formation. Bars indicate mean ± s.d. of 10 wells.


B Knockdown of SIRT2 causes a decrease in the sphere formation capacity of GB16 . ( n = 3 ) .

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B Knockdown of SIRT2 causes a decrease in the sphere formation capacity of GB16. (n = 3).


C mRNA levels of the indicated genes in GB2 , GB4 and GB16 cells infected with a lentivirus expressing an shRNA targeting SIRT2 were measured by qRT - PCR . The results were normalized with the values for GAPDH . Bars indicate mean ± s . d . ( n = 3 - 4 ) .

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C mRNA levels of the indicated genes in GB2, GB4 and GB16 cells infected with a lentivirus expressing an shRNA targeting SIRT2 were measured by qRT-PCR. The results were normalized with the values for GAPDH. Bars indicate mean ± s.d. (n = 3-4).


The sphere formation capacity of CD133 - positive and CD133 - negative cells sorted by FACS directly from a tumor sample . GB17 was infected with a control ( Empty ) or shSIRT2 - expressing ( shS2 # 1 ) lentivirus . Bars indicate mean ± s . d . of 8 wells .

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The sphere formation capacity of CD133-positive and CD133-negative cells sorted by FACS directly from a tumor sample. GB17 was infected with a control (Empty) or shSIRT2-expressing (shS2 #1) lentivirus. Bars indicate mean ± s.d. of 8 wells.


E The sphere formation capacity of CD133 - positive and CD133 - negative cells sorted by FACS directly from a tumor sample . ( Left panel ) GB18 was treated with AGK2 ( 10 μM ) or DMSO . ( Right panel ) Secondary sphere formation of GB18 was examined in the absence of AGK2 . Bars indicate mean ± s . d . of 8 wells .

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E The sphere formation capacity of CD133-positive and CD133-negative cells sorted by FACS directly from a tumor sample. (Left panel) GB18 was treated with AGK2 (10 μM) or DMSO. (Right panel) Secondary sphere formation of GB18 was examined in the absence of AGK2. Bars indicate mean ± s.d. of 8 wells.


A Kaplan - Meier overall survival curves of mice transplanted with GB2 cells ( Left panel ) , GB13 cells ( Middle panel ) or GB16 cells ( Right panel ) infected with the indicated lentivirus ( 1 × 104 cells , 7 mice per group ) . The y - axis indicates the percent survival .

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A Kaplan-Meier overall survival curves of mice transplanted with GB2 cells (Left panel), GB13 cells (Middle panel) or GB16 cells (Right panel) infected with the indicated lentivirus (1 × 104 cells, 7 mice per group). The y-axis indicates the percent survival.


B Mice were transplanted with the indicated number of GB2 cells infected with a control ( Empty ) or shSIRT2 - expressing ( shS2 # 1 ) lentivirus . Six weeks after transplantation , mice ( 3 or 4 animals , see number of dots ) were sacrificed and the expression levels of human β - actin mRNA were quantified by qRT - PCR .

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B Mice were transplanted with the indicated number of GB2 cells infected with a control (Empty) or shSIRT2-expressing (shS2 #1) lentivirus. Six weeks after transplantation, mice (3 or 4 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA were quantified by qRT-PCR.


C H & E staining of tumors that were developed in mice implanted with GB16 cells that had been infected with a control ( Empty ) or shSIRT2 - expressing ( shS2 # 1 ) lentivirus . Scale bar , 1 mm . An image with higher magnification is shown on the right ( scale bar , 50 μm ) .

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C H&E staining of tumors that were developed in mice implanted with GB16 cells that had been infected with a control (Empty) or shSIRT2-expressing (shS2#1) lentivirus. Scale bar, 1 mm. An image with higher magnification is shown on the right (scale bar, 50 μm).


D The effect of AK7 on the deacetylation of acetyl - tubulin ( Left panel ) and the proliferation of GB2 cells ( Right panel ) . ( Left panel ) Immunoblotting analysis of the effect of AK7 ( 20 μM ) on deacetylation of acetyl - tubulin / tubulin were performed on day 3 in Right panel . Bars indicate mean ± s . d . ( n = 3 ) .

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D The effect of AK7 on the deacetylation of acetyl-tubulin (Left panel) and the proliferation of GB2 cells (Right panel). (Left panel) Immunoblotting analysis of the effect of AK7 (20 μM) on deacetylation of acetyl-tubulin/tubulin were performed on day 3 in Right panel. Bars indicate mean ± s.d. (n = 3).


E Ten days after intracranial transplantation of GB2 cells ( 1 . 0 × 104 cells ) , AK7 was intraperitoneally administrated for 4 weeks ( 15mg / kg , twice / week ) . After eight weeks , mice ( 4 or 5 animals , see number of dots ) were sacrificed and the expression levels of human β - actin mRNA was quantified by qRT - PCR .

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E Ten days after intracranial transplantation of GB2 cells (1.0 × 104 cells), AK7 was intraperitoneally administrated for 4 weeks (15mg/kg, twice/week). After eight weeks, mice (4 or 5 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA was quantified by qRT-PCR.


B Immunoblotting analysis of p73 in GB2 cells infected with a control ( Empty ) or shTP73 - expressing lentivirus for 96 h .

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B Immunoblotting analysis of p73 in GB2 cells infected with a control (Empty) or shTP73-expressing lentivirus for 96 h.


C Growth curve of GB16 cells infected with the indicated lentiviruses . Bars indicate mean ± s . d . ( n = 3 ) .

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C Growth curve of GB16 cells infected with the indicated lentiviruses. Bars indicate mean ± s.d. (n = 3).


Ectopic expression of the ∆ N isoform of p73β suppresses the expression levels of PUMA and GADD45 induced by SIRT2 knockdown in GB2 cells . Four days after lentiviral infection , the expression levels of PUMA and GADD45 mRNA were measured by qRT - PCR . Bars indicate mean ± s . d . ( n = 3 ) .

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Ectopic expression of the ∆N isoform of p73β suppresses the expression levels of PUMA and GADD45 induced by SIRT2 knockdown in GB2 cells. Four days after lentiviral infection, the expression levels of PUMA and GADD45 mRNA were measured by qRT-PCR. Bars indicate mean ± s.d. (n = 3).


E GB2 and GB4 cells [ 5 × 104 and 2 × 104 cells , respectively ( indicated by the dashed lines ) ] were infected with the indicated lentiviruses . After 96 h , the number of viable cells was counted . Bars indicate mean ± s . d . ( n = 4 ) .

E GB2 and GB4 cells [5 × 104 and 2 × 104 cells, respectively (indicated by the dashed lines)] were infected with the indicated lentiviruses. After 96 h, the number of viable cells was counted. Bars indicate mean ± s.d. (n = 4).


F HEK293 cells were transfected with TAp73α along with ΔNp73β . PUMA mRNA was measured by qRT - PCR analyses . Bars indicate mean ± s . d . ( n = 3 ) .

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F HEK293 cells were transfected with TAp73α along with ΔNp73β. PUMA mRNA was measured by qRT-PCR analyses. Bars indicate mean ± s.d. (n = 3).


GB2 cells ( 5 × 104 cells , dashed line ) were infected with the indicated lentiviruses , respectively . After 96 h , the number of viable cells was counted . shTP73 corresponds to both α and β isoforms of p73 . Bars indicate mean ± s . d . ( n = 4 ) .

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GB2 cells (5 × 104 cells, dashed line) were infected with the indicated lentiviruses, respectively. After 96 h, the number of viable cells was counted. shTP73 corresponds to both α and β isoforms of p73. Bars indicate mean ± s.d. (n = 4).


H GB2 cells were transfected with TAp73α along with NLS - SIRT2 ( nuclear - localizing mutant of SIRT2 ) or 3mut ( deacetylase - inactive , nuclear - localizing mutant of SIRT2 ) and a reporter construct consisting of the promoter region of PUMA fused to a luciferase gene ( Left panel ) . Reporter activities were determined by Dual luciferase assays ( Right panel ) . Bars indicate mean ± s . d . ( n = 3 ) .

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H GB2 cells were transfected with TAp73α along with NLS-SIRT2 (nuclear-localizing mutant of SIRT2) or 3mut (deacetylase-inactive, nuclear-localizing mutant of SIRT2) and a reporter construct consisting of the promoter region of PUMA fused to a luciferase gene (Left panel). Reporter activities were determined by Dual luciferase assays (Right panel). Bars indicate mean ± s.d. (n = 3).


I GB2 cells were electroporated with TAp73α along with NLS - SIRT2 ( nuclear - localizing mutant of SIRT2 ) or 3mut ( deacetylase - inactive , nuclear - localizing mutant of SIRT2 ) . The expression of PUMA and GADD45 was measured by qRT - PCR analysis . Bars indicate mean ± s . d . ( n = 3 )

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I GB2 cells were electroporated with TAp73α along with NLS-SIRT2 (nuclear-localizing mutant of SIRT2) or 3mut (deacetylase-inactive, nuclear-localizing mutant of SIRT2). The expression of PUMA and GADD45 was measured by qRT-PCR analysis. Bars indicate mean ± s.d. (n = 3)


A Cells transfected with FLAG - tagged p73 isoforms were treated with 20 μM AGK2 or DMSO for 6 h and subjected to immunoprecipitation with anti - FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG .

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A Cells transfected with FLAG-tagged p73 isoforms were treated with 20 μM AGK2 or DMSO for 6 h and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG.


Cells transfected with the indicated constructs were treated with 20 μM AGK2 or DMSO , respectively , for 6 h and subjected to immunoprecipitation with anti - FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG .

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Cells transfected with the indicated constructs were treated with 20 μM AGK2 or DMSO, respectively, for 6 h and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG.


E HEK293T cells transfected with FLAG - tagged TAp73α were treated with 20 μM AGK2 ( pre - AGK2 + ) or DMSO ( pre - AGK2 - ) for 6 h . p73 was purified by immunoprecipitation and incubated with recombinant SIRT2 ( 10 U ) , NAD ( 1 mM ) and / or AGK2 ( 3 . 5 μM ) as indicated .

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E HEK293T cells transfected with FLAG-tagged TAp73α were treated with 20 μM AGK2 (pre-AGK2 +) or DMSO (pre-AGK2 -) for 6 h. p73 was purified by immunoprecipitation and incubated with recombinant SIRT2 (10 U), NAD (1 mM) and/or AGK2 (3.5 μM) as indicated.


F qRT - PCR analysis of PUMA and GADD45 mRNA in GB2 cells infected with the indicated lentiviruses . Bars indicate mean ± s . e . m . ( n = 4 - 5 ) .

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F qRT-PCR analysis of PUMA and GADD45 mRNA in GB2 cells infected with the indicated lentiviruses. Bars indicate mean ± s.e.m. (n = 4-5).


G Expression of p73 in ( F ) was determined by immunoblotting with anti - p73 antibody . RFP was used as a control .

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G Expression of p73 in (F) was determined by immunoblotting with anti-p73 antibody. RFP was used as a control.


H GB glioblastima neurospheres [ 5 × 104 cells ( indicated by the dashed line ) ] were infected with the indicated lentiviruses . After 96 h , the number of viable cells was counted . Bars indicate mean ± s . d . ( n = 4 ) .

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H GB glioblastima neurospheres [5 × 104 cells (indicated by the dashed line)] were infected with the indicated lentiviruses. After 96 h, the number of viable cells was counted. Bars indicate mean ± s.d. (n = 4).


GB2 cells infected with the indicated lentiviruses were intracranially transplanted into immunocompromised mice . After 8 weeks , mice ( 3 to 5 animals , see number of dots ) were sacrificed and the expression levels of human β - actin mRNA were quantified by qRT - PCR .

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GB2 cells infected with the indicated lentiviruses were intracranially transplanted into immunocompromised mice. After 8 weeks, mice (3 to 5 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA were quantified by qRT-PCR.


B ) Log ( odds enrichment ) scores for charged GKs in the flanks of the 30 % strongest versus the 30 % weakest APRs in the E . coli cytoplasmic proteome . Whiskers indicate 95 % confidence intervals for no enrichment as determined through bootstrapping analysis .

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B) Log(odds enrichment) scores for charged GKs in the flanks of the 30 % strongest versus the 30 % weakest APRs in the E. coli cytoplasmic proteome. Whiskers indicate 95 % confidence intervals for no enrichment as determined through bootstrapping analysis.


C ) Percentual difference in non - synonymous mutation rate in E . coli for GK versus background i . e . versus the corresponding residue in non - gatekeeper positions .

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C) Percentual difference in non-synonymous mutation rate in E. coli for GK versus background i.e. versus the corresponding residue in non-gatekeeper positions.


D ) Percentual difference in non - synonymous mutation rate in mammalian species for GK versus background i . e . versus the corresponding residue in non - gatekeeper positions .

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D) Percentual difference in non-synonymous mutation rate in mammalian species for GK versus background i.e. versus the corresponding residue in non-gatekeeper positions.


B ) Time - resolved Dynamic Light Scattering ( DLS ) analysis indicating average particle size over time for each of the peptides in the β - Gal peptide set ( aggregating core sequence SVIIWSLGN ) .

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B) Time-resolved Dynamic Light Scattering (DLS) analysis indicating average particle size over time for each of the peptides in the β-Gal peptide set (aggregating core sequence SVIIWSLGN).


C ) ThT binding kinetics of the β - Gal peptide set . Background - corrected fluorescence over time is shown for 3 independent repeats .

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C) ThT binding kinetics of the β-Gal peptide set. Background-corrected fluorescence over time is shown for 3 independent repeats.


D ) pFTAA binding kinetics of the β - Gal peptide set . Background - corrected fluorescence over time is shown for 3 independent repeats .

D) pFTAA binding kinetics of the β-Gal peptide set. Background-corrected fluorescence over time is shown for 3 independent repeats.


E ) End - point solubility analysis quantifying the concentration of peptide remaining in the supernatant following ultracentrifugation for the β - Gal peptide set . Bars depict mean concentration , whiskers indicate standard deviation ( n = 4 ) . Statistical significance was determined through two - way ANOVA with Bonferroni ' s correction for multiple comparisons . P - values for significant differences are indicated .

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E) End-point solubility analysis quantifying the concentration of peptide remaining in the supernatant following ultracentrifugation for the β-Gal peptide set. Bars depict mean concentration, whiskers indicate standard deviation (n=4). Statistical significance was determined through two-way ANOVA with Bonferroni's correction for multiple comparisons. P-values for significant differences are indicated.


F ) TEM images of the β - Gal peptide set after 3 days incubation . Top panels give an overview image of one square in a TEM grid , lower panel shows enlarged image ( magnification 12000X ) . Scale bars indicate 12 µm ( top row ) or 1 µm ( bottom row ) .

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F) TEM images of the β-Gal peptide set after 3 days incubation. Top panels give an overview image of one square in a TEM grid, lower panel shows enlarged image (magnification 12000X). Scale bars indicate 12 µm (top row) or 1 µm (bottom row).


H ) Violin and boxplots showing the distribution of end - point solubilities across 11 aggregating cores , grouped by GK . The dotted line indicates a concentration of µM , corresponding to completely insoluble peptides .

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H) Violin and boxplots showing the distribution of end-point solubilities across 11 aggregating cores, grouped by GK. The dotted line indicates a concentration of µM, corresponding to completely insoluble peptides.


I ) Solubility analysis of the in vitro solubility screen performed by Niwa et al ( Niwa et al , 2009 ) . Left panel shows combined box - and violin plots depicting the solubility distributions of proteins containing at least one strong APR ( TANGO score > 70 ) not protected by any negative GKs ( red ) versus proteins where all strong APRs are flanked by at least one negative charge ( blue ) . The right panel shows a control analysis , in which proteins were classified by the occurrence of at least one strong APR ( TANGO score > 70 ) not flanked by any positive GKs ( red ) versus proteins where all strong APRs are flanked by at least one positive charge ( blue ) . The upper boxplot whiskers indicate the largest value no further than 1 . 5 times the Inter - Quartile Range ( IQR ) from the upper hinge , the lower whiskers show the smallest value at least 1 . 5 * IQR from the lower hinge . Notches extend 1 . 58 * IQR / sqrt ( n ) from either side of the median and represent a 95 % confidence interval for the median value . Statistical significance was determined through Kruskal - Wallis test with a post - hoc Wilcoxon analysis for pairwise testing . P - values are indicated .

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I) Solubility analysis of the in vitro solubility screen performed by Niwa et al (Niwa et al, 2009). Left panel shows combined box- and violin plots depicting the solubility distributions of proteins containing at least one strong APR (TANGO score > 70) not protected by any negative GKs (red) versus proteins where all strong APRs are flanked by at least one negative charge (blue). The right panel shows a control analysis, in which proteins were classified by the occurrence of at least one strong APR (TANGO score > 70) not flanked by any positive GKs (red) versus proteins where all strong APRs are flanked by at least one positive charge (blue). The upper boxplot whiskers indicate the largest value no further than 1.5 times the Inter-Quartile Range (IQR) from the upper hinge, the lower whiskers show the smallest value at least 1.5 * IQR from the lower hinge. Notches extend 1.58 * IQR / sqrt(n) from either side of the median and represent a 95% confidence interval for the median value. Statistical significance was determined through Kruskal-Wallis test with a post-hoc Wilcoxon analysis for pairwise testing. P-values are indicated.


D ) Time - resolved Dynamic Light Scattering ( DLS ) analysis showing average particle radius over time for the β - Gal peptides flanked by both natural and non - natural GKs , at increasing NaCl concentrations ( indicated in µM ) . Structures of the amino acids flanking the aggregating core are indicated on the right - hand side , the sequence of the aggregating core is indicated below .

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D) Time-resolved Dynamic Light Scattering (DLS) analysis showing average particle radius over time for the β-Gal peptides flanked by both natural and non-natural GKs, at increasing NaCl concentrations (indicated in µM). Structures of the amino acids flanking the aggregating core are indicated on the right-hand side, the sequence of the aggregating core is indicated below.


A ) DnaK binding to cellulose - bound peptide array . Sequences of the aggregating cores are indicated in the top of the panel , the GKs flanking them in each instance are indicated above the blots .

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A) DnaK binding to cellulose-bound peptide array. Sequences of the aggregating cores are indicated in the top of the panel, the GKs flanking them in each instance are indicated above the blots.


C ) Representative Western blot of in vitro translation analysis of folding - incompetent model constructs . Bands show Total ( T ) and Soluble ( S ) fractions after centrifugation at 21 , 000 g for 30 mins . " + Dnak " indicates addition of either DnaK mix - containing DnaK , DnaJ and GrpE .

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C) Representative Western blot of in vitro translation analysis of folding-incompetent model constructs. Bands show Total (T) and Soluble (S) fractions after centrifugation at 21,000 g for 30 mins. "+ Dnak" indicates addition of either DnaK mix - containing DnaK, DnaJ and GrpE.


D ) Bar plots showing mean solubility as determined through quantification of western blot in ( C ) , whiskers indicate standard deviation ( n = 4 ) . Statistically significant differences between the - DnaK and + DnaK conditions were determined through two - way ANOVA with Bonferroni ' s correction for multiple comparisons . P - values for significant differences are indicated .

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D) Bar plots showing mean solubility as determined through quantification of western blot in (C), whiskers indicate standard deviation (n=4). Statistically significant differences between the -DnaK and +DnaK conditions were determined through two-way ANOVA with Bonferroni's correction for multiple comparisons. P-values for significant differences are indicated.


E ) Representative western blot of the solubility analysis of GK variants upon overexpression in E . coli K12 . Bands show Soluble ( Sol ) and Pellet fractions after centrifugation at 17 , 100 g for 15 mins . " - DnaK " indicates expression in E . coli K12 DnaK knockout line , " + Dnak " indicates co - expression of pKJE7 , encoding DnaK , DnaJ and GrpE .

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E) Representative western blot of the solubility analysis of GK variants upon overexpression in E. coli K12. Bands show Soluble (Sol) and Pellet fractions after centrifugation at 17,100 g for 15 mins. "-DnaK" indicates expression in E. coli K12 DnaK knockout line, "+ Dnak" indicates co-expression of pKJE7, encoding DnaK, DnaJ and GrpE.


F ) Barplots showing mean solubility as determined through quantification of western blot bands in ( E ) , whiskers indicate standard deviation ( n = 7 , except for the AA construct , where n = 4 ) . Statistical significance was determined through two - way ANOVA followed by Tukey ' s multiple comparisons test . P - values are indicated .

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F) Barplots showing mean solubility as determined through quantification of western blot bands in (E), whiskers indicate standard deviation (n=7, except for the AA construct, where n=4). Statistical significance was determined through two-way ANOVA followed by Tukey's multiple comparisons test. P-values are indicated.


G ) Structured Illumination Microsopy ( SIM ) images of E . coli after three hours of expression of one of the GK variants . GFP fluorescence is shown in green . " ΔDnaK " indicates expression in E . coli K12 DnaK knockout line , " + Dnak " indicates co - expression of pKJE7 , encoding DnaK , DnaJ and GrpE .

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G) Structured Illumination Microsopy (SIM) images of E. coli after three hours of expression of one of the GK variants. GFP fluorescence is shown in green. "ΔDnaK" indicates expression in E. coli K12 DnaK knockout line, "+ Dnak" indicates co-expression of pKJE7, encoding DnaK, DnaJ and GrpE.


A Western blot analysis of B16F1 WT and two independent XIAPKO cell lines . Actin was used as a loading control .

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A Western blot analysis of B16F1 WT and two independent XIAPKO cell lines. Actin was used as a loading control.


B Subcutaneous melanoma tumour growth in BL / 6 WT mice ( n = 5 ) using B16F1 WT and XIAPKO ( clone1 and 2 ) .

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B Subcutaneous melanoma tumour growth in BL/6 WT mice (n=5) using B16F1 WT and XIAPKO (clone1 and 2).


C Representative H & E ( scale bar 50 µm ) and fluorescent images of cl - Caspase 3 staining in B16 WT and XIAPKO tumours . Nuclei were stained with DAPI ( blue ) . Scale bar 100 µm . Quantification thereof ( right panel ) . The average of cl - Caspase 3 positive cells from 5 randomly selected areas of the tumour ( 5 mice per genotype ) was represented . Dots represent individual mice ( n = 5 ) .

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C Representative H&E (scale bar 50 µm) and fluorescent images of cl-Caspase 3 staining in B16 WT and XIAPKO tumours. Nuclei were stained with DAPI (blue). Scale bar 100 µm. Quantification thereof (right panel). The average of cl-Caspase 3 positive cells from 5 randomly selected areas of the tumour (5 mice per genotype) was represented. Dots represent individual mice (n=5).


D qRT - PCR analysis ( PrimePCRTM Assay ) of different cyto - / chemokines in B16F1 WT cells plotted vs B16F1 - XIAPKO cells ( clone1 and 2 ) . Samples that are at least 2 - fold up - regulated are shown in red , samples that are at least 2 - fold down - regulated are shown in green .

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D qRT-PCR analysis (PrimePCRTM Assay) of different cyto-/chemokines in B16F1 WT cells plotted vs B16F1-XIAPKO cells (clone1 and 2). Samples that are at least 2-fold up-regulated are shown in red, samples that are at least 2-fold down-regulated are shown in green.


E IL8 measurement in the supernatant of the indicated cell lines after 48 h transfection with siScr ( control ) or siXIAP and Western blot analysis of the corresponding cell lysates ( bottom ) .

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E IL8 measurement in the supernatant of the indicated cell lines after 48 h transfection with siScr (control) or siXIAP and Western blot analysis of the corresponding cell lysates (bottom).


F IL8 measurement in the supernatant of the indicated cell lines after 24 h of seeding the cells .

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F IL8 measurement in the supernatant of the indicated cell lines after 24 h of seeding the cells.


G qRT - PCR analysis ( PrimePCRTM Assay ) of different cyto - / chemokines in HEK293T cells overexpressing myc - XIAP plotted vs HEK293T cells overexpressing myc - XIAPΔRING . Samples that are at least 2 - fold up - regulated are shown in red , samples that are at least 2 - fold down - regulated are shown in green .

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G qRT-PCR analysis (PrimePCRTM Assay) of different cyto-/chemokines in HEK293T cells overexpressing myc-XIAP plotted vs HEK293T cells overexpressing myc-XIAPΔRING. Samples that are at least 2-fold up-regulated are shown in red, samples that are at least 2-fold down-regulated are shown in green.


H Human cytokines array analysis of HEK293T cells transfected with myc - XIAP or myc - XIAPΔRING . Medium was changed after 16 h and the supernatant was collected after 48 h . Pre - spotted nitrocellulose membranes were incubated with the supernatants overnight at 4 ° C .

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H Human cytokines array analysis of HEK293T cells transfected with myc-XIAP or myc-XIAPΔRING. Medium was changed after 16 h and the supernatant was collected after 48 h. Pre-spotted nitrocellulose membranes were incubated with the supernatants overnight at 4°C.


A Western blot analysis of cell lysates of BLM and BLM - XIAPKO cells ( input ) and XIAP - IP . Data are representative of two experiments .

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A Western blot analysis of cell lysates of BLM and BLM-XIAPKO cells (input) and XIAP-IP. Data are representative of two experiments.


B Confocal microscopic analysis of transfected HCT116 cells with GFP - XIAP for 16 h and stained for TAB1 or RIPK2 ( red ) . Nuclei were stained with DAPI ( blue ) .

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B Confocal microscopic analysis of transfected HCT116 cells with GFP-XIAP for 16 h and stained for TAB1 or RIPK2 (red). Nuclei were stained with DAPI (blue).


C Cell - free ubiquitylation assay of recombinant GST - RIPK2 by Flag - XIAP after addition of respective E1 / E2 and recombinant ubiquitin . Poly - ubiquitylated RIPK2 and XIAP were detected by Western blotting .

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C Cell-free ubiquitylation assay of recombinant GST-RIPK2 by Flag-XIAP after addition of respective E1/E2 and recombinant ubiquitin. Poly-ubiquitylated RIPK2 and XIAP were detected by Western blotting.


D Western blot analysis of cell lysates of BLM , BLM - XIAPKO , SK - Mel28 , SK - Mel28 - XIAPKO , B16 , and B16 - XIAPKO cells ( input ) and RIPK2 - IP . Poly - ubiquitin is detected using anti - ubiquitin antibody .

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D Western blot analysis of cell lysates of BLM, BLM-XIAPKO, SK-Mel28, SK-Mel28-XIAPKO, B16, and B16-XIAPKO cells (input) and RIPK2-IP. Poly-ubiquitin is detected using anti-ubiquitin antibody.


E IL8 measurement in the supernatant of transfected BLM ( left panel ) or SK - Mel28 ( right panel ) cells with siScr ( control ) , siXIAP , siTAB1 or siRIPK2 after 48 h . Western blot analysis of the respective cell lysates of BLM cells ( left bottom ) or SK - Mel28 cells ( right bottom ) transfected with the indicated siRNA .

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E IL8 measurement in the supernatant of transfected BLM (left panel) or SK-Mel28 (right panel) cells with siScr (control), siXIAP, siTAB1 or siRIPK2 after 48 h. Western blot analysis of the respective cell lysates of BLM cells (left bottom) or SK-Mel28 cells (right bottom) transfected with the indicated siRNA.


F IL8 measurement in the supernatant of BLM or SK - Mel28 treated with 2 µM d89 ( BIR2 antagonist ) or DMSO as a negative control for 24 h .

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F IL8 measurement in the supernatant of BLM or SK-Mel28 treated with 2 µM d89 (BIR2 antagonist) or DMSO as a negative control for 24 h.


G IL8 measurement in the supernatant of BLM or SK - Mel28 treated with 20 µM birinapant ( Bir ) , 2 µM BV6 ( both BIR3 antagonist ) or DMSO as a negative control for 24 h .

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G IL8 measurement in the supernatant of BLM or SK-Mel28 treated with 20 µM birinapant (Bir), 2 µM BV6 (both BIR3 antagonist) or DMSO as a negative control for 24 h.


A Intensities of CD177 and XIAP immunostainings in tumour sections from 55 patients were visually scored . The numbers of melanomas with different intensities were plotted into the table as a percentage of the total . Specific staining intensity for XIAP and CD177 , corresponding to relative amounts of infiltrated neutrophils , was arbitrarily set as the following : - , not expressed ; + , low ; + + , moderate ; and + + + , strong expression . Representative IHC pictures ( right panel ) .

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A Intensities of CD177 and XIAP immunostainings in tumour sections from 55 patients were visually scored. The numbers of melanomas with different intensities were plotted into the table as a percentage of the total. Specific staining intensity for XIAP and CD177, corresponding to relative amounts of infiltrated neutrophils, was arbitrarily set as the following: -, not expressed; +, low; ++, moderate; and +++, strong expression. Representative IHC pictures (right panel).


B Representative IF ( neutrophils staining ) in B16F1 WT , XIAPKO , TAB1KO or RIPK2KO tumours ( Scale bar 50 µm ) and quantification of Ly6G + cells . The sum of Ly6G - positive cells from 5 randomly selected areas of the tumour ( 5 mice per genotype ) was represented . Dots represent individual mice ( n = 5 ) . C B16F1 WT , XIAPKO ( clone 2 ) subcutaneous melanoma tumour growth in BL / 6 WT mice ( n = 5 ) . Mice were intra - peritoneal injected with IgG antibody as a negative control or Ly6G antibody 1 day prior to the subcutaneous injection of the B16F1 WT or XIAPKO cells .

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B Representative IF (neutrophils staining) in B16F1 WT, XIAPKO, TAB1KO or RIPK2KO tumours (Scale bar 50 µm) and quantification of Ly6G+ cells. The sum of Ly6G-positive cells from 5 randomly selected areas of the tumour (5 mice per genotype) was represented. Dots represent individual mice (n=5). C B16F1 WT, XIAPKO (clone 2) subcutaneous melanoma tumour growth in BL/6 WT mice (n=5). Mice were intra-peritoneal injected with IgG antibody as a negative control or Ly6G antibody 1 day prior to the subcutaneous injection of the B16F1 WT or XIAPKO cells.


D Representative IF ( neutrophils ) scale bar 50 µm and quantification of Ly6G + cells in tumours from C . The sum of Ly6G - positive cells from 5 randomly selected areas of the tumour ( 4 mice per genotype ) was represented . Dots represent individual mice ( n = 4 ) .

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D Representative IF (neutrophils) scale bar 50 µm and quantification of Ly6G+ cells in tumours from C. The sum of Ly6G-positive cells from 5 randomly selected areas of the tumour (4 mice per genotype) was represented. Dots represent individual mice (n=4).


Gene - trap - based genetic screen in haploid human cells for regulators of p38 kinase activation ( p38 Thr180 / Tyr182 phosphorylation ) in response to hyperosmotic shock ( 500 mM Sorbitol , 1 h ) . Per gene ( dots ) , the frequency of gene - trap insertions in the ' high ' phospho - p38 population divided by the frequency of insertions in the ' low ' population is plotted as mutation index ( y - axis ) against the total number of insertions assigned to the gene ( x - axis ) . Significant negative and positive regulators are colored in orange and blue , respectively ( two - sided Fisher ' s exact test , false discovery rate - corrected , p ≤ 0 . 05 ) .

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Gene-trap-based genetic screen in haploid human cells for regulators of p38 kinase activation (p38 Thr180/Tyr182 phosphorylation) in response to hyperosmotic shock (500 mM Sorbitol, 1 h). Per gene (dots), the frequency of gene-trap insertions in the 'high' phospho-p38 population divided by the frequency of insertions in the 'low' population is plotted as mutation index (y-axis) against the total number of insertions assigned to the gene (x-axis). Significant negative and positive regulators are colored in orange and blue, respectively (two-sided Fisher's exact test, false discovery rate-corrected, p≤0.05).


Human U2OS cells and cells deleted for ZAK ( ∆ ZAK ) were treated with anisomycin ( 1 h ) or 500 mM sorbitol ( 1 h ) . Lysates were analyzed by immunoblotting as indicated .

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Human U2OS cells and cells deleted for ZAK (∆ZAK) were treated with anisomycin (1 h) or 500 mM sorbitol (1 h). Lysates were analyzed by immunoblotting as indicated.


Unique gene - trap insertions ( dots ) mapped to the genomic ZAK locus ( x - axis ) identified in the low ( blue ) and high ( orange ) channel of two individual haploid genetic screens for stress - induced p38 activation and Anisomycin ( Brockmann et al . , 2017 ) ) . The total number of identified insertions was similar for each channel within the individual screens . For visual purposes , insertion dots were spread on the y - axis and exons in the ZAK gene body schematic have been scaled up ( compared to introns ) .

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Unique gene-trap insertions (dots) mapped to the genomic ZAK locus (x-axis) identified in the low (blue) and high (orange) channel of two individual haploid genetic screens for stress-induced p38 activation and Anisomycin(Brockmann et al., 2017)). The total number of identified insertions was similar for each channel within the individual screens. For visual purposes, insertion dots were spread on the y-axis and exons in the ZAK gene body schematic have been scaled up (compared to introns).


U2OS / S - HA - ZAKβ cells were induced for expression with doxycycline ( DOX ) and subjected to the indicated drugs and treatments ( 1 h ) . Lysates were analyzed by immunoblotting with the indicated antibodies . Sorb . , sorbitol ; Ani . , anisomycin ; Ars . , arsenite .

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U2OS/S-HA-ZAKβ cells were induced for expression with doxycycline (DOX) and subjected to the indicated drugs and treatments (1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. Sorb., sorbitol; Ani., anisomycin; Ars., arsenite.


U2OS cells stably expressing WT and kinase - dead ( KD ) versions of S - HA - ZAKβ were pre - treated with ZAK inhibitor ( ZAKi - 0 . 5 h ) and 500 mM sorbitol ( 1 h ) as indicated . Whole Cell Extracts ( WCE ) were analyzed by immunoblotting , and strep - pulldown material was used in a kinase assay , separated by SDS - PAGE and analyzed by autoradiography .

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U2OS cells stably expressing WT and kinase-dead (KD) versions of S-HA-ZAKβ were pre-treated with ZAK inhibitor (ZAKi - 0.5 h) and 500 mM sorbitol (1 h) as indicated. Whole Cell Extracts (WCE) were analyzed by immunoblotting, and strep-pulldown material was used in a kinase assay, separated by SDS-PAGE and analyzed by autoradiography.


U2OS and ∆ ZAK cells rescued with S - HA - ZAKβ , were DOX - induced and treated with 500 mM sorbitol ( 1 h ) as indicated . Lysates were analyzed by immunoblotting with the indicated antibodies .

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U2OS and ∆ZAK cells rescued with S-HA-ZAKβ, were DOX-induced and treated with 500 mM sorbitol (1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies.


U2OS and ∆ ZAK cells were transfected with the indicated siRNAs and treated with sorbitol ( 500 mM , 1 h ) as indicated . Lysates were analyzed by immunoblotting with the indicated antibodies . T1 , TAK1 ; M2 , MEKK2 .

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U2OS and ∆ZAK cells were transfected with the indicated siRNAs and treated with sorbitol (500 mM, 1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies. T1, TAK1; M2, MEKK2.


U2OS cells stably expressing Strep - HA - ZAKβ or - ZAKα were treated with sorbitol ( 500 mM , 1 h ) as indicated . Cells were fixed , immunostained with HA antibody and counter - stained with DAPI . Data information : scale bars , 10 μm .

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U2OS cells stably expressing Strep-HA-ZAKβ or -ZAKα were treated with sorbitol (500 mM, 1 h) as indicated. Cells were fixed, immunostained with HA antibody and counter-stained with DAPI. Data information: scale bars, 10 μm.


U2OS cells stably expressing ZAKβ - GFP were treated with sorbitol ( 500 mM , 1 h ) , pre - extracted , fixed and immunostained with Lamin A / C antibody . Lower right : Intensity distribution graph showing fluorescence intensities along the magenta line . As in ( B ) except that Hela cells were transfected with ZAKbeta - GFP and immunostained with 53BP1 antibody . Data information : scale bars , 10 μm .

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U2OS cells stably expressing ZAKβ-GFP were treated with sorbitol (500 mM, 1 h), pre-extracted, fixed and immunostained with Lamin A/C antibody. Lower right: Intensity distribution graph showing fluorescence intensities along the magenta line. As in (B) except that Hela cells were transfected with ZAKbeta-GFP and immunostained with 53BP1 antibody. Data information: scale bars, 10 μm.


Cells from ( B ) were imaged by live cell fluorescence microscopy . Sorbitol ( final concentration 500 mM ) was added after acquisition of the first frame . Data information : scale bars , 10 μm .

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Cells from (B) were imaged by live cell fluorescence microscopy. Sorbitol (final concentration 500 mM) was added after acquisition of the first frame. Data information: scale bars, 10 μm.


As in ( D ) , except that cells were co - transfected with mCherry - FAK . Inserts show higher magnification of the yellow and red regions , white arrow indicates the direction of stress fibers . Data information : scale bars , 10 μm .

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As in (D), except that cells were co-transfected with mCherry-FAK. Inserts show higher magnification of the yellow and red regions, white arrow indicates the direction of stress fibers. Data information: scale bars, 10 μm.


U2OS cells were transfected with constructs from ( F ) and imaged by live cell fluorescence microscopy . Sorbitol ( final concentration 500 mM ) was added after acquisition of the first frame . Data information : scale bars , 10 μm .

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U2OS cells were transfected with constructs from (F) and imaged by live cell fluorescence microscopy. Sorbitol (final concentration 500 mM) was added after acquisition of the first frame. Data information: scale bars, 10 μm.


U2OS and ∆ ZAK cells stably expressing the indicated Strep - HA - tagged ZAKβ constructs were treated with sorbitol ( 500 mM , 5 min ) . Lysates were analyzed by immunoblotting with the indicated antibodies .

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U2OS and ∆ZAK cells stably expressing the indicated Strep-HA-tagged ZAKβ constructs were treated with sorbitol (500 mM, 5 min). Lysates were analyzed by immunoblotting with the indicated antibodies.