Primary brown adipocytes were transfected with MPC1 siRNA ( MPC1 KD ) or Scramble RNA ( Scramble ) . ( C ) Representative Western Blot analysis of MPC1 , MPC2 and actin . ( D ) Quantification of MPC1 and MPC2 expression normalized to actin from Western Blots in ( C ) from n = 3 individual experiments .

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Primary brown adipocytes were transfected with MPC1 siRNA (MPC1 KD) or Scramble RNA (Scramble). (C) Representative Western Blot analysis of MPC1, MPC2 and actin. (D) Quantification of MPC1 and MPC2 expression normalized to actin from Western Blots in (C) from n=3 individual experiments.


Primary brown adipocytes were transfected with MPC1 siRNA ( MPC1 KD ) or Scramble RNA ( Scramble ) . ( E ) mRNA levels of MPC1 and MPC2 from n = 3 individual experiments .

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Primary brown adipocytes were transfected with MPC1 siRNA (MPC1 KD) or Scramble RNA (Scramble). (E) mRNA levels of MPC1 and MPC2 from n=3 individual experiments.


Primary brown adipocytes were transfected with MPC1 siRNA ( MPC1 KD ) or Scramble RNA ( Scramble ) . ( F ) Representative OCR traces of differentiated primary brown adipocytes averaging 6 technical replicates . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine . Norepinephrine ( NE ; 1 µM ) , oligomycin A ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) and antimycin A ( AA ; 4 µM ) were injected where indicated . ( G ) Quantification of basal , NE - stimulated and Eto - sensitive OCR as shown in ( F ) from n = 7 individual experiments . Data were normalized to Scramble RNA for each individual experiment . ( H ) Quantification of basal OCR in response to 100 nM UK5099 treatment in Scramble RNA of MPC1 siRNA transfected cells from n = 5 individual experiments . Data were normalized to vehicle for each experiment .

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Primary brown adipocytes were transfected with MPC1 siRNA (MPC1 KD) or Scramble RNA (Scramble). (F) Representative OCR traces of differentiated primary brown adipocytes averaging 6 technical replicates. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (G) Quantification of basal, NE-stimulated and Eto-sensitive OCR as shown in (F) from n=7 individual experiments. Data were normalized to Scramble RNA for each individual experiment. (H) Quantification of basal OCR in response to 100 nM UK5099 treatment in Scramble RNA of MPC1 siRNA transfected cells from n=5 individual experiments. Data were normalized to vehicle for each experiment.


( A , B ) Requirement for glutamine in UK5099 - induced energy expenditure . Primary brown adipocytes were pre - treated with vehicle ( DMSO ) or 100 nM UK5099 for 2 h . OCR were measured in presence of either 5 mM glucose and 3 mM glutamine or 5 mM glucose alone . Norepinephrine ( NE ; 1 µM ) , oligomycin A ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) and antimycin A ( AA ; 4 µM ) were injected where indicated . ( A ) Representative OCR traces averaging 6 technical replicates . ( B ) Quantification of non - stimulated OCR , NE - stimulated OCR , ATP - linked OCR , mitochondrial proton leak and etomoxir - sensitive OCR after vehicle or UK5099 treatment as shown in ( A ) from n = 4 individual experiments .

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(A,B) Requirement for glutamine in UK5099-induced energy expenditure. Primary brown adipocytes were pre-treated with vehicle (DMSO) or 100 nM UK5099 for 2 h. OCR were measured in presence of either 5 mM glucose and 3 mM glutamine or 5 mM glucose alone. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (A) Representative OCR traces averaging 6 technical replicates. (B) Quantification of non-stimulated OCR, NE-stimulated OCR, ATP-linked OCR, mitochondrial proton leak and etomoxir-sensitive OCR after vehicle or UK5099 treatment as shown in (A) from n=4 individual experiments.


( C ) Effect of glutaminase inhibitor on UK5099 - induced energy expenditure . Primary brown adipocytes were pre - treated with either vehicle ( DMSO ) , 100 nM UK5099 , or 100 nM UK5099 + 2 . 5 µM CB839 for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle , UK5099 or UK5099 + CB839 from n = 5 individual experiments . Data were normalized to vehicle for each experiment .

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(C) Effect of glutaminase inhibitor on UK5099-induced energy expenditure. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, or 100 nM UK5099 + 2.5 µM CB839 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099 or UK5099 + CB839 from n=5 individual experiments. Data were normalized to vehicle for each experiment.


( D ) Effect of MPC inhibition on glutamate / aspartate ratio measurement . Glutamate / aspartate ratio was quantified using GC - MS as a measure of glutamine catabolism . Primary brown adipocytes were treated for 24 h with vehicle ( DMSO ) or 10 µM UK5099 from n = 6 individual experiments . * *

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(D) Effect of MPC inhibition on glutamate/aspartate ratio measurement. Glutamate/aspartate ratio was quantified using GC-MS as a measure of glutamine catabolism. Primary brown adipocytes were treated for 24 h with vehicle (DMSO) or 10 µM UK5099 from n=6 individual experiments. **


( B ) Effect of transaminase inhibition on UK5099 - induced energy expenditure . Brown adipocytes were pre - treated with vehicle ( DMSO ) , 50 - 100 nM UK5099 , 1 mM aminooxyacetic acid ( AOA ) , or a combination of UK5099 and AOA for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of the tested compounds . Data show quantification of non - stimulated OCR from n = 5 individual experiments .

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(B) Effect of transaminase inhibition on UK5099-induced energy expenditure. Brown adipocytes were pre-treated with vehicle (DMSO), 50-100 nM UK5099, 1 mM aminooxyacetic acid (AOA), or a combination of UK5099 and AOA for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of the tested compounds. Data show quantification of non-stimulated OCR from n=5 individual experiments.


( C - E ) Effect OGC1 downregulation on UK5099 - induced energy expenditure . Primary brown adipocytes were transduced with Scramble RNA ( Scramble ) or shOGC1 ( OGC1 KD ) adenovirus . Cells were pre - treated for 2 h with vehicle ( DMSO ) or 100 nM UK5099 before OCR measurements . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099 . Norepinephrine ( NE ; 1 µM , ) oligomycin A ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) and antimycin A ( AA ; 4 µM ) were injected where indicated . ( C ) Representative OCR traces averaging 4 technical replicates . ( D ) Quantification of non - stimulated OCR as measured in ( C ) from n = 6 individual experiments . ( E ) Quantification of Eto - sensitive OCR as measured in ( C ) from n = 6 individual experiments . Data were normalized to vehicle for each experiment .

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(C-E) Effect OGC1 downregulation on UK5099-induced energy expenditure. Primary brown adipocytes were transduced with Scramble RNA (Scramble) or shOGC1 (OGC1 KD) adenovirus. Cells were pre-treated for 2 h with vehicle (DMSO) or 100 nM UK5099 before OCR measurements. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Norepinephrine (NE; 1 µM,) oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (C) Representative OCR traces averaging 4 technical replicates. (D) Quantification of non-stimulated OCR as measured in (C) from n=6 individual experiments. (E) Quantification of Eto-sensitive OCR as measured in (C) from n=6 individual experiments. Data were normalized to vehicle for each experiment.


( F - H ) Effect of Aralar1 downregulation on UK5099 - induced energy expenditure . Primary brown adipocytes were transfected with Scramble RNA ( Scramble ) or siRNA for Aralar1 ( Aralar1 KD ) . Cells were pre - treated for 2 h with vehicle ( DMSO ) or 100 nM UK5099 before OCR measurements . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099 . Norepinephrine ( NE ; 1 µM ) , oligomycin a ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) and antimycin a ( AA ; 4 µM ) were injected where indicated . ( F ) Representative OCR traces averaging 4 technical replicates . ( G ) Quantification of non - stimulated OCR as measured ( G ) from n = 4 individual experiments . Data were normalized to vehicle for each experiment . ( H ) Quantification of Eto - sensitive OCR as measured in ( F ) from n = 4 individual experiments .

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(F-H) Effect of Aralar1 downregulation on UK5099-induced energy expenditure. Primary brown adipocytes were transfected with Scramble RNA (Scramble) or siRNA for Aralar1 (Aralar1 KD). Cells were pre-treated for 2 h with vehicle (DMSO) or 100 nM UK5099 before OCR measurements. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Norepinephrine (NE; 1 µM), oligomycin a (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin a (AA; 4 µM) were injected where indicated. (F) Representative OCR traces averaging 4 technical replicates. (G) Quantification of non-stimulated OCR as measured (G) from n=4 individual experiments. Data were normalized to vehicle for each experiment. (H) Quantification of Eto-sensitive OCR as measured in (F) from n=4 individual experiments.


( I ) Brown adipocytes transfected with Scramble RNA or Aralar1 siRNA were treated for 24 hours with vehicle ( DMSO ) or 10 µM UK5099 . Data shows quantification of the ratio of aspartate to glutamate abundance as measured by GC - MS from n = 3 individual experiments .

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(I) Brown adipocytes transfected with Scramble RNA or Aralar1 siRNA were treated for 24 hours with vehicle (DMSO) or 10 µM UK5099. Data shows quantification of the ratio of aspartate to glutamate abundance as measured by GC-MS from n=3 individual experiments.


( A , B ) Effect of UK5099 on energy expenditure contributed by ATP demand and by mitochondrial proton leak in non - stimulated brown adipocytes . Primary brown adipocytes were pre - treated with vehicle ( DMSO ) or 100 nM UK5099 for 2 hours . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099 . Oligomycin A ( Oligo ; 4 µM ) , mitochondrial uncoupler FCCP ( 2 µM ) and antimycin A ( AA ; 4 µM ) were injected where indicated . ( A ) Representative OCR traces averaging 6 technical replicates . ( B ) Quantification of mitochondrial proton leak and ATP - linked OCR ( oligomycin sensitive ) as measured in ( A ) from n = 9 individual experiments . Data were normalized to vehicle for each individual experiment .

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(A,B) Effect of UK5099 on energy expenditure contributed by ATP demand and by mitochondrial proton leak in non-stimulated brown adipocytes. Primary brown adipocytes were pre-treated with vehicle (DMSO) or 100 nM UK5099 for 2 hours. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Oligomycin A (Oligo; 4 µM), mitochondrial uncoupler FCCP (2 µM) and antimycin A (AA; 4 µM) were injected where indicated. (A) Representative OCR traces averaging 6 technical replicates. (B) Quantification of mitochondrial proton leak and ATP-linked OCR (oligomycin sensitive) as measured in (A) from n=9 individual experiments. Data were normalized to vehicle for each individual experiment.


( C ) To determine the contribution of ATP demand to increased energy expenditure under UK5099 cells were permeabilized and cytosolic ADP concentrations were clamped . OCR were measured in the presence of 5 mM pyruvate , 3 mM malate , 0 . 1 mM palmitoyl - CoA , 0 . 5 mM carnitine and 5 mM ADP . Cells were treated with either 100 nM , 1 µM or 10 µM UK5099 . Data shows maximal state 3 OCR normalized to maximal complex 4 activity from n = 3 individual experiments .

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(C) To determine the contribution of ATP demand to increased energy expenditure under UK5099 cells were permeabilized and cytosolic ADP concentrations were clamped. OCR were measured in the presence of 5 mM pyruvate, 3 mM malate, 0.1 mM palmitoyl-CoA, 0.5 mM carnitine and 5 mM ADP. Cells were treated with either 100 nM, 1 µM or 10 µM UK5099. Data shows maximal state 3 OCR normalized to maximal complex 4 activity from n=3 individual experiments.


( E ) Effect of MPC inhibition on glycerol 3 - phosphate ( G3P ) and dihydroxyacetone phosphate ( DHAP ) generation . G3P / DHAP is used as a measure of acylglycerol synthesis . Brown adipocytes were treated with either vehicle , 100 nM UK5099 or 1 µM norepinephrine ( NE ) for 24 h from n = 4 individual experiments .

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(E) Effect of MPC inhibition on glycerol 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) generation. G3P/DHAP is used as a measure of acylglycerol synthesis. Brown adipocytes were treated with either vehicle, 100 nM UK5099 or 1 µM norepinephrine (NE) for 24 h from n=4 individual experiments.


( F , G ) Contribution of lipolysis to UK5099 induced energy demand . Primary brown adipocytes were pre - treated with either vehicle ( DMSO ) , 100 nM UK5099 , 40 µM Atglistatin or a combination of Atglistatin with UK5099 for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle , UK5099 , or Atglistatin . ( F ) Quantification of non - stimulated OCR from n = 4 individual experiments . ( G ) Quantification of ATP - linked respiration from n = 4 individual experiments . Data were normalized to vehicle for each individual experiment .

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(F,G) Contribution of lipolysis to UK5099 induced energy demand. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, 40 µM Atglistatin or a combination of Atglistatin with UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099, or Atglistatin. (F) Quantification of non-stimulated OCR from n=4 individual experiments. (G) Quantification of ATP-linked respiration from n=4 individual experiments. Data were normalized to vehicle for each individual experiment.


( H , I ) Contribution of TAG synthesis to UK5099 induced energy demand . Primary brown adipocytes were pre - treated with either vehicle ( DMSO ) , 100 nM UK5099 , 1 µM DGAT1 and 1 µM DGAT2 or a combination of DGAT1 / 2 inhibitors with UK5099 for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle , UK5099 , DGAT1 / 2 inhibitors , or UK5099 + DGAT1 / 2 inhibitors . ( H ) Quantification of non - stimulated OCR from n = 6 individual experiments . ( I ) Quantification of ATP - linked OCR from n = 6 individual experiments . Data were normalized to vehicle for each individual experiment .

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(H,I) Contribution of TAG synthesis to UK5099 induced energy demand. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, 1 µM DGAT1 and 1 µM DGAT2 or a combination of DGAT1/2 inhibitors with UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099, DGAT1/2 inhibitors, or UK5099 + DGAT1/2 inhibitors. (H) Quantification of non-stimulated OCR from n=6 individual experiments. (I) Quantification of ATP-linked OCR from n=6 individual experiments. Data were normalized to vehicle for each individual experiment.


( J , K ) Contribution of ACS to UK5099 induced energy demand . Primary brown adipocytes were pre - treated with either vehicle ( DMSO ) , 100 nM UK5099 , 5 µM Triacsin C or a combination of 5 µM Triacsin C with UK5099 for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle , UK5099 , Triacsin C , or UK5099 + Triacsin C . ( J ) Quantification of non - stimulated OCR from n = 4 individual experiments . ( K ) Quantification of ATP - linked OCR from n = 4 individual experiments . Data were normalized to vehicle for each individual experiment .

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(J,K) Contribution of ACS to UK5099 induced energy demand. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, 5 µM Triacsin C or a combination of 5 µM Triacsin C with UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099, Triacsin C, or UK5099 + Triacsin C. (J) Quantification of non-stimulated OCR from n=4 individual experiments. (K) Quantification of ATP-linked OCR from n=4 individual experiments. Data were normalized to vehicle for each individual experiment.


( A ) Dually labeled D . discoideum amoebae producing MCS components fused to GFP , and either calnexin ( CnxA ) - mCherry ( pAW012 ) , P4C - mCherry ( pWS032 ) , or AmtA - mCherry were infected ( MOI 5 , 2 h ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) , fixed with 4 % PFA , and imaged by confocal fluorescence microscopy . Merged images are shown . Scale bars : 3 μm ( insets : 1 μm ) .

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(A) Dually labeled D. discoideum amoebae producing MCS components fused to GFP, and either calnexin (CnxA)-mCherry (pAW012), P4C-mCherry (pWS032), or AmtA-mCherry were infected (MOI 5, 2 h) with mCerulean-producing L. pneumophila JR32 (pNP99), fixed with 4 % PFA, and imaged by confocal fluorescence microscopy. Merged images are shown. Scale bars: 3 μm (insets: 1 μm).


( B - D ) The Pearson ' s correlation coefficients were generated using Coloc 2 from Fiji ( ImageJ ) and are shown for MCS components fused to GFP with respect to ( B ) CnxA - mCherry ( n = 146 ) , ( C ) P4C - mCherry ( n = 138 ) , or ( D ) AmtA - mCherry ( n = 159 ) . Data represent mean and SEM of three independent biological replicates ( statistics refer to JR32 - infected D . discoideum producing GFP - Sac1 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) .

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(B-D) The Pearson's correlation coefficients were generated using Coloc 2 from Fiji (ImageJ) and are shown for MCS components fused to GFP with respect to (B) CnxA-mCherry (n=146), (C) P4C-mCherry (n=138), or (D) AmtA-mCherry (n=159). Data represent mean and SEM of three independent biological replicates (statistics refer to JR32-infected D. discoideum producing GFP-Sac1; *P<0.05; **P<0.01; ***P<0.001, Student`s t-test).


( A ) Dually labeled D . discoideum Ax3 producing mCherry - Vap ( pSV048 ) and OSBP8 - GFP ( pMIB89 ) or GFP - OSBP11 ( pMIB39 ) were infected ( MOI 5 , 1 - 8 h ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm ( insets : 1 μm ) .

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(A) Dually labeled D. discoideum Ax3 producing mCherry-Vap (pSV048) and OSBP8-GFP (pMIB89) or GFP-OSBP11 (pMIB39) were infected (MOI 5, 1-8 h) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm (insets: 1 μm).


( B ) The Pearson ' s correlation coefficient was generated using Coloc 2 from Fiji ( ImageJ ) and is shown for OSBP8 or OSBP11 fused to GFP with respect to mCherry - Vap ( n = 100 - 150 each per time point ) . Data represent mean and SEM of three independent biological replicates ( * * P < 0 . 01 , Student ` s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The Pearson's correlation coefficient was generated using Coloc 2 from Fiji (ImageJ) and is shown for OSBP8 or OSBP11 fused to GFP with respect to mCherry-Vap (n=100-150 each per time point). Data represent mean and SEM of three independent biological replicates (**P<0.01, Student`s t-test).


( A ) D . discoideum Ax3 , or the ∆ vap , ∆ osbG , ∆ osbH , or ∆ osbK mutant strains producing GFP ( pDM317 ) or GFP fusions of the MCS components were infected ( MOI 1 ) with mCherry - producing L . pneumophila JR32 ( pNP102 ) , and intracellular replication was assessed by relative fluorescence units ( RFU ) . Mean and SEM of three independent biological replicates are shown ( statistics refer to JR32 infected D . discoideum Ax3 producing GFP ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) . To improve clarity , the different mutant strains are shown in separate graphs , each depicting the same data for Ax3 / GFP .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) D. discoideum Ax3, or the ∆vap, ∆osbG, ∆osbH, or ∆osbK mutant strains producing GFP (pDM317) or GFP fusions of the MCS components were infected (MOI 1) with mCherry-producing L. pneumophila JR32 (pNP102), and intracellular replication was assessed by relative fluorescence units (RFU). Mean and SEM of three independent biological replicates are shown (statistics refer to JR32 infected D. discoideum Ax3 producing GFP; *P<0.05; **P<0.01; ***P<0.001, Student`s t-test). To improve clarity, the different mutant strains are shown in separate graphs, each depicting the same data for Ax3/GFP.


( B ) D . discoideum Ax3 producing CnxA - GFP ( pAW016 ) , GFP - Sac1 ( pLS037 ) , GFP - Sac1 _ C383S ( pSV015 ) or GFP - Sac1 _ ΔTMD ( pSV034 ) were infected ( MOI 1 ) with mCherry - producing L . pneumophila JR32 ( pNP102 ) , and intracellular replication was assessed by relative fluorescence units ( RFU ) . Mean and SEM of three independent biological replicates are shown ( statistics refer to JR32 infected D . discoideum Ax3 producing GFP - Sac1 ; * * * P < 0 . 001 , Student ` s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) D. discoideum Ax3 producing CnxA-GFP (pAW016), GFP-Sac1 (pLS037), GFP-Sac1_C383S (pSV015) or GFP-Sac1_ΔTMD (pSV034) were infected (MOI 1) with mCherry-producing L. pneumophila JR32 (pNP102), and intracellular replication was assessed by relative fluorescence units (RFU). Mean and SEM of three independent biological replicates are shown (statistics refer to JR32 infected D. discoideum Ax3 producing GFP-Sac1; ***P<0.001, Student`s t-test).


( C ) RAW 264 . 7 macrophages were treated with increasing concentrations of OSW - 1 ( 0 - 100 nM , 1 - 60 h ) , infected ( MOI 1 ) with GFP - producing L . pneumophila JR32 ( pNT28 ) , and intracellular replication was assessed by relative fluorescence units ( RFU ) . Mean and SEM of three independent biological replicates are shown ( statistics refer to infected cells treated with 0 nM OSW - 1 ; * P < 0 . 05 ; * * * P < 0 . 001 , Student ` s t - test ) .

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

(C) RAW 264.7 macrophages were treated with increasing concentrations of OSW-1 (0-100 nM, 1-60 h), infected (MOI 1) with GFP-producing L. pneumophila JR32 (pNT28), and intracellular replication was assessed by relative fluorescence units (RFU). Mean and SEM of three independent biological replicates are shown (statistics refer to infected cells treated with 0 nM OSW-1; *P<0.05; ***P<0.001, Student`s t-test).


( A ) Dually labeled D . discoideum Ax3 , ∆ vap , ∆ osbG , ∆ osbH , ∆ osbK or ∆ sey1 mutants producing P4C - mCherry ( pWS032 ) and CnxA - GFP ( pAW016 ) , or Ax3 producing P4C - mCherry and either GFP - Sac1 ( pLS037 ) , GFP - Sac1 _ C383S ( pSV015 ) or GFP - Sac1 _ ΔTMD ( pSV034 ) were infected ( MOI 5 , 2 - 16 h ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm . ( B ) LCV area was measured using ImageJ ( n = 100 - 200 per condition from 3 independent biological replicates ) . Means and SEM of single cells are shown ( * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dually labeled D. discoideum Ax3, ∆vap, ∆osbG, ∆osbH, ∆osbK or ∆sey1 mutants producing P4C-mCherry (pWS032) and CnxA-GFP (pAW016), or Ax3 producing P4C-mCherry and either GFP-Sac1 (pLS037), GFP-Sac1_C383S (pSV015) or GFP-Sac1_ΔTMD (pSV034) were infected (MOI 5, 2-16 h) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (B) LCV area was measured using ImageJ (n=100-200 per condition from 3 independent biological replicates). Means and SEM of single cells are shown (**P<0.01; ***P<0.001, Student`s t-test).


( A ) Dually labeled D . discoideum Ax3 , ∆ vap , ∆ osbG , ∆ osbH , ∆ osbK or ∆ sey1 mutants producing P4C - mCherry ( pWS032 ) and GFP - Sac1 ( pLS037 ) were infected ( MOI 5 , 2 - 16 h ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm . ( B , C ) Imaging flow cytometry ( IFC ) analysis of dually labeled D . discoideum Ax3 , ∆ vap , ∆ osbG , ∆ osbH , ∆ osbK or ∆ sey1 mutants producing P4C - mCherry ( pWS032 ) and either GFP - Sac1 ( pLS037 ) or GFP - Sac1 _ ΔTMD ( pSV034 ) , infected ( MOI 5 , 2 h ) with mPlum - producing L . pneumophila JR32 ( pAW014 ) . Data information ( B , C , : Data represent mean and SEM of three independent biological replicates ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dually labeled D. discoideum Ax3, ∆vap, ∆osbG, ∆osbH, ∆osbK or ∆sey1 mutants producing P4C-mCherry (pWS032) and GFP-Sac1 (pLS037) were infected (MOI 5, 2-16 h) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (B, C) Imaging flow cytometry (IFC) analysis of dually labeled D. discoideum Ax3, ∆vap, ∆osbG, ∆osbH, ∆osbK or ∆sey1 mutants producing P4C-mCherry (pWS032) and either GFP-Sac1 (pLS037) or GFP-Sac1_ΔTMD (pSV034), infected (MOI 5, 2 h) with mPlum-producing L. pneumophila JR32 (pAW014). Data information (B, C, : Data represent mean and SEM of three independent biological replicates (*P<0.05; **P<0.01; ***P<0.001, Student`s t-test).


( D ) Dually labeled D . discoideum Ax3 producing GFP fusions of MCS components and either mCherry - Sac1 ( pSV044 ) or mCherry - Sac1 _ ΔTMD ( pSV045 ) were infected ( MOI 5 , 2 h ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm . ( E ) IFC analysis of dually labeled D . discoideum Ax3 producing GFP fusions of MCS components and either mCherry - Sac1 ( pSV044 ) or mCherry - Sac1 _ ΔTMD ( pSV045 ) , infected ( MOI 5 , 2 h ) with mPlum - producing L . pneumophila JR32 ( pAW014 ) . Quantification of ( B ) P4C - mCherry ( C ) Sac1 - GFP or ( E ) GFP fusions of MCS components localizing to LCVs at 2 h p . i . Due to the lower resolution of IFC , " LCVs " designates the LCV limiting membrane and tightly attached ER . Number of events per sample , n = 5000 . Data information E ) : Data represent mean and SEM of three independent biological replicates ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dually labeled D. discoideum Ax3 producing GFP fusions of MCS components and either mCherry-Sac1 (pSV044) or mCherry-Sac1_ΔTMD (pSV045) were infected (MOI 5, 2 h) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (E) IFC analysis of dually labeled D. discoideum Ax3 producing GFP fusions of MCS components and either mCherry-Sac1 (pSV044) or mCherry-Sac1_ΔTMD (pSV045), infected (MOI 5, 2 h) with mPlum-producing L. pneumophila JR32 (pAW014). Quantification of (B) P4C-mCherry (C) Sac1-GFP or (E) GFP fusions of MCS components localizing to LCVs at 2 h p.i. Due to the lower resolution of IFC, "LCVs" designates the LCV limiting membrane and tightly attached ER. Number of events per sample, n = 5000. Data information E): Data represent mean and SEM of three independent biological replicates (*P<0.05; **P<0.01; ***P<0.001, Student`s t-test).


( A ) Dually labeled D . discoideum Ax3 producing GFP - D4H * ( pSV046 ) or P4C - GFP ( pWS034 ) and P4C - mCherry ( pWS032 ) , CnxA - mCherry ( pAW012 ) , or AmtA - mCherry were infected ( MOI 5 , 15 - 120 min ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm . ( B - C ) The Pearson ' s correlation coefficient was generated using Coloc 2 from Fiji ( ImageJ ) and is shown for ( B ) AmtA - mCherry with respect to P4C - GFP or GFP - D4H * ( n = 100 - 150 per time point ) , and ( C ) GFP - D4H * with respect to P4C - mCherry or CnxA - mCherry ( n = 100 - 150 each per time point ) . Data represent mean and SEM of three independent biological replicates ( * P < 0 . 05 , Student ` s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dually labeled D. discoideum Ax3 producing GFP-D4H* (pSV046) or P4C-GFP (pWS034) and P4C-mCherry (pWS032), CnxA-mCherry (pAW012), or AmtA-mCherry were infected (MOI 5, 15 - 120 min) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (B-C) The Pearson's correlation coefficient was generated using Coloc 2 from Fiji (ImageJ) and is shown for (B) AmtA-mCherry with respect to P4C-GFP or GFP-D4H* (n=100-150 per time point), and (C) GFP-D4H* with respect to P4C-mCherry or CnxA-mCherry (n=100-150 each per time point). Data represent mean and SEM of three independent biological replicates (*P<0.05, Student`s t-test).


( D ) Single labeled D . discoideum Ax3 producing either CnxA - GFP ( pAW016 ) or P4C - GFP ( pWS034 ) were infected ( MOI 5 , 15 - 120 min ) with mCherry - producing L . pneumophila JR32 ( pNP102 ) , fixed with 4 % PFA and stained with filipin . Merged images for the analyzed time points are shown . Scale bars : 3 μm . ( E ) The Pearson ' s correlation coefficient was generated using Coloc 2 from Fiji ( ImageJ ) and is shown for filipin with respect to CnxA - GFP or P4C - GFP ( n = 80 - 90 each per time point ) . Data represent mean and SEM of the means of three independent biological replicates ( * * * P < 0 . 001 , Student ` s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Single labeled D. discoideum Ax3 producing either CnxA-GFP (pAW016) or P4C-GFP (pWS034) were infected (MOI 5, 15-120 min) with mCherry-producing L. pneumophila JR32 (pNP102), fixed with 4 % PFA and stained with filipin. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (E) The Pearson's correlation coefficient was generated using Coloc 2 from Fiji (ImageJ) and is shown for filipin with respect to CnxA-GFP or P4C-GFP (n=80-90 each per time point). Data represent mean and SEM of the means of three independent biological replicates (***P<0.001, Student`s t-test).


( A ) Dually labeled D . discoideum Ax3 producing P4C - mCherry ( pWS032 ) and either CnxA - GFP ( pAW016 ) , GFP - Sac1 ( pLS037 ) , or GFP - Sac1 _ ΔTMD ( pSV034 ) were infected ( MOI 5 , 1 - 8 h ) with mCerulean - producing L . pneumophila JR32 , ΔlepB or ΔsidC ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm .

(A) Dually labeled D. discoideum Ax3 producing P4C-mCherry (pWS032) and either CnxA-GFP (pAW016), GFP-Sac1 (pLS037), or GFP-Sac1_ΔTMD (pSV034) were infected (MOI 5, 1-8 h) with mCerulean-producing L. pneumophila JR32, ΔlepB or ΔsidC (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm.


( C ) Imaging flow cytometry ( IFC ) analysis of dually labeled D . discoideum Ax3 producing P4C - mCherry ( pWS032 ) and either CnxA - GFP ( pAW016 ) , GFP - Sac1 ( pLS037 ) , or GFP - Sac1 _ ΔTMD ( pSV034 ) , infected ( MOI 5 , 1 - 8 h ) with mPlum - producing L . pneumophila JR32 ( pAW014 ) . Number of events per sample , n = 5000 . Data represent mean and SEM of three independent biological replicates ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Imaging flow cytometry (IFC) analysis of dually labeled D. discoideum Ax3 producing P4C-mCherry (pWS032) and either CnxA-GFP (pAW016), GFP-Sac1 (pLS037), or GFP-Sac1_ΔTMD (pSV034), infected (MOI 5, 1-8 h) with mPlum-producing L. pneumophila JR32 (pAW014). Number of events per sample, n = 5000. Data represent mean and SEM of three independent biological replicates (*P<0.05; **P<0.01; ***P<0.001, Student`s t-test).


A U2OS cells were transfected with Flag - NAT10 or control vectors . Forty - eight hours later , cells were harvested and whole cell extracts were immunoprecipitated with Flag - antibody affinity resin . The NAT10 - binding proteins were resolved by SDS - PAGE and detected by silver staining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 U2OS cells were transfected with Flag-NAT10 or control vectors. Forty-eight hours later, cells were harvested and whole cell extracts were immunoprecipitated with Flag-antibody affinity resin. The NAT10-binding proteins were resolved by SDS-PAGE and detected by silver staining.


B - F U2OS , HCT116 p53 + / + or HCT116 p53 - / - cell lysates were immunoprecipitated with control IgG , anti - NAT10 ( B and F ) , anti - p53 ( C ) and anti - Mdm2 ( D and E ) antibodies . The immunoprecipitates were subsequently immunoblotted with the indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F U2OS, HCT116 p53+/+ or HCT116 p53-/- cell lysates were immunoprecipitated with control IgG, anti-NAT10 (B and F), anti-p53 (C) and anti-Mdm2 (D and E) antibodies. The immunoprecipitates were subsequently immunoblotted with the indicated antibodies.


G Purified NAT10 was incubated with GST , GST - p53 or GST - Mdm2 proteins coupled to Glutathione Sepharose 4B . Proteins retained on the Sepharose were then analyzed by western blot using the antibodies as indicated . The amount of GST fusion proteins are shown in the lower panel .

G Purified NAT10 was incubated with GST, GST-p53 or GST-Mdm2 proteins coupled to Glutathione Sepharose 4B. Proteins retained on the Sepharose were then analyzed by western blot using the antibodies as indicated. The amount of GST fusion proteins are shown in the lower panel.


H Full - length GST - p53 fusion protein , its deletion mutants or GST protein was used to pull - down experiments with purified NAT10 protein . The levels of the GST fusion proteins are shown in the left 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 O O O O O O O O O O O O

H Full-length GST-p53 fusion protein, its deletion mutants or GST protein was used to pull-down experiments with purified NAT10 protein. The levels of the GST fusion proteins are shown in the left panel.


I GST pull - down assay was performed using purified GST - NAT10 deletion mutants or GST protein and overexpressed Flag - p53 or Mdm2 protein in HEK293T cells . Schematic diagram represents the constructs of GST - NAT10 deletion mutants ( 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 O O O O O O O O O O O O O O O O

I GST pull-down assay was performed using purified GST-NAT10 deletion mutants or GST protein and overexpressed Flag-p53 or Mdm2 protein in HEK293T cells. Schematic diagram represents the constructs of GST-NAT10 deletion mutants (right panel).


A Recombinant His - p53 was incubated with or without purified Flag - NAT10 as described in Experimental Procedures . The reaction products were resolved by SDS - PAGE and immunoblotted using anti - acetyl - lysine antibodies . Flag - PCAF was used as a positive control .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 Recombinant His-p53 was incubated with or without purified Flag-NAT10 as described in Experimental Procedures. The reaction products were resolved by SDS-PAGE and immunoblotted using anti-acetyl-lysine antibodies. Flag-PCAF was used as a positive control.


B In vitro acetylation was performed and the acetylated p53 was detected as in A .

B In vitro acetylation was performed and the acetylated p53 was detected as in A.


C In vitro acetylation assay was performed as described in A . The reaction products were resolved by SDS - PAGE and acetylated p53 was purified from the SDS - PAGE and subjected to mass spectrometry analysis .

O O O O O O O O O O O O O O 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 vitro acetylation assay was performed as described in A. The reaction products were resolved by SDS-PAGE and acetylated p53 was purified from the SDS-PAGE and subjected to mass spectrometry analysis.


D His - p53 or His - p53 - K120R fusion protein was incubated with purified NAT10 or NAT10 GE mutant as described in Experimental Procedures . Reaction mixtures were subjected to western blot using the site specific monoclonal anti - Ac - p53 - K120 antibody .

O O O O O O O O O O O O O O O O O O O O O O O O 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 His-p53 or His-p53-K120R fusion protein was incubated with purified NAT10 or NAT10 GE mutant as described in Experimental Procedures. Reaction mixtures were subjected to western blot using the site specific monoclonal anti-Ac-p53-K120 antibody.


E H1299 cells were transfected with the indicated vectors . Total proteins and the anti - Flag antibody M2 - specific immunoprecipitates were analyzed by western blot using the indicated antibodies .

O O O O O O O O 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 H1299 cells were transfected with the indicated vectors. Total proteins and the anti-Flag antibody M2-specific immunoprecipitates were analyzed by western blot using the indicated antibodies.


A U2OS cells were transfected with the indicated siRNAs . Seventy - two hours later , cell lysates were prepared and proteins from lysates were subjected to western blot using the indicated antibodies .

O O O O O O O O O O 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 U2OS cells were transfected with the indicated siRNAs. Seventy-two hours later, cell lysates were prepared and proteins from lysates were subjected to western blot using the indicated antibodies.


B U2OS cells were transfected as in A . Seventy - two hours later , total mRNAs were extracted and subjected to RT - qPCR for the indicated genes .

O O O O O O 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 U2OS cells were transfected as in A. Seventy-two hours later, total mRNAs were extracted and subjected to RT-qPCR for the indicated genes.


C U2OS cells were transfected with increasing amount of plasmid DNA expressing NAT10 . Proteins from whole cell extracts were subjected to western blot using the indicated antibodies .

O O O O O 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 U2OS cells were transfected with increasing amount of plasmid DNA expressing NAT10. Proteins from whole cell extracts were subjected to western blot using the indicated antibodies.


D U2OS cells were transfected with control or NAT10 siRNAs . Seventy - two hours later , cells were treated with cycloheximide ( CHX ) and harvested at the indicated time points . Proteins from cell lysates were subjected to immunoblot for the evaluation of NAT10 and p53 ( upper panels ) . Beta - actin was evaluated as a loading control ( lower panel ) . p53 half - life ( t1 / 2 ) obtained from three independent experiments was presented as mean ± SEM at the bottom .

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

D U2OS cells were transfected with control or NAT10 siRNAs. Seventy-two hours later, cells were treated with cycloheximide (CHX) and harvested at the indicated time points. Proteins from cell lysates were subjected to immunoblot for the evaluation of NAT10 and p53 (upper panels). Beta-actin was evaluated as a loading control (lower panel). p53 half-life (t1/2) obtained from three independent experiments was presented as mean ± SEM at the bottom.


E U2OS cells were treated with MG132 ( 10μM ) for 4 hours after transfected with the indicated siRNAs . Proteins from cell extracts were detected by western blot for the indicated proteins .

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

E U2OS cells were treated with MG132 (10μM) for 4 hours after transfected with the indicated siRNAs. Proteins from cell extracts were detected by western blot for the indicated proteins.


F HCT116 p53 + / + cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest . p53 was immunoprecipitated with anti - p53 polyclonal antibody and immunoblotted with anti - Ub antibody .

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

F HCT116 p53+/+ cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest. p53 was immunoprecipitated with anti-p53 polyclonal antibody and immunoblotted with anti-Ub antibody.


G p53 and Mdm2 double null MEF cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest . Proteins from whole cell extracts were subjected to western blot using the indicated antibodies .

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

G p53 and Mdm2 double null MEF cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest. Proteins from whole cell extracts were subjected to western blot using the indicated antibodies.


H U2OS cells were transfected with vectors as indicated . Forty - eight hours later , cells were harvested and proteins from whole cell extracts were subjected to western blot using the indicated antibodies .

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H U2OS cells were transfected with vectors as indicated. Forty-eight hours later, cells were harvested and proteins from whole cell extracts were subjected to western blot using the indicated antibodies.


A HCT116 cells were transfected with increasing amount of DNA expressing NAT10 . Forty - eight hours later , whole cell lysates were prepared and proteins from lysates were analyzed by western blot using the indicated antibodies .

A HCT116 cells were transfected with increasing amount of DNA expressing NAT10. Forty-eight hours later, whole cell lysates were prepared and proteins from lysates were analyzed by western blot using the indicated antibodies.


B HCT116 cells were transfected with Flag - NAT10 and harvested at indicated time points after treated with CHX . Proteins from cell lysates were immunoblotted with the antibodies as indicated ( left panel ) . Relative Mdm2 protein levels standardized by β - actin are shown in the right . Mdm2 half - life ( t1 / 2 ) obtained from three independent experiments was presented as mean ± SEM at the bottom .

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B HCT116 cells were transfected with Flag-NAT10 and harvested at indicated time points after treated with CHX. Proteins from cell lysates were immunoblotted with the antibodies as indicated (left panel). Relative Mdm2 protein levels standardized by β-actin are shown in the right. Mdm2 half-life (t1/2) obtained from three independent experiments was presented as mean ± SEM at the bottom.


C HCT116 cells transfected with the indicated constructs were harvested after treatment with MG132 for 4 hours . The Mdm2 protein was immunoprecipitated with anti - Mdm2 antibody and immunoblotted with anti - HA antibody .

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C HCT116 cells transfected with the indicated constructs were harvested after treatment with MG132 for 4 hours. The Mdm2 protein was immunoprecipitated with anti-Mdm2 antibody and immunoblotted with anti-HA antibody.


D In vitro ubiquitination was performed with recombinant GST - Mdm2 ( left ) or GST - Mdm2 C464A mutant ( right ) and Flag - NAT10 or Flag - NAT10 GE in the presence of E1 , E2 and Ub in vitro as described in Experimental Procedures . The reaction products were resolved by SDS - PAGE , followed by western blot for evaluation of the indicated proteins .

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D In vitro ubiquitination was performed with recombinant GST-Mdm2 (left) or GST-Mdm2 C464A mutant (right) and Flag-NAT10 or Flag-NAT10 GE in the presence of E1, E2 and Ub in vitro as described in Experimental Procedures. The reaction products were resolved by SDS-PAGE, followed by western blot for evaluation of the indicated proteins.


E Mdm2 is ubiquitinated by NAT10 in a dose - dependent manner . In vitro ubiquitination assay were carried out with Mdm2 and different doses of purified Flag - NAT10 as described in D .

E Mdm2 is ubiquitinated by NAT10 in a dose-dependent manner. In vitro ubiquitination assay were carried out with Mdm2 and different doses of purified Flag-NAT10 as described in D.


G U2OS cells transfected with NAT10 siRNA - 3 ( targeting 3 ' - UTR of NAT10 mRNA ) or control siRNA were co - expressed different NAT10 deletion mutants . Cell lysates were extracted and proteins from lysates were subjected to western blot using the indicated antibodies . Relative p53 protein levels standardized by β - actin are shown in the lower .

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

G U2OS cells transfected with NAT10 siRNA-3 (targeting 3'-UTR of NAT10 mRNA) or control siRNA were co-expressed different NAT10 deletion mutants. Cell lysates were extracted and proteins from lysates were subjected to western blot using the indicated antibodies. Relative p53 protein levels standardized by β-actin are shown in the lower.


H In vitro ubiquitination reaction was conducted using recombinant GST - Mdm2 C464A and full - length Flag - NAT10 or Flag - NAT10 - D5 . Ubiquitinated Mdm2 was detected as in D .

H In vitro ubiquitination reaction was conducted using recombinant GST-Mdm2 C464A and full-length Flag-NAT10 or Flag-NAT10-D5. Ubiquitinated Mdm2 was detected as in D.


I HCT116 cells were transfected with the indicated constructs . Cells were harvested after CHX treatment for the indicated times and cell lysates were prepared . Mdm2 protein levels were evaluated by western blot . Relative Mdm2 protein levels standardized by β - actin are shown in the right .

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I HCT116 cells were transfected with the indicated constructs. Cells were harvested after CHX treatment for the indicated times and cell lysates were prepared. Mdm2 protein levels were evaluated by western blot. Relative Mdm2 protein levels standardized by β-actin are shown in the right.


A U2OS cells were treated with 10nM actinomycin D and cell extracts were prepared at indicated time points . Proteins from extracts were analyzed by western blot using the indicated antibodies .

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A U2OS cells were treated with 10nM actinomycin D and cell extracts were prepared at indicated time points. Proteins from extracts were analyzed by western blot using the indicated antibodies.


B U2OS and H1299 cells were treated with actinomycin D for 12 hours . Cells were harvested and proteins from cell lysates were subjected to western blot probed with the indicated antibodies .

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B U2OS and H1299 cells were treated with actinomycin D for 12 hours. Cells were harvested and proteins from cell lysates were subjected to western blot probed with the indicated antibodies.


C U2OS cells transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points . Proteins from cell lysates were subjected to western blot .

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C U2OS cells transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points. Proteins from cell lysates were subjected to western blot.


D HCT116 cells were treated with 10nM of actinomycin D or 4 μM of doxorubicin ( Dox ) for 12 hours . Cells were harvested and total proteins were immunoprecipitated with anti - NAT10 antibody . Immunoprecipitates were analyzed by western blot for the indicated proteins .

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D HCT116 cells were treated with 10nM of actinomycin D or 4 μM of doxorubicin (Dox) for 12 hours. Cells were harvested and total proteins were immunoprecipitated with anti-NAT10 antibody. Immunoprecipitates were analyzed by western blot for the indicated proteins.


E HCT116 cells were treated with actinomycin D for the indicated times . Cells were fixed and immuno - stained as indicated . Immunofluorescence images were taken by confocal microscopy . Scale bar represents 10 μM .

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E HCT116 cells were treated with actinomycin D for the indicated times. Cells were fixed and immuno-stained as indicated. Immunofluorescence images were taken by confocal microscopy. Scale bar represents 10 μM.


F H1299 cells were transfected with the indicated vectors . After treated with actinomycin D , cells were harvested and the whole cell extracts were subjected to western blot using the indicated antibodies .

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F H1299 cells were transfected with the indicated vectors. After treated with actinomycin D, cells were harvested and the whole cell extracts were subjected to western blot using the indicated antibodies.


G H1299 cells were transfected with GFP - NAT10 vectors . After treated with actinomycin D for 12 hours , cells were fixed and nuclei were stained with DAPI . Bar represents 10μM .

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G H1299 cells were transfected with GFP-NAT10 vectors. After treated with actinomycin D for 12 hours, cells were fixed and nuclei were stained with DAPI. Bar represents 10μM.


H HCT116 cells transfected with the indicated constructs were treated with actinomycin D or doxorubicin . Western blot was performed with total cell extracts and Mdm2 - specific immunoprecipitates for the indicated proteins .

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H HCT116 cells transfected with the indicated constructs were treated with actinomycin D or doxorubicin. Western blot was performed with total cell extracts and Mdm2-specific immunoprecipitates for the indicated proteins.


I HCT116 cells were transfected with the indicated constructs . Cells were harvested after treatment with actinomycin D . p53 protein was immunoprecipitated with an anti - p53 polyclonal antibody and immunoblotted with p53 monoclonal antibody ( DO - 1 ) .

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I HCT116 cells were transfected with the indicated constructs. Cells were harvested after treatment with actinomycin D. p53 protein was immunoprecipitated with an anti-p53 polyclonal antibody and immunoblotted with p53 monoclonal antibody (DO-1).


A p21 promoter luciferase reporter plasmid was cotransfected with the indicated siRNAs into U2OS cells . Luciferase activity was determined as described in Experimental Procedure .

O O 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 p21 promoter luciferase reporter plasmid was cotransfected with the indicated siRNAs into U2OS cells. Luciferase activity was determined as described in Experimental Procedure.


B U2OS cells were transfected with siRNAs as indicated . Seventy - two hours later , cells were harvested after treated with actinomycin D for 12 hours . Relative mRNA levels for the indicated genes were analyzed by RT - qPCR .

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B U2OS cells were transfected with siRNAs as indicated. Seventy-two hours later, cells were harvested after treated with actinomycin D for 12 hours. Relative mRNA levels for the indicated genes were analyzed by RT-qPCR.


C - E U2OS cells ( C and D ) and H1299 cells ( D ) transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points . Cell cycle profiles were determined by FACS . Results from three independent experiments are shown as mean ± SEM . ( two - tailed Student ' s t test )

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C-E U2OS cells (C and D) and H1299 cells (D) transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points. Cell cycle profiles were determined by FACS. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student's t test)


F U2OS cells were transfected with the indicated siRNAs . Cells were irradiated with 50 J / m2 UV . Forty - eight hours later , apoptotic cells were determined . Quantification of apoptotic cells is presented . ( two - tailed Student ' s t test )

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F U2OS cells were transfected with the indicated siRNAs. Cells were irradiated with 50 J/m2 UV. Forty-eight hours later, apoptotic cells were determined. Quantification of apoptotic cells is presented. (two-tailed Student's t test)


G U2OS cells transfected with the indicated siRNAs were plated . Cell numbers were determined as described in Experimental Procedures at the indicated time points .

G U2OS cells transfected with the indicated siRNAs were plated. Cell numbers were determined as described in Experimental Procedures at the indicated time points.


H Colony formation assay were performed with U2OS cells transfected with the indicated siRNAs . The right panel shows the quantification of formed colonies . * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t test )

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H Colony formation assay were performed with U2OS cells transfected with the indicated siRNAs. The right panel shows the quantification of formed colonies. **p<0.01, ***p<0.001 (two-tailed Student's t test)


A Twenty pairs of fresh frozen CRC tissues and corresponding normal tissues were lysed and total RNAs were extracted and subjected to RT - qPCR . ( N , Normal tissue ; T , Tumor tissue ) * * p < 0 . 01 ( non - parametric Mann - Whitney t - test )

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A Twenty pairs of fresh frozen CRC tissues and corresponding normal tissues were lysed and total RNAs were extracted and subjected to RT-qPCR. (N, Normal tissue; T, Tumor tissue) **p<0.01 (non-parametric Mann-Whitney t-test)


B Immunohistochemical staining of NAT10 in CRC tissues and normal colon tissues . NAT10 - specific immuno - complex was detected with DAB as shown as brown color . Quantification of NAT10 - positive or NAT10 - negative CRC cases is shown in the lower table .

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B Immunohistochemical staining of NAT10 in CRC tissues and normal colon tissues. NAT10-specific immuno-complex was detected with DAB as shown as brown color. Quantification of NAT10-positive or NAT10-negative CRC cases is shown in the lower table.


C Cell extracts were prepared from different human colon epithelial cell lines as indicated at the top . Proteins from the extracts were subjected to western blot for the evaluation of NAT10 and p53 .

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C Cell extracts were prepared from different human colon epithelial cell lines as indicated at the top. Proteins from the extracts were subjected to western blot for the evaluation of NAT10 and p53.


D CRISPR - Cas9 system mediated NAT10 gene editing . The genome targeting for knockdown NAT10 was performed as described in Experimental Procedures . The HCT116 NAT10 knockdown cell lines were harvested and western blot was performed on the cell lysates for the indicated proteins .

O O O O O O O O O O O O O O O O O O O O O O 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 CRISPR-Cas9 system mediated NAT10 gene editing. The genome targeting for knockdown NAT10 was performed as described in Experimental Procedures. The HCT116 NAT10 knockdown cell lines were harvested and western blot was performed on the cell lysates for the indicated proteins.


E HCT116 Ctrl or c2 - 4 cells with NAT10 disruption were treated with actinomycin D and harvested at the indicated time points . Cell cycle profiles were determined by FACS . Results from three independent experiments are shown as mean ± SEM . ( two - tailed Student ' s t test )

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E HCT116 Ctrl or c2-4 cells with NAT10 disruption were treated with actinomycin D and harvested at the indicated time points. Cell cycle profiles were determined by FACS. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student's t test)


F HCT116 Ctrl cells or c2 - 4 cells with NAT10 disruption were treated with actinomycin D Cells were harvested at the indicated time points and lysed . The total proteins were analyzed by western blot using the indicated antibodies .

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F HCT116 Ctrl cells or c2-4 cells with NAT10 disruption were treated with actinomycin D Cells were harvested at the indicated time points and lysed. The total proteins were analyzed by western blot using the indicated antibodies.


G HCT116 Ctrl or c2 - 4 cells were irradiated with 80 J / m2 UV . Forty - eight hours later , apoptotic cells were determined . Results from three independent experiments are shown as mean ± SEM . ( two - tailed Student ' s t test )

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

G HCT116 Ctrl or c2-4 cells were irradiated with 80 J/m2 UV. Forty-eight hours later, apoptotic cells were determined. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student's t test)


H Colony formation assay were performed with HCT116 cells ( Ctrl , c1 - 1 and c2 - 4 ) . The right panel shows the quantification of formed colonies . ( two - tailed Student ' s t test )

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H Colony formation assay were performed with HCT116 cells (Ctrl, c1-1 and c2-4). The right panel shows the quantification of formed colonies. (two-tailed Student's t test)


I HCT116 c1 - 1 and c2 - 4 cell lines were plated and cell growth curves were determined as described in Fig 6F . Error bars represent the SEM of the triplicate experiments .

I HCT116 c1-1 and c2-4 cell lines were plated and cell growth curves were determined as described in Fig 6F. Error bars represent the SEM of the triplicate experiments.


Human osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control Human osteosarcoma U2OS stably expressing CALR - GFP and H2B - RFP were treated as described above and images were acquired once per hour for 12 h ( A ) . For one representative experiment among three , the mean ± SEM of the average area of high CALR dots ( normalized to the control at each timepoint ) of quadruplicates is shown ( B ) . Values are depicted as the area under the curve ± SD of

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Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control Human osteosarcoma U2OS stably expressing CALR-GFP and H2B-RFP were treated as described above and images were acquired once per hour for 12 h (A). For one representative experiment among three, the mean ± SEM of the average area of high CALR dots (normalized to the control at each timepoint) of quadruplicates is shown (B). Values are depicted as the area under the curve ± SD of triplicates


Human osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control Treated U2OS cells stably expressing HMGB1 - GFP and H2B - RFP images were acquired every hour for 24 h ( D ) . For one representative experiment among three , the mean ± SEM of the green fluorescence intensity in the nucleus ( normalized to the control at each timepoint ) of quadruplicates is depicted ( E ) . For each cell , the speed of nuclear release ( difference of HMGB1 nuclear green fluorescence intensity between two time points ) was calculated . Values are depicted as the average speed of the nuclear release ± SD of

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Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control Treated U2OS cells stably expressing HMGB1-GFP and H2B-RFP images were acquired every hour for 24 h (D). For one representative experiment among three, the mean ± SEM of the green fluorescence intensity in the nucleus (normalized to the control at each timepoint) of quadruplicates is depicted (E). For each cell, the speed of nuclear release (difference of HMGB1 nuclear green fluorescence intensity between two time points) was calculated. Values are depicted as the average speed of the nuclear release ± SD of quadruplicates


Human osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control U2OS cells were treated for 6 , 12 or 24 h and ATP was stained with quinacrine ( G ) . The number of quinacrine negative cells was assessed based on the distribution of cellular green fluorescence intensity in MTX versus control conditions . For one representative experiment among three the mean ± SD of quadruplicate assessments is

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Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS cells were treated for 6, 12 or 24 h and ATP was stained with quinacrine (G). The number of quinacrine negative cells was assessed based on the distribution of cellular green fluorescence intensity in MTX versus control conditions. For one representative experiment among three the mean ± SD of quadruplicate assessments is shown


Human osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control U2OS wild - type cells were treated with MTX or DACT as described above for 6 h . Then medium was refreshed and 24 h later , type I interferon response was assessed by transferring the supernatant on HT29 MX1 - GFP reporter cells lines cells for additional 48 h . Human type 1α interferon ( IFNα1 ) was also added on the cells as an additional positive control . Images were acquired by fluorescence microscopy and the number of positive cells was assessed based on the distribution of cellular green fluorescence intensity in IFNα1 versus control conditions ( I ) . The percentage of MX1 positive cells was calculated and the mean ± SEM of five independent experiments is

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Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS wild-type cells were treated with MTX or DACT as described above for 6 h. Then medium was refreshed and 24 h later, type I interferon response was assessed by transferring the supernatant on HT29 MX1-GFP reporter cells lines cells for additional 48 h. Human type 1α interferon (IFNα1) was also added on the cells as an additional positive control. Images were acquired by fluorescence microscopy and the number of positive cells was assessed based on the distribution of cellular green fluorescence intensity in IFNα1 versus control conditions (I). The percentage of MX1 positive cells was calculated and the mean ± SEM of five independent experiments is depicted


Human osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control U2OS wild - type cells were treated as mentioned above for 6 h and then medium was refreshed . Twenty - four hours later , cells were collected and surface - exposed calreticulin ( CALR ) was stained with an antibody specific for CALR . DAPI was used as an exclusion dye and cells were acquired by flow cytometry ( K ) . The percentage of CALR + cells among viable ( DAPI - ) ones are depicted . The mean ± SEM of six independent experiments is

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Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS wild-type cells were treated as mentioned above for 6 h and then medium was refreshed. Twenty-four hours later, cells were collected and surface-exposed calreticulin (CALR) was stained with an antibody specific for CALR. DAPI was used as an exclusion dye and cells were acquired by flow cytometry (K). The percentage of CALR+ cells among viable (DAPI-) ones are depicted. The mean ± SEM of six independent experiments is depicted


Human osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control U2OS cells were treated as described above for 24 h and the concentration of HMGB1 released in the supernatant was quantified with an ELISA kit , then normalized to control . The mean ± SEM of four independent experiments is shown .

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Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS cells were treated as described above for 24 h and the concentration of HMGB1 released in the supernatant was quantified with an ELISA kit, then normalized to control. The mean ± SEM of four independent experiments is shown.


Human osteosarcoma U2OS cells were treated with dactinomycin ( DACT ) at 0 . 5 or 1 µM , or with mitoxantrone ( MTX ) between 1 and 6 µM as positive control U2OS were treated as described above for 24 h . Concentration of secreted ATP in the supernatant was quantified with a luciferase - based bioluminescence kit . The mean ± SD of quadruplicates from one representative among three experiments is depicted .

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Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS were treated as described above for 24 h. Concentration of secreted ATP in the supernatant was quantified with a luciferase-based bioluminescence kit. The mean ± SD of quadruplicates from one representative among three experiments is depicted.


Human osteosarcoma U2OS cells were treated with different concentrations of dactinomycin ( DACT ) ( 0 . 25 , 0 . 5 or 1 µM ) for 6 h . Thapsigargin ( THAPS ) at 3 µM was used as a positive control . After fixation , cells were stained with phospho - eIF2α ( Ser51 ) specific antibody followed by an AlexaFluor - 647 secondary antibody , nuclei were counterstained with Hoechst 33342 and phosphorylation was assessed by fluorescence microscopy . Images were segmented , analyzed and the red cytoplasmic fluorescence intensity was measured . Images are shown for untreated control cells ( Ctr ) , THAPS and DACT at 1

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Human osteosarcoma U2OS cells were treated with different concentrations of dactinomycin (DACT) (0.25, 0.5 or 1 µM) for 6 h. Thapsigargin (THAPS) at 3 µM was used as a positive control. After fixation, cells were stained with phospho-eIF2α (Ser51) specific antibody followed by an AlexaFluor-647 secondary antibody, nuclei were counterstained with Hoechst 33342 and phosphorylation was assessed by fluorescence microscopy. Images were segmented, analyzed and the red cytoplasmic fluorescence intensity was measured. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM


U2OS cells stably expressing ATF4 - reporter were treated as described above for 12 h . The expression and nuclear translocation of ATF4 was assessed by fluorescence microscopy and the nuclear green fluorescence intensity was quantified . Images are shown for untreated control cells ( Ctr ) , THAPS and DACT at 1

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U2OS cells stably expressing ATF4-reporter were treated as described above for 12 h. The expression and nuclear translocation of ATF4 was assessed by fluorescence microscopy and the nuclear green fluorescence intensity was quantified. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM


U2OS stably expressing ATF6 - GFP were treated as described above and nuclear translocation of ATF6 was represented as the ratio of nuclear versus cytoplasmic green fluorescence intensity . Images are shown for untreated control cells ( Ctr ) , THAPS and DACT at 1

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U2OS stably expressing ATF6-GFP were treated as described above and nuclear translocation of ATF6 was represented as the ratio of nuclear versus cytoplasmic green fluorescence intensity. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM


U2OS cells stably expressing venus in frame with alternatively spliced XBP1 ( sXBP1 ) were treated as described above for 12 h . De novo expressed venus was measured intracellular . Images are shown for untreated control cells ( Ctr ) , THAPS and DACT at 1

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U2OS cells stably expressing venus in frame with alternatively spliced XBP1 (sXBP1) were treated as described above for 12 h. De novo expressed venus was measured intracellular. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM


U2OS wild - type and knock out for eIF2α kinases 1 , 2 , 3 and 4 cells were treated with 3 µM THAPS as a positive control for EIF2AK3 - mediated eIF2α phosphorylation or with 1 µM DACT for 6 h . After fixation , cells were stained for peIF2α as described above and cytoplasmic intensity was quantified . Images are shown for untreated control cells ( Ctr ) , THAPS and DACT at 1

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U2OS wild-type and knock out for eIF2α kinases 1, 2, 3 and 4 cells were treated with 3 µM THAPS as a positive control for EIF2AK3-mediated eIF2α phosphorylation or with 1 µM DACT for 6 h. After fixation, cells were stained for peIF2α as described above and cytoplasmic intensity was quantified. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM


U2OS CALR - RFP were injected subcutaneously ( s . c ) in the flank of nu / nu mice . Tumors were further injected with PBS ( Ctr ) ( n = 5 ) , 0 . 5 mg / kg tunicamycin ( TM ) for 6 h ( n = 3 ) or 0 . 5 mg / kg DACT for 6 h ( n = 4 ) or 24 h ( n = 3 ) . Tumor slices were cut and stained with a peIF2α antibody followed by an AlexaFluor - 647 secondary antibody and counterstained with Hoechst 33342 . Two slices per tumor were imaged for their DAPI , RFP and Cy5 signals . Out - of - focused images were removed from the dataset leading to the analysis of the following conditions : 8 x Ctr , 5 x TM , 8 x DACT 6 h and 6 x for DACT 24 h . PeIF2α was quantified measuring Cy5 intensity in the cytoplasm ( K , L ) and CALR translocation by measuring the coefficient variation ( CV ) of the RFP signal in the cytoplasm ( M , N ) . Representative images of peIF2α are shown for Ctr , TM and DACT at 6 h ( K ) , whereas images of CALR are shown for Ctr and DACT at 24

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U2OS CALR-RFP were injected subcutaneously (s.c) in the flank of nu/nu mice. Tumors were further injected with PBS (Ctr) (n=5), 0.5 mg/kg tunicamycin (TM) for 6 h (n=3) or 0.5 mg/kg DACT for 6 h (n=4) or 24 h (n=3). Tumor slices were cut and stained with a peIF2α antibody followed by an AlexaFluor-647 secondary antibody and counterstained with Hoechst 33342. Two slices per tumor were imaged for their DAPI, RFP and Cy5 signals. Out-of-focused images were removed from the dataset leading to the analysis of the following conditions: 8 x Ctr, 5 x TM, 8 x DACT 6 h and 6 x for DACT 24 h. PeIF2α was quantified measuring Cy5 intensity in the cytoplasm (K, L) and CALR translocation by measuring the coefficient variation (CV) of the RFP signal in the cytoplasm (M, N). Representative images of peIF2α are shown for Ctr, TM and DACT at 6 h (K), whereas images of CALR are shown for Ctr and DACT at 24 h


Mouse fibrosarcoma MCA205 cells were stained with CellTracker Orange ( CMTMR ) and treated for 24 h with 1 µM dactinomycin ( DACT ) or 500 µM oxaliplatin ( OXA ) as a positive control . Then , untreated or dying MCA205 were co - cultured with differentiated bone marrow - derived dendritic cells ( BMDCs ) for 4 h at 37 ° C or at 4 ° C . Cells were collected and dendritic cells were stained with CD11c specific antibody before analysis by flow cytometry The percentage of CMTMR and CD11c double positive cells among all CD11c + cells are

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Mouse fibrosarcoma MCA205 cells were stained with CellTracker Orange (CMTMR) and treated for 24 h with 1 µM dactinomycin (DACT) or 500 µM oxaliplatin (OXA) as a positive control. Then, untreated or dying MCA205 were co-cultured with differentiated bone marrow-derived dendritic cells (BMDCs) for 4 h at 37 °C or at 4 °C. Cells were collected and dendritic cells were stained with CD11c specific antibody before analysis by flow cytometry The percentage of CMTMR and CD11c double positive cells among all CD11c+ cells are indicated


MCA205 cells were treated for 24 h with 500 µM oxaliplatin ( OXA ) or with 0 . 25 , 0 . 5 or 1 µM dactinomycin ( DACT ) . Then , untreated or dying MCA205 were co - cultured with BMDCs for 24 h at 37 ° C . As a positive control , BMDCs were co - cultured with 1 μg / mL LPS and 100 ng / mL IFNγ . The increase in the percentages of MHCII + and CD86 + cells among CD11c + cells was quantified with respect to untreated controls .

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MCA205 cells were treated for 24 h with 500 µM oxaliplatin (OXA) or with 0.25, 0.5 or 1 µM dactinomycin (DACT). Then, untreated or dying MCA205 were co-cultured with BMDCs for 24 h at 37 °C. As a positive control, BMDCs were co-cultured with 1 μg/mL LPS and 100 ng/mL IFNγ. The increase in the percentages of MHCII+ and CD86+ cells among CD11c+ cells was quantified with respect to untreated controls.


1 x 106 mouse fibrosarcoma MCA205 cells were treated in vitro with 1 µM dactinomycin ( DACT ) . Next , tumor size was measured regularly and individual tumor growths of DACT - vaccinated versus Ctr mice are

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1 x 106 mouse fibrosarcoma MCA205 cells were treated in vitro with 1 µM dactinomycin (DACT). Next, tumor size was measured regularly and individual tumor growths of DACT-vaccinated versus Ctr mice are depicted


1 x 106 mouse fibrosarcoma MCA205 cells were treated in vitro with 1 µM dactinomycin ( DACT ) . Overall survival is depicted and p - values ( * * * p < 0 . 001 . ) were calculated with a Log - Rank

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1 x 106 mouse fibrosarcoma MCA205 cells were treated in vitro with 1 µM dactinomycin (DACT). Overall survival is depicted and p-values (*** p<0.001.) were calculated with a Log-Rank test


3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously ( s . c ) into the flank of immunocompetent syngeneic Balb / c mice When tumors became palpable , the mice were injected intraperitoneally ( i . p . ) with injectable solution ( Ctr ) or with 0 . 5 mg / kg dactinomycin ( DACT ) . A second injection of chemotherapy was performed four days later . Tumor size was assessed regularly and individual tumor growth curves of DACT versus

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

3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously (s.c) into the flank of immunocompetent syngeneic Balb/c mice When tumors became palpable, the mice were injected intraperitoneally (i.p.) with injectable solution (Ctr) or with 0.5 mg/kg dactinomycin (DACT). A second injection of chemotherapy was performed four days later. Tumor size was assessed regularly and individual tumor growth curves of DACT versus Ctr


3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously ( s . c ) into the flank of immunocompetent syngeneic Balb / c mice When tumors became palpable , the mice were injected intraperitoneally ( i . p . ) with injectable solution ( Ctr ) or with 0 . 5 mg / kg dactinomycin ( DACT ) . A second injection of chemotherapy was performed four days later . DACT + anti - CD4 / CD8 versus anti - CD - 4 / anti - CD - 8 Mean tumor area for each group was calculated and significances were tested using a type II ANOVA test Overall survival is depicted and p - values were calculated with a Log - Rank test Stars indicate the p - values of each treatment versus Ctr and hashes indicate the p - values of the DACT + anti - CD - 4 / anti - CD - 8 versus DACT

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3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously (s.c) into the flank of immunocompetent syngeneic Balb/c mice When tumors became palpable, the mice were injected intraperitoneally (i.p.) with injectable solution (Ctr) or with 0.5 mg/kg dactinomycin (DACT). A second injection of chemotherapy was performed four days later. DACT + anti-CD4/CD8 versus anti-CD-4/anti-CD-8 Mean tumor area for each group was calculated and significances were tested using a type II ANOVA test Overall survival is depicted and p-values were calculated with a Log-Rank test Stars indicate the p-values of each treatment versus Ctr and hashes indicate the p-values of the DACT + anti-CD-4/anti-CD-8 versus DACT alone


3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously ( s . c ) into the flank of immunocompetent syngeneic Balb / c mice When tumors became palpable , the mice were injected intraperitoneally ( i . p . ) with injectable solution ( Ctr ) or with 0 . 5 mg / kg dactinomycin ( DACT ) . A second injection of chemotherapy was performed four days later . DACT + anti - PD - 1 versus anti - PD - 1 are depicted . Overall survival is depicted and p - values were calculated with a Log - Rank test Stars indicate the p - values of each treatment versus Ctr and of DACT + anti - PD - 1 versus anti - PD - 1

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

3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously (s.c) into the flank of immunocompetent syngeneic Balb/c mice When tumors became palpable, the mice were injected intraperitoneally (i.p.) with injectable solution (Ctr) or with 0.5 mg/kg dactinomycin (DACT). A second injection of chemotherapy was performed four days later. DACT + anti-PD-1 versus anti-PD-1 are depicted. Overall survival is depicted and p-values were calculated with a Log-Rank test Stars indicate the p-values of each treatment versus Ctr and of DACT + anti-PD-1 versus anti-PD-1 alone


3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously ( s . c ) into the flank of immunocompetent syngeneic Balb / c mice When tumors became palpable , the mice were injected intraperitoneally ( i . p . ) with injectable solution ( Ctr ) or with 0 . 5 mg / kg dactinomycin ( DACT ) . A second injection of chemotherapy was performed four days later . Five naïve mice or the eleven mice that were cured by treatment with DACT alone or in combination with PD - 1 blockade were ( re ) challenged with WEHI 164 cells , and individual tumor growths ( I , J ) , as well as overall survival ( * * * p < 0 . 001 , Log - Rank test ) ( K ) , were monitored .

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3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously (s.c) into the flank of immunocompetent syngeneic Balb/c mice When tumors became palpable, the mice were injected intraperitoneally (i.p.) with injectable solution (Ctr) or with 0.5 mg/kg dactinomycin (DACT). A second injection of chemotherapy was performed four days later. Five naïve mice or the eleven mice that were cured by treatment with DACT alone or in combination with PD-1 blockade were (re)challenged with WEHI 164 cells, and individual tumor growths (I, J), as well as overall survival (*** p<0.001, Log-Rank test) (K), were monitored.


Mice bearing WEHI 164 sarcomas were treated by systemic ( intraperitoneal , i . p . ) injections of DACT or PBS as a control ( Ctr ) , and tumor were excised 9 days later for the quantitation of mRNA coding for IFNγ Two independent experiments were conducted with a total of 22 mice in the Ctr group and 23 mice in the DACT - treated group , with each data point indicating one tumor . The cycle threshold of the qRT - PCR of IFNγ was normalized to the one of the housekeeping gene peptidylpropyl isomerase A ( Ppia ) in each mouse and results are shown normalized with respect to controls as a dot plot depicting mean ± SD . The p - value ( * p < 0 . 05 ) was calculated by means of a Student ' s t test ( B ) .

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Mice bearing WEHI 164 sarcomas were treated by systemic (intraperitoneal, i.p.) injections of DACT or PBS as a control (Ctr), and tumor were excised 9 days later for the quantitation of mRNA coding for IFNγ Two independent experiments were conducted with a total of 22 mice in the Ctr group and 23 mice in the DACT-treated group, with each data point indicating one tumor. The cycle threshold of the qRT-PCR of IFNγ was normalized to the one of the housekeeping gene peptidylpropyl isomerase A (Ppia) in each mouse and results are shown normalized with respect to controls as a dot plot depicting mean ± SD . The p-value (* p<0.05) was calculated by means of a Student's t test (B).


Balb / c mice with palpable WEHI 164 sarcomas ( n = 7 mice in Ctr and anti - IFNγ groups ; n = 8 mice in DACT and DACT + anti - IFNγ groups ) received two injections of DACT - based chemotherapy ( 0 . 5 mg / kg ) as well as multiple injections ( 3 times per week ) of neutralizing IFNγ - specific antibody ( C ) . Tumor growth curves are shown for individual mice ( D ) and as means ( E ) . Statistical difference between DACT - treated tumors and respective controls , which was calculated with a type II ANOVA ( * p < 0 . 05 ) , is lost upon IFNγ neutralization ( E ) . Overall survival of mice is also indicated with statistics to respective controls calculated with a Log - Rank test ( * p < 0 . 05 ) ( F ) .

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Balb/c mice with palpable WEHI 164 sarcomas (n=7 mice in Ctr and anti-IFNγ groups; n=8 mice in DACT and DACT+anti-IFNγ groups) received two injections of DACT-based chemotherapy (0.5 mg/kg) as well as multiple injections (3 times per week) of neutralizing IFNγ-specific antibody (C). Tumor growth curves are shown for individual mice (D) and as means (E). Statistical difference between DACT-treated tumors and respective controls, which was calculated with a type II ANOVA (* p<0.05), is lost upon IFNγ neutralization (E). Overall survival of mice is also indicated with statistics to respective controls calculated with a Log-Rank test (* p<0.05) (F).