figf3 ( a ) Left : lipid binding of EndoB1 is inhibited by mutating the five lysine / arginine residues between amino acids 176 and 183 of EndoB1 to glutamic acid ( 5E mutant ) . Right : purified GST protein was included as a negative control . S , supernatant fraction ; P , pellet fraction that contains the lipid - bound proteins . ( b , c ) The 5E mutant of EndoB1 fails to dimerize ( b ) in 293T cells . b , c ) The 5E mutant of EndoB1 fails to dimerize ( b ) and interact with UVRAG ( c ) in 293T cells . ( d ) Overexpression of the 5E mutant of EndoB1 blocks starvation - induced autophagy in cortical neurons . Data are means ± s . e . m . ; n = 3 . ( e ) Mutation of Thr 145 to alanine ( T145A ) or glutamic acid ( T145E ) has negligible effect on the lipid - binding property of EndoB1 . Conversely , the 5E mutant is less able to bind lipid . The right panel shows quantified data as means ± s . e . m . ; n = 3 . ( f ) Starvation increases co - localization of GFP - EndoB1 ( green ) with Atg5 ( red ) in neurons . The T145A mutation of GFP - EndoB1 does not affect its co - localization with Atg5 - positive entities . Arrows denote GFP - EndoB1 - and Atg5 - positive vesicles . The right panel shows quantified data as means ± s . e . m . ; n = 9 . Uncropped images of blots are shown in Supplementary Fig . S8 .

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

figf3(a) Left: lipid binding of EndoB1 is inhibited by mutating the five lysine/arginine residues between amino acids 176 and 183 of EndoB1 to glutamic acid (5E mutant). Right: purified GST protein was included as a negative control. S, supernatant fraction; P, pellet fraction that contains the lipid-bound proteins.(b,c) The 5E mutant of EndoB1 fails to dimerize (b) in 293T cells.b,c) The 5E mutant of EndoB1 fails to dimerize (b) and interact with UVRAG (c) in 293T cells.(d) Overexpression of the 5E mutant of EndoB1 blocks starvation-induced autophagy in cortical neurons. Data are means ± s.e.m.; n=3.(e) Mutation of Thr 145 to alanine (T145A) or glutamic acid (T145E) has negligible effect on the lipid-binding property of EndoB1. Conversely, the 5E mutant is less able to bind lipid. The right panel shows quantified data as means ± s.e.m.; n=3.(f) Starvation increases co-localization of GFP-EndoB1 (green) with Atg5 (red) in neurons. The T145A mutation of GFP-EndoB1 does not affect its co-localization with Atg5-positive entities. Arrows denote GFP-EndoB1- and Atg5- positive vesicles. The right panel shows quantified data as means ± s.e.m.; n=9. Uncropped images of blots are shown in Supplementary Fig. S8.


figf4 ( a ) Overexpression of Cdk5 / p35 enhances EndoB1 dimerization in 293T cells . Samples were run on the same gel and spliced together to create the image shown ( b ) Thr 145 phosphorylation of EndoB1 facilitates its dimerization in 293T cells . ( c ) Diminished interaction between EndoB1 and UVRAG in Cdk5 − / − mouse brain lysates . ( d , e ) Thr 145 phosphorylation of EndoB1 enhances its interaction with UVRAG ( d ) in 293T cells . ( d , e ) Thr 145 phosphorylation of EndoB1 enhances its interaction with UVRAG ( d ) and Beclin 1 ( e ) in 293T cells . ( f , g ) Gel - filtration analysis on the membrane fraction of mouse brain lysates . Fractions obtained were subjected to immunoblot analysis ( f ) , or immunoprecipitation followed by immunoblotting ( g ) . Uncropped images of blots are shown in Supplementary Fig . S8 .

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figf4(a) Overexpression of Cdk5/p35 enhances EndoB1 dimerization in 293T cells. Samples were run on the same gel and spliced together to create the image shown(b) Thr 145 phosphorylation of EndoB1 facilitates its dimerization in 293T cells.(c) Diminished interaction between EndoB1 and UVRAG in Cdk5−/− mouse brain lysates.(d,e) Thr 145 phosphorylation of EndoB1 enhances its interaction with UVRAG (d) in 293T cells.(d,e) Thr 145 phosphorylation of EndoB1 enhances its interaction with UVRAG (d) and Beclin 1 (e) in 293T cells.(f,g) Gel-filtration analysis on the membrane fraction of mouse brain lysates. Fractions obtained were subjected to immunoblot analysis (f), or immunoprecipitation followed by immunoblotting (g). Uncropped images of blots are shown in Supplementary Fig. S8.


figf5 ( a ) MPTP administration decreases the numbers of tyrosine hydroxylase ( TH ) - and cresyl violet ( CV ) - positive neurons in the substantia nigra . Representative images of tyrosine hydroxylase - stained brain sections are shown . Right panels show quantified data as means ± s . e . m . ; n = 9 for tyrosine hydroxylase and n = 3 for cresyl violet . ( b ) MPTP administration upregulates the levels of LC3 - II and Thr 145 - phosphorylated EndoB1 in the midbrain . Lower panels show quantified data as means ± s . e . m . ; n = 3 . ( c ) Knockdown of EndoB1 or Cdk5 inhibits MPP + - induced autophagy in cultured neurons . Data are means ± s . e . m . ; n = 5 . ( d ) Cdk5 mediates the MPP + - induced EndoB1 phosphorylation at Thr 145 and the MPP + - induced increase in the LC3 - II level in neurons . ( e ) Overexpression of EndoB1T145A attenuates MPP + - induced autophagy in neurons . Data are means ± s . e . m . ; n = 7 . ( f ) MPTP fails to increase LC3 - II and Thr 145 - phosphorylated EndoB1 levels in the midbrain of p35 − / − mice . The concomitant reduction in tyrosine hydroxylase level triggered by MPTP injection is also markedly attenuated . Right panels show quantified data as means ± s . e . m . ; n = 3 . Uncropped images of blots are shown in Supplementary Fig . S8 .

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figf5(a) MPTP administration decreases the numbers of tyrosine hydroxylase (TH)- and cresyl violet (CV)-positive neurons in the substantia nigra. Representative images of tyrosine hydroxylase-stained brain sections are shown. Right panels show quantified data as means ± s.e.m.; n=9 for tyrosine hydroxylase and n=3 for cresyl violet.(b) MPTP administration upregulates the levels of LC3-II and Thr 145-phosphorylated EndoB1 in the midbrain. Lower panels show quantified data as means ± s.e.m.; n=3.(c) Knockdown of EndoB1 or Cdk5 inhibits MPP+-induced autophagy in cultured neurons. Data are means ± s.e.m.; n=5.(d) Cdk5 mediates the MPP+-induced EndoB1 phosphorylation at Thr 145 and the MPP+-induced increase in the LC3-II level in neurons.(e) Overexpression of EndoB1T145A attenuates MPP+-induced autophagy in neurons. Data are means ± s.e.m.; n=7.(f) MPTP fails to increase LC3-II and Thr 145-phosphorylated EndoB1 levels in the midbrain of p35−/− mice. The concomitant reduction in tyrosine hydroxylase level triggered by MPTP injection is also markedly attenuated. Right panels show quantified data as means ± s.e.m.; n=3. Uncropped images of blots are shown in Supplementary Fig. S8.


figf6 ( a ) Upregulation of the levels of LC3 - II and Thr 145 - phosphorylated EndoB1 in the cerebellum and striatum of α - synucleinA53T transgenic mice . Right panels show quantified data as means ± s . e . m . ; n = 3 . b ) Overexpression of α - synucleinA53T mutant upregulates LC3 - II level and Thr 145 - phosphorylated EndoB1 in cultured neurons . Lower panel shows quantified data as means ± s . e . m . ; n = 3 . ( c ) Knockdown of EndoB1 or Cdk5 inhibits α - synucleinA53T mutant - induced autophagy induction in neurons . Data are means ± s . e . m . ; n = 7 . ( d ) Cdk5 mediates α - synucleinA53T mutant - induced Thr 145 phosphorylation of EndoB1 and the increase of LC3 - II in neurons . ( e ) Overexpression of EndoB1T145A attenuates α - synucleinA53T mutant - induced autophagy in neurons . Data are means ± s . e . m . ; n = 7 . Uncropped images of blots are shown in Supplementary Fig . S8 .

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

figf6(a) Upregulation of the levels of LC3-II and Thr 145-phosphorylated EndoB1 in the cerebellum and striatum of α-synucleinA53T transgenic mice. Right panels show quantified data as means ± s.e.m.; n=3.b) Overexpression of α-synucleinA53T mutant upregulates LC3-II level and Thr 145-phosphorylated EndoB1 in cultured neurons. Lower panel shows quantified data as means ± s.e.m.; n=3.(c) Knockdown of EndoB1 or Cdk5 inhibits α-synucleinA53T mutant-induced autophagy induction in neurons. Data are means ± s.e.m.; n=7.(d) Cdk5 mediates α-synucleinA53T mutant-induced Thr 145 phosphorylation of EndoB1 and the increase of LC3-II in neurons.(e) Overexpression of EndoB1T145A attenuates α-synucleinA53T mutant-induced autophagy in neurons. Data are means ± s.e.m.; n=7. Uncropped images of blots are shown in Supplementary Fig. S8.


figf7 ( a ) Pretreatment with 3 - MA , but not z - VAD - fmk , significantly reduces MPP + - induced neuronal death . Data are means ± s . e . m . ; n = 6 . ( b ) Knockdown of Atg5 expression abrogates MPP + - induced neuronal loss , whereas it reduces neuronal survival in untreated cells . Data are means ± s . e . m . ; n = 3 . ( c ) Knockdown of EndoB1 or Cdk5 expression abolishes MPP + - induced neuronal death . Data are means ± s . e . m . ; n = 8 . ( d ) Overexpression of EndoB1T145A inhibits MPP + - induced neuronal death . Data are means ± s . e . m . ; n = 5 . ( e ) Pretreatment with 3 - MA , but not z - VAD - fmk , reduces α - synucleinA53T mutant - induced neuronal death . Data are means ± s . e . m . ; n = 6 . ( f ) Knockdown of Atg5 expression abolishes α - synucleinA53T mutant - induced neuronal death , whereas it reduces neuronal survival in vector - transfected cells . Data are means ± s . e . m . ; n = 3 . ( g ) Knockdown of EndoB1 or Cdk5 expression abolishes α - synucleinA53T mutant - induced neuronal death . Data are means ± s . e . m . ; n = 4 . ( h ) Expression of EndoB1T145A inhibits α - synucleinA53T mutant - induced neuronal death . Data are means ± s . e . m . ; n = 6 .

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

figf7(a) Pretreatment with 3-MA, but not z-VAD-fmk, significantly reduces MPP+-induced neuronal death. Data are means ± s.e.m.; n=6.(b) Knockdown of Atg5 expression abrogates MPP+-induced neuronal loss, whereas it reduces neuronal survival in untreated cells. Data are means ± s.e.m.; n=3.(c) Knockdown of EndoB1 or Cdk5 expression abolishes MPP+-induced neuronal death. Data are means ± s.e.m.; n=8.(d) Overexpression of EndoB1T145A inhibits MPP+-induced neuronal death. Data are means ± s.e.m.; n=5.(e) Pretreatment with 3-MA, but not z-VAD-fmk, reduces α-synucleinA53T mutant-induced neuronal death. Data are means ± s.e.m.; n=6.(f) Knockdown of Atg5 expression abolishes α-synucleinA53T mutant-induced neuronal death, whereas it reduces neuronal survival in vector-transfected cells. Data are means ± s.e.m.; n=3.(g) Knockdown of EndoB1 or Cdk5 expression abolishes α-synucleinA53T mutant-induced neuronal death. Data are means ± s.e.m.; n=4.(h) Expression of EndoB1T145A inhibits α-synucleinA53T mutant-induced neuronal death. Data are means ± s.e.m.; n=6.


Figure 1A , Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times . B , H1299 cells transfected with a control siRNA ( siCont . ) or an RNF20 - specific siRNA ( siRNF20 ) and a wild - type H2B ( H2BWT ) or a K120R - mutated H2B ( H2BK120R ) were treated with 12 μM erastin ( + ) or untreated ( - ) for 24 h . Representative phase - contrast images were recorded ( magnification , × 20 ) , and the surviving cells were counted . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 , * * : p < 0 . 01 . C , Lipid ROS levels were assessed by flow cytometry after C11 - BODIPY staining in cells treated Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 , * * : p < 0 . 01 . D , H1299 cells treated were subjected to transmission electron microscopy . Representative images are shown . E , Control H1299 cells ( transfected with siCont . or H2BWT ) and H2Bub1 - depleted H1299 cells ( transfected with siRNF20 or H2BK120R ) were treated with erastin either with or without a ferroptosis - specific inhibitor , ferrostatin - 1 ( 2 μM ) , for 24 h . Representative phase - contrast images were recorded ( magnification , × 20 ) , and the surviving cells were counted . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

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Figure 1A, Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times.B, H1299 cells transfected with a control siRNA (siCont.) or an RNF20-specific siRNA (siRNF20) and a wild-type H2B (H2BWT) or a K120R-mutated H2B (H2BK120R) were treated with 12 μM erastin (+) or untreated (-) for 24 h. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.C, Lipid ROS levels were assessed by flow cytometry after C11-BODIPY staining in cells treated Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.D, H1299 cells treated were subjected to transmission electron microscopy. Representative images are shown.E, Control H1299 cells (transfected with siCont. or H2BWT) and H2Bub1-depleted H1299 cells (transfected with siRNF20 or H2BK120R) were treated with erastin either with or without a ferroptosis-specific inhibitor, ferrostatin-1 (2 μM), for 24 h. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


Figure 2A , qRT - PCR ( left ) and Western blot ( right ) analyses of H1299 cells transfected with a control siRNA ( siCont . ) or an RNF20 - specific siRNA ( siRNF20 ) and a wild - type H2B ( H2BWT ) or a K120R - mutated H2B ( H2BK120R ) for 24 h . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 , * * : p < 0 . 01 . B , Chromatin immunoprecipitation ( ChIP ) assay was carried out with anti - H2Bub1 antibodies in H1299 cells ( left ) or 293T cells either untreated or treated with 20 μM erastin for 24 h ( right ) . The intergenic region was used as a negative control for the occupancy of H2Bub1 . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 , * * : p < 0 . 01 . C , Intracellular GSH levels were examined in H1299 cells treated as indicated , and bar graphs are shown . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 , * * : p < 0 . 01 . D , H1299 cells transfected as indicated were treated with 12 μM erastin ( + ) or untreated ( - ) for 24 h . Representative phase - contrast images were recorded ( magnification , × 20 ) . E , Surviving cells from the assay shown in ( D ) were counted . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 , * * : p < 0 . 01 . G , Affected metal ion - binding genes were selected and subjected to cluster analysis . H , Labile iron levels were assessed by flow cytometry with a standard method in H1299 cells . I , Labile iron levels examined were quantified . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

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

Figure 2A, qRT-PCR (left) and Western blot (right) analyses of H1299 cells transfected with a control siRNA (siCont.) or an RNF20-specific siRNA (siRNF20) and a wild-type H2B (H2BWT) or a K120R-mutated H2B (H2BK120R) for 24 h. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.B, Chromatin immunoprecipitation (ChIP) assay was carried out with anti-H2Bub1 antibodies in H1299 cells (left) or 293T cells either untreated or treated with 20 μM erastin for 24 h (right). The intergenic region was used as a negative control for the occupancy of H2Bub1. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.C, Intracellular GSH levels were examined in H1299 cells treated as indicated, and bar graphs are shown. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.D, H1299 cells transfected as indicated were treated with 12 μM erastin (+) or untreated (-) for 24 h. Representative phase-contrast images were recorded (magnification, ×20). E, Surviving cells from the assay shown in (D) were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.G, Affected metal ion-binding genes were selected and subjected to cluster analysis.H, Labile iron levels were assessed by flow cytometry with a standard method in H1299 cells.I, Labile iron levels examined were quantified. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


Figure 3Western blot analysis was performed with H1299 or Hep3B cells transfected with a Flag - tagged p53 or an empty plasmid ( Cont . ) for 48 h . Western blot analysis was performed with H1299 cells transfected with a Flag - tagged p53 or an empty plasmid ( Cont . ) for 48 h . C , Immunofluorescence ( IF ) assay for H1299 cells transfected with a Flag - tagged p53 ( Flag - p53 ) or an empty plasmid ( Cont . ) for 48 h . Representative images are shown ( magnification , × 40 ) . Arrowhead indicates a Flag - p53 overexpressing cell . D , Western blot analysis was performed with H1299 transfected with a Flag - tagged p53 or an empty vector ( Cont . ) for 48 h . E , 293T cells transfected with a control siRNA ( siCont . ) and three p53 - specific siRNAs ( sip53 ) were subjected to Western blot analysis . F , SMMC cells were treated with 100 μM etoposide ( + ) or untreated ( - ) for 24 h , and Western blot analysis was performed . , Western blot analysis of A549 cells and H1299 cells . H , H1299 cells transfected with a Flag - tagged p53 or an empty plasmid ( Cont . ) were analyzed by micrococcal nuclease ( MNase ) digestion assay . I , Western blot analysis of H1299 cells transfected with a wild - type p53 ( p53 - WT ) or two " gain - of - function " mutants ( p53 - R273H and p53 - R175H ) for 48 h .

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Figure 3Western blot analysis was performed with H1299 or Hep3B cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) for 48 h.Western blot analysis was performed with H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) for 48 h.C, Immunofluorescence (IF) assay for H1299 cells transfected with a Flag-tagged p53 (Flag-p53) or an empty plasmid (Cont.) for 48 h. Representative images are shown (magnification, ×40). Arrowhead indicates a Flag-p53 overexpressing cell.D, Western blot analysis was performed with H1299 transfected with a Flag-tagged p53 or an empty vector (Cont.) for 48 h.E, 293T cells transfected with a control siRNA (siCont.) and three p53-specific siRNAs (sip53) were subjected to Western blot analysis.F, SMMC cells were treated with 100 μM etoposide (+) or untreated (-) for 24 h, and Western blot analysis was performed., Western blot analysis of A549 cells and H1299 cells.H, H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) were analyzed by micrococcal nuclease (MNase) digestion assay.I, Western blot analysis of H1299 cells transfected with a wild-type p53 (p53-WT) or two "gain-of-function" mutants (p53-R273H and p53-R175H) for 48 h.


Figure 4A , Coimmunoprecipitation ( Co - IP ) analysis was performed in A549 cells with antibodies against p53 or USP7 . B , H1299 cells were transfected with an empty vector ( Cont . ) , a Flag - tagged p53 , a USP7 - specific shRNA , or the last two plasmids together for 48 h . Western blot analysis was then performed . C , Nuclear and cytosolic extracts were differentially prepared from H1299 cells transfected with a Flag - tagged p53 or an empty plasmid ( Cont . ) . Western blot analysis was then performed . D , H1299 cells transfected with a Myc - tagged USP7 or a Myc - tagged USP7 together with a Flag - tagged p53 were subjected to IF analysis ( upper ) . A549 cells transfected with a p53 - specific siRNA or a control siRNA ( siCont . ) were examined by IF analysis , and the RNAi efficiency is shown ( lower ) . Representative images are shown ( magnification , × 40 ) . F , IF analysis of A549 cells transfected with a Myc - tagged wild - type USP7 ( Myc - USP7 - WT ) or three Myc - tagged truncated mutants . Representative images are shown ( magnification , × 40 ) . G , H1299 cells were transfected with a Flag - tagged p53 or an empty vector together with a USP7 - specific siRNA or a control siRNA as indicated for 48 h . Cells were then harvested and subjected to a MNase digestion assay .

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Figure 4A, Coimmunoprecipitation (Co-IP) analysis was performed in A549 cells with antibodies against p53 or USP7.B, H1299 cells were transfected with an empty vector (Cont.), a Flag-tagged p53, a USP7-specific shRNA, or the last two plasmids together for 48 h. Western blot analysis was then performed.C, Nuclear and cytosolic extracts were differentially prepared from H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.). Western blot analysis was then performed.D, H1299 cells transfected with a Myc-tagged USP7 or a Myc-tagged USP7 together with a Flag-tagged p53 were subjected to IF analysis (upper). A549 cells transfected with a p53-specific siRNA or a control siRNA (siCont.) were examined by IF analysis, and the RNAi efficiency is shown (lower). Representative images are shown (magnification, ×40).F, IF analysis of A549 cells transfected with a Myc-tagged wild-type USP7 (Myc-USP7-WT) or three Myc-tagged truncated mutants. Representative images are shown (magnification, ×40).G, H1299 cells were transfected with a Flag-tagged p53 or an empty vector together with a USP7-specific siRNA or a control siRNA as indicated for 48 h. Cells were then harvested and subjected to a MNase digestion assay.


Figure 5A , Co - IP analysis was performed in 293T cells with antibodies against p53 or H2Bub1 . Peptide pull - down assay was performed with synthesized peptidesD , Co - IP analysis for H1299 cells transfected with a Flag - tagged p53 or an empty plasmid ( Cont . ) treated with a USP7 - specific inhibitor P5091 .

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Figure 5A, Co-IP analysis was performed in 293T cells with antibodies against p53 or H2Bub1.Peptide pull-down assay was performed with synthesized peptidesD, Co-IP analysis for H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) treated with a USP7-specific inhibitor P5091.


Figure 6A , Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times . B , H1299 cells were transfected with Flag - tagged p53 for 24 h and then treated with 12 μM erastin or untreated for another 24 h . Co - IP analysis was performed with antibodies against Flag . C , H1299 cells transfected with Flag - tagged p53 were treated with erastin or untreated for 24 h . Nuclear and cytosolic extracts were differentially prepared and subjected to Western blot analysis . D , 293T cells transfected with a p53 - specific siRNA , a USP7 - specific siRNA , or a control siRNA ( siCont . ) were treated with 20 μM erastin or untreated for 24 h as indicated . Western blot analysis was then performed . , qRT - PCR ( above ) and Western blot ( below ) analyses for H1299 cells transfected with a Flag - tagged p53 or an empty plasmid together with a USP7 - specific siRNA or a control siRNA ( siCont . ) as indicated . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . F , ChIP analysis of H1299 cells transfected with a Flag - tagged p53 or an empty plasmid together with a USP7 - specific siRNA or a control siRNA ( siCont . ) as indicated . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . G , H1299 cells transfected with Flag - tagged p53 were treated with 12 μM erastin or untreated for 24 h . ChIP analysis with antibodies against Flag or USP7 was performed . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . H , 293T cells transfected with a p53 - specific siRNA , a USP7 - specific siRNA or a control were treated with 20 μM erastin or untreated for 24 h . ChIP analysis was performed with anti - H2Bub1 antibodies . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats .

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Figure 6A, Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times.B, H1299 cells were transfected with Flag-tagged p53 for 24 h and then treated with 12 μM erastin or untreated for another 24 h. Co-IP analysis was performed with antibodies against Flag.C, H1299 cells transfected with Flag-tagged p53 were treated with erastin or untreated for 24 h. Nuclear and cytosolic extracts were differentially prepared and subjected to Western blot analysis.D, 293T cells transfected with a p53-specific siRNA, a USP7-specific siRNA, or a control siRNA (siCont.) were treated with 20 μM erastin or untreated for 24 h as indicated. Western blot analysis was then performed., qRT-PCR (above) and Western blot (below) analyses for H1299 cells transfected with a Flag-tagged p53 or an empty plasmid together with a USP7-specific siRNA or a control siRNA (siCont.) as indicated. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.F, ChIP analysis of H1299 cells transfected with a Flag-tagged p53 or an empty plasmid together with a USP7-specific siRNA or a control siRNA (siCont.) as indicated. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.G, H1299 cells transfected with Flag-tagged p53 were treated with 12 μM erastin or untreated for 24 h. ChIP analysis with antibodies against Flag or USP7 was performed. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.H, 293T cells transfected with a p53-specific siRNA, a USP7-specific siRNA or a control were treated with 20 μM erastin or untreated for 24 h. ChIP analysis was performed with anti-H2Bub1 antibodies. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.


Figure 7A , H1299 cells transfected with a Flag - tagged p53 or an empty plasmid together with a USP7 - specific siRNA or a control siRNA ( siCont . ) as were treated with 12 μM erastin ( + ) or untreated ( - ) for 24 h as indicated . Representative phase - contrast images were recorded ( magnification , × 20 ) , and the surviving cells were counted . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 . B , H1299 cells treated were subjected to transmission electron microscopy . Representative images are shown . Arrow head indicates condensed and marginated chromatin . C , Lipid ROS levels were examined in cells treated Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 .

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

Figure 7A, H1299 cells transfected with a Flag-tagged p53 or an empty plasmid together with a USP7-specific siRNA or a control siRNA (siCont.) as were treated with 12 μM erastin (+) or untreated (-) for 24 h as indicated. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05.B, H1299 cells treated were subjected to transmission electron microscopy. Representative images are shown. Arrow head indicates condensed and marginated chromatin.C, Lipid ROS levels were examined in cells treated Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05.


Figure 1A ) Left panel : experimental strategy . Hematopoietic stem cells ( HSPC ) and progenitors were characterized and isolated using specific sets of surface markers . All cells express CD34 . Upon differentiation , progenitors start to express CD38 in contrast to HSPC ( blue ) . Progenitors engaged in the myeloid ( magenta ) and lymphoid ( green ) pathways express respectively CD33 and CD19 markers . Right panel : FACS gating strategy to isolate HSPC ( further referred as CD38 - ( blue box ) , and cells engaged in myeloid and lymphoid differentiation pathways ( referred to as CD33 + [ magenta box ] and CD19 + [ green box ] , respectively ) . B ) For each population , equatorial Z stacks of two representative nuclei are shown in the left panel ( chromatin is shown in blue ( DAPI staining ) and nuclear envelop in white ( Sun2 staining ) . Scale bar : 5 µm . The corresponding 3D reconstructions are presented in the right panel . Convex and non - convex surfaces are shown in blue and yellow , respectively . C ) Nucleus - invagination ratio is defined as the ratio of the non - convex area over the total nucleus surface area . Nuclei are larger and deformed in CD33 + ( magenta , n = 106 ) than CD38 - and CD19 + cells ( blue [ n = 120 ] and green [ n = 115 ] , respectively ; 3 independent experiments ; Bars correspond to mean ± SD ; * * * * : p < 0 . 0001 , Mann - Whitney test ; n . s : non significant ) .

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Figure 1A) Left panel: experimental strategy. Hematopoietic stem cells (HSPC) and progenitors were characterized and isolated using specific sets of surface markers. All cells express CD34. Upon differentiation, progenitors start to express CD38 in contrast to HSPC (blue). Progenitors engaged in the myeloid (magenta) and lymphoid (green) pathways express respectively CD33 and CD19 markers. Right panel: FACS gating strategy to isolate HSPC (further referred as CD38- (blue box), and cells engaged in myeloid and lymphoid differentiation pathways (referred to as CD33+ [magenta box] and CD19+ [green box], respectively).B) For each population, equatorial Z stacks of two representative nuclei are shown in the left panel (chromatin is shown in blue (DAPI staining) and nuclear envelop in white (Sun2 staining). Scale bar: 5 µm. The corresponding 3D reconstructions are presented in the right panel. Convex and non-convex surfaces are shown in blue and yellow, respectively. C) Nucleus-invagination ratio is defined as the ratio of the non-convex area over the total nucleus surface area. Nuclei are larger and deformed in CD33+ (magenta, n=106) than CD38- and CD19+ cells (blue [n=120] and green [n=115], respectively; 3 independent experiments; Bars correspond to mean ± SD; ****: p<0.0001, Mann-Whitney test; n.s: non significant).


Figure 2A ) For each population ( CD38 - and CD33 + cells ) , Z stacks of two representative cells are shown in the left panel . Microtubules are shown in green , actin in magenta , centrosome in white and chromatin in blue . In the middle panel , inverted image the corresponding actin staining is shown . In the left panel , the corresponding 3D projection of microtubules is presented as inverted image . Scale bar 5 µm . B ) Microtubules are tightly associated with the nucleus . Z stacks of representative CD38 - and CD33 + cells are shown . Microtubules are shown in green , nucleus envelope ( Sun2 immuno - staining ) in magenta and chromatin in blue . Scale bar 5 µm . C ) As schematized , centrosome - to - nucleus center distance ( d ) over nucleus center - to - nucleus convex envelope ( R ) ratio is calculated to extract relative position of the centrosome to the center . This parameter is significantly lower in myeloid progenitors ( n = 129 ) than in HSPC ( n = 200 ; 3 independent experiments ; * * * * ; p < 0 . 0001 . Mann - Whitney test ) indicating that centrosome becomes internalized within an invaginated region upon differentiation . D ) p150 glued associates with the nuclear membrane of HSPC ( CD38 - ) and myeloid progenitors ( CD33 + ) cells . Z stacks of representative cells are shown . Image contrast has been inverted . Stainings show DAPI , Sun2 and p150 glued . Scale bar 5µm . E ) The dynein adaptor BicD2 associates with the nuclear membrane of HSPC ( CD38 - ) and myeloid progenitors ( CD33 + ) cells . Z stacks of representative cells are shown . Image contrast has been inverted . Stainings show DAPI and BicD2 . Scale bar 5µm .

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

Figure 2A) For each population (CD38- and CD33+ cells), Z stacks of two representative cells are shown in the left panel. Microtubules are shown in green, actin in magenta, centrosome in white and chromatin in blue. In the middle panel, inverted image the corresponding actin staining is shown. In the left panel, the corresponding 3D projection of microtubules is presented as inverted image. Scale bar 5 µm.B) Microtubules are tightly associated with the nucleus. Z stacks of representative CD38- and CD33+ cells are shown. Microtubules are shown in green, nucleus envelope (Sun2 immuno-staining) in magenta and chromatin in blue. Scale bar 5 µm.C) As schematized, centrosome-to-nucleus center distance (d) over nucleus center-to-nucleus convex envelope (R) ratio is calculated to extract relative position of the centrosome to the center. This parameter is significantly lower in myeloid progenitors (n=129) than in HSPC (n=200; 3 independent experiments; ****; p<0.0001. Mann-Whitney test) indicating that centrosome becomes internalized within an invaginated region upon differentiation.D) p150 glued associates with the nuclear membrane of HSPC (CD38-) and myeloid progenitors (CD33+) cells. Z stacks of representative cells are shown. Image contrast has been inverted. Stainings show DAPI, Sun2 and p150 glued . Scale bar 5µm.E) The dynein adaptor BicD2 associates with the nuclear membrane of HSPC (CD38-) and myeloid progenitors (CD33+) cells. Z stacks of representative cells are shown. Image contrast has been inverted. Stainings show DAPI and BicD2. Scale bar 5µm.


Figure 3B ) The nucleus is progressively deformed over time in culture . For each time point , equatorial Z stacks of two representative cells are presented . Left panel : inverted image of chromatin ( DAPI ) . Right panel : microtubules are shown in green and chromatin in blue . Scale bar : 5 µm . C ) Nucleus volume ( left panel ) and invagination ratio ( right panel ) increase to attain levels similar to those of freshly isolated CD33 + cells ( 24 h , n = 165 , 4 donors ; 48 h , n = 143 , 3 donors ; and 72h , n = 192 , 4donors ; CD33 + , n = 227 , 5 donors ; Bars correspond to mean ± SD ; n . s : non significant ; * * * : p < 0 . 001 , * * * * : p < 0 . 0001 ; Mann Whitney test ) . D ) For each condition ( CTL , untreated cells ; + Bleb , blebbistatin - treated cells ; + Bleb + NZ , blebbistatin and nocodazole - treated cells ; + TX , taxol - treated cells ; and + CilioD , ciliobrevin - D - treated cells ) Z stacks of 2 representative cells are displayed . Microtubules are shown in green , chromatin in blue , centrosome in white and actin in magenta . Asterisks indicate regions where microtubules are not in contact with the nucleus . Scale bar 5 μm . F ) Quantifications of nuclear volume ( left panel ) , invagination ratio ( right panel ) in the indicated conditions . Non - treated cells at 48 hours ( n = 137 , 3 donors ) and 72 hours ( n = 143 , 3 donors ) ; and blebbistatin - treated cells ( n = 96 / 3 donors ) , blebbistatin and nocodazole treated cells ( n = 66 , 2 donors ) , taxol - treated cells ( n = 122 , 3 donors ) , and ciliobrevin - D - treated cells ( n = 128 , 3 donors ) . Bars correspond to mean ± SD ; * * * * : p < 0 . 0001 . * * : p < 0 . 01 , n . s : non significant ; Mann - Whitney test .

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Figure 3B) The nucleus is progressively deformed over time in culture. For each time point, equatorial Z stacks of two representative cells are presented. Left panel: inverted image of chromatin (DAPI). Right panel: microtubules are shown in green and chromatin in blue. Scale bar: 5 µm. C) Nucleus volume (left panel) and invagination ratio (right panel) increase to attain levels similar to those of freshly isolated CD33+ cells (24 h, n=165, 4 donors; 48 h, n=143, 3 donors; and 72h, n=192, 4donors; CD33+, n=227, 5 donors; Bars correspond to mean ± SD; n.s: non significant; ***: p<0.001, ****: p<0.0001; Mann Whitney test). D) For each condition (CTL, untreated cells; +Bleb, blebbistatin-treated cells; +Bleb+NZ, blebbistatin and nocodazole-treated cells; +TX, taxol-treated cells; and +CilioD, ciliobrevin-D-treated cells) Z stacks of 2 representative cells are displayed. Microtubules are shown in green, chromatin in blue, centrosome in white and actin in magenta. Asterisks indicate regions where microtubules are not in contact with the nucleus. Scale bar 5 μm.F) Quantifications of nuclear volume (left panel), invagination ratio (right panel) in the indicated conditions. Non-treated cells at 48 hours (n=137, 3 donors) and 72 hours (n=143, 3 donors); and blebbistatin-treated cells (n= 96/3 donors), blebbistatin and nocodazole treated cells (n=66, 2 donors), taxol-treated cells (n=122, 3 donors), and ciliobrevin-D-treated cells (n=128, 3 donors). Bars correspond to mean ± SD; ****: p<0.0001. **: p<0.01, n.s: non significant; Mann-Whitney test.


Figure 4A ) Spatial distribution of lamin A / C in the nuclei of HSPC ( CD38 - ) , myeloid progenitors ( CD33 + ) and lymphoid progenitors ( CD19 + ) . Inverted images of equatorial Z plane of DAPI and lamin A / C are shown . Scale bar : 5 μm . B ) Line scans of lamin A / C signal along nucleus contour of representative cells are presented for each cell population . The corresponding variations in lamin A / C intensity and nucleus curvature are plotted in blue and in red , respectively . Negative curvature values correspond to local nuclear invaginations . Red asterisk underlines local nucleus invaginations . C ) Dispersion of A / C signal along the nucleus periphery is similarly low in CD38 - ( n = 17 , 3 donors ) , CD33 + ( n = 61 , 3 donors ) and CD19 + ( n = 11 , 3 donors ) indicative of an homogenous distribution of the protein along the nuclear membrane . ( Bars correspond to mean ± SD ; n . s : non significant ; Mann Whitney test ) . D ) Dispersion of lamin B signal along the nucleus periphery is similarly low in CD38 - ( n = 131 , 3 donors ) , and CD19 + ( n = 74 , 3 donors ) indicative of a homogenous distribution of the protein along the nuclear membrane . But is significantly increased in CD33 + ( n = 146 , 3 donors ) . Bars correspond to mean ± SD ; * * * * : p < 0 . 0001 ; Mann Whitney test . E - F - G ) Spatial distribution of lamin B and local measurements of lamin B intensity and curvature of nuclear membrane in CD38 - ( E ) , CD33 + ( F ) and CD19 + ( G ) cells . Inverted images of the equatorial Z plane of DAPI ( left panel ) and lamin B ( right panel ) of a representative nucleus are shown for each population . Scale bar : 5 μm . The variations in lamin B signal intensity and curvature are plotted in blue in red , respectively . Asterisks indicate local invaginations of the nuclear membrane . In the bottom graphs , positive and negative curvatures are shown in grey and red , respectively . The absence of curvature ( curvature = 0 ) is highlighted as a dashed line . In CD33 + cells , negative curvature of the nuclear membrane are associated with low lamin B intensities .

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

Figure 4A) Spatial distribution of lamin A/C in the nuclei of HSPC (CD38-), myeloid progenitors (CD33+) and lymphoid progenitors (CD19+). Inverted images of equatorial Z plane of DAPI and lamin A/C are shown. Scale bar: 5 μm. B) Line scans of lamin A/C signal along nucleus contour of representative cells are presented for each cell population. The corresponding variations in lamin A/C intensity and nucleus curvature are plotted in blue and in red, respectively. Negative curvature values correspond to local nuclear invaginations. Red asterisk underlines local nucleus invaginations. C) Dispersion of A/C signal along the nucleus periphery is similarly low in CD38- (n=17, 3 donors), CD33+ (n=61, 3 donors) and CD19+ (n=11, 3 donors) indicative of an homogenous distribution of the protein along the nuclear membrane. (Bars correspond to mean ± SD; n.s: non significant; Mann Whitney test). D) Dispersion of lamin B signal along the nucleus periphery is similarly low in CD38- (n=131, 3 donors), and CD19+ (n=74, 3 donors) indicative of a homogenous distribution of the protein along the nuclear membrane. But is significantly increased in CD33+ (n=146, 3 donors). Bars correspond to mean ± SD; ****:p<0.0001; Mann Whitney test. E-F-G) Spatial distribution of lamin B and local measurements of lamin B intensity and curvature of nuclear membrane in CD38- (E), CD33+ (F) and CD19+ (G) cells. Inverted images of the equatorial Z plane of DAPI (left panel) and lamin B (right panel) of a representative nucleus are shown for each population. Scale bar: 5 μm. The variations in lamin B signal intensity and curvature are plotted in blue in red, respectively. Asterisks indicate local invaginations of the nuclear membrane. In the bottom graphs, positive and negative curvatures are shown in grey and red, respectively. The absence of curvature (curvature =0) is highlighted as a dashed line. In CD33+ cells, negative curvature of the nuclear membrane are associated with low lamin B intensities.


Figure 5A ) Spatial distribution of H3K9me3 in CD38 - , CD33 + and CD19 + cells . For each population , two representative nuclei are presented . Inverted images of equatorial Z plane of DAPI ( left panel ) and H3K9me3 ( right panel ) are shown . Scale bar : 5 µm . B ) Spatial distribution of H3K9me3 at the indicated time points of differentiation of CD38 - cells . For each population , two representative nuclei are presented . Inverted images of equatorial Z plane of DAPI ( left panel ) and H3K9me3 ( right panel ) are shown . Scale bar : 5 µm . C ) For each cell , the raw DAPI image at the equatorial Z stack is used to extract the nucleus contour and measure H3K9Me3 intensity ( in arbitrary units [ a . u ] ) along the contour ( lower row ) . D ) H3K9me3 loss from the nucleus periphery occurs locally in nuclear invaginations . Line scans of H3K9me3 signal along nucleus contour of 2 representative cells are presented in the upper panel for each time point . Asterisks indicate the local loss of H3K9me3 . The corresponding variations in H3K9Me3 intensity and curvature are plotted in blue in red , respectively . Negative curvature values correspond to local nuclear invaginations . E ) Local curvature of the nucleus envelope as a function of H3K9Me3 local intensity at the indicated time points of differentiation . Positive and negative curvatures are shown in grey and red , respectively . The absence of curvature is highlighted as a dashed line . During differentiation , negatively curved domains increase in number and size and display low H3K9me3 intensities . F ) H3K9me3 redistribution during differentiation . H3K9me3 signal dispersion at the nucleus periphery is low in CD38 - ( n = 137 , 3 donors ) indicating that H3K9me3 is homogenously distributed at the nucleus periphery . Upon differentiation , H3K9me3 signal dispersion remains homogenous after 24 hours in culture ( n = 115 , 3 donors ) , but progressively becomes heterogeneous ( 48 h : n = 139 , 3 donors ) to attain a level at 72 h ( n = 148 , 3 donors ) similar to that in freshly collected CD33 + cells ( n = 124 , 3 donors ) , indicative of H3K9me3 redistribution from the nucleus periphery . Bars correspond to mean ± SD ; * * * : p < 0 . 001 ; * * : p < 0 . 01 ; n . s : non significant ; Mann Whitney test . G ) Microtubule perturbations impair H3K9me3 redistribution during HSPC differentiation . Microtubule - modifying agents were added to culture medium between 48 and 72 hours . Cells were fixed and analyzed after 72 hours of culture . For each indicated treatment , one representative nucleus is presented . Inverted images of equatorial Z stacks of DAPI ( left panel ) and H3K9me3 ( right panel ) are shown . Scale bar : 5 µm . H ) H3K9me3 signal dispersion significantly increases in non - treated cells between 48h ( n = 139 / 3 donors ) and 72h ( n = 148 / 3 donors ) of culture . At 72h , the dispersion is similar in non - treated and blebbistatin - treated cells ( n = 124 / 3 donors ) . By contrast , H3K9me3 signal dispersion is significantly reduced compared with non - treated cells after treatment with nocodazole and blebbistatin ( n = 55 , 2donors ) , taxol ( n = 109 , 3 donors ) and ciliobrevin - D ( n = 144 , 3donors ) to a level attained by non - treated cells at 48 hours , demonstrating that microtubules affect H3K9me3 redistribution during HSPC differentiation . Bars correspond to mean ± SD ; * * * * : p < 0 . 0001 ; n . s : non significant ; Mann Whitney test .

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

Figure 5A) Spatial distribution of H3K9me3 in CD38-, CD33+ and CD19+ cells. For each population, two representative nuclei are presented. Inverted images of equatorial Z plane of DAPI (left panel) and H3K9me3 (right panel) are shown. Scale bar: 5 µm.B) Spatial distribution of H3K9me3 at the indicated time points of differentiation of CD38- cells. For each population, two representative nuclei are presented. Inverted images of equatorial Z plane of DAPI (left panel) and H3K9me3 (right panel) are shown. Scale bar: 5 µm.C) For each cell, the raw DAPI image at the equatorial Z stack is used to extract the nucleus contour and measure H3K9Me3 intensity (in arbitrary units [a.u]) along the contour (lower row).D) H3K9me3 loss from the nucleus periphery occurs locally in nuclear invaginations. Line scans of H3K9me3 signal along nucleus contour of 2 representative cells are presented in the upper panel for each time point. Asterisks indicate the local loss of H3K9me3. The corresponding variations in H3K9Me3 intensity and curvature are plotted in blue in red, respectively. Negative curvature values correspond to local nuclear invaginations.E) Local curvature of the nucleus envelope as a function of H3K9Me3 local intensity at the indicated time points of differentiation. Positive and negative curvatures are shown in grey and red, respectively. The absence of curvature is highlighted as a dashed line. During differentiation, negatively curved domains increase in number and size and display low H3K9me3 intensities.F) H3K9me3 redistribution during differentiation. H3K9me3 signal dispersion at the nucleus periphery is low in CD38- (n=137, 3 donors) indicating that H3K9me3 is homogenously distributed at the nucleus periphery. Upon differentiation, H3K9me3 signal dispersion remains homogenous after 24 hours in culture (n=115, 3 donors), but progressively becomes heterogeneous (48 h: n=139, 3 donors) to attain a level at 72 h (n= 148, 3 donors) similar to that in freshly collected CD33+ cells (n=124, 3 donors), indicative of H3K9me3 redistribution from the nucleus periphery. Bars correspond to mean ± SD; ***:p<0.001; **:p<0.01; n.s: non significant; Mann Whitney test.G) Microtubule perturbations impair H3K9me3 redistribution during HSPC differentiation. Microtubule-modifying agents were added to culture medium between 48 and 72 hours. Cells were fixed and analyzed after 72 hours of culture. For each indicated treatment, one representative nucleus is presented. Inverted images of equatorial Z stacks of DAPI (left panel) and H3K9me3 (right panel) are shown. Scale bar: 5 µm. H) H3K9me3 signal dispersion significantly increases in non-treated cells between 48h (n=139/3 donors) and 72h (n=148/3 donors) of culture. At 72h, the dispersion is similar in non-treated and blebbistatin-treated cells (n=124/ 3 donors). By contrast, H3K9me3 signal dispersion is significantly reduced compared with non-treated cells after treatment with nocodazole and blebbistatin (n=55, 2donors), taxol (n= 109, 3 donors) and ciliobrevin-D (n=144, 3donors) to a level attained by non-treated cells at 48 hours, demonstrating that microtubules affect H3K9me3 redistribution during HSPC differentiation. Bars correspond to mean ± SD; ****:p<0.0001; n.s: non significant; Mann Whitney test.


Figure 6A ) Microtubules perturbations impact the trancriptome of differentiating cells . Volcano - plot representation of down - regulated ( blue ) and up - regulated genes ( red ) in non - treated / control cells relative to ciliobrevin - D treated cells . Each circle represents the fold change ( FC ) of the quantity of mRNA and the significance of the change based on three independent replicates . Expressed genes with a false discovery rate ( FDR ) lower than 0 . 05 are shown in grey , whereas genes with a FDR greater than 0 . 05 are shown in blue or red . Genes significantly mis - regulated i . e . with an absolute value of FC higher than 1 . 8 are shown as full disks . Names of genes of interest are indicated within the graph . Genes that are in the signature of the myeloid - differentiation pathway are shown in magenta . Genes that are the signature of the megakaryocyte - differentiation pathway are shown in green . B ) Heatmap representation and hierarchical clustering of gene expression changes between non - treated and ciliobrevin - D - treated cells collected from 3 donors in each condition . The color code corresponds to a log2 scale of the differential expression levels .

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

Figure 6A) Microtubules perturbations impact the trancriptome of differentiating cells. Volcano-plot representation of down-regulated (blue) and up-regulated genes (red) in non-treated/control cells relative to ciliobrevin-D treated cells. Each circle represents the fold change (FC) of the quantity of mRNA and the significance of the change based on three independent replicates. Expressed genes with a false discovery rate (FDR) lower than 0.05 are shown in grey, whereas genes with a FDR greater than 0.05 are shown in blue or red. Genes significantly mis-regulated i.e. with an absolute value of FC higher than 1.8 are shown as full disks. Names of genes of interest are indicated within the graph. Genes that are in the signature of the myeloid-differentiation pathway are shown in magenta. Genes that are the signature of the megakaryocyte-differentiation pathway are shown in green. B) Heatmap representation and hierarchical clustering of gene expression changes between non-treated and ciliobrevin-D-treated cells collected from 3 donors in each condition. The color code corresponds to a log2 scale of the differential expression levels.


Figure 1 ( B ) Wing size variation in absolute centroid size ( CS ) and relative CS ( adjusted for body size ) . ( C ) Reproducibility of RNA - seq experiment . Pairwise Spearman correlation coefficients between RNA levels showed higher correlations among biological replicates than among non - replicates . ( D ) Comparable variability of RNA and protein levels for genes quantified at both levels . ( E ) RNAs of the genes quantified at both RNA and protein levels have the largest means and smallest variabilities among the whole RNAs quantified . ( F ) Weak correlation between RNA and protein levels . The median Spearman correlation coefficient was 0 . 1 and only 18 % of genes compared showed a positive correlation with statistical significance ( p < 0 . 05 ) .

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

Figure 1(B) Wing size variation in absolute centroid size (CS) and relative CS (adjusted for body size).(C) Reproducibility of RNA-seq experiment. Pairwise Spearman correlation coefficients between RNA levels showed higher correlations among biological replicates than among non-replicates.(D) Comparable variability of RNA and protein levels for genes quantified at both levels.(E) RNAs of the genes quantified at both RNA and protein levels have the largest means and smallest variabilities among the whole RNAs quantified.(F) Weak correlation between RNA and protein levels. The median Spearman correlation coefficient was 0.1 and only 18 % of genes compared showed a positive correlation with statistical significance (p < 0.05).


Figure 2 ( B ) Association of RNAs with the whole wing size variation that encompasses both sexes . Volcano plot of p - values against the slope of the fitted lines . The horizontal lines indicate 10 and 20 % FDR thresholds . ( C ) Association of RNAs with between - line wing size variation ( absolute / relative CS , adjusted for sex ) . ( E ) Association of canonical growth regulator transcripts with wing size ( absCS and relCS ) . P - values from the tests for association type 2 ( ANCOVA ) and type 3 ( ANOVA on sex ) are plotted . The dashed and dotted lines indicate 10 % and 20 % FDR thresholds , respectively . Note that canonical growth genes mostly exhibit type - 3 association . ( F ) Sexually dimorphic expression pattern of canonical growth genes . Plots of RNA levels confirm type - 3 association with wing size . ( G ) Exceptional growth genes that are associated with between - line size variation ( association type 2 ) .

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

Figure 2(B) Association of RNAs with the whole wing size variation that encompasses both sexes. Volcano plot of p-values against the slope of the fitted lines. The horizontal lines indicate 10 and 20 % FDR thresholds.(C) Association of RNAs with between-line wing size variation (absolute/ relative CS, adjusted for sex).(E) Association of canonical growth regulator transcripts with wing size (absCS and relCS). P-values from the tests for association type 2 (ANCOVA) and type 3 (ANOVA on sex) are plotted. The dashed and dotted lines indicate 10% and 20% FDR thresholds, respectively. Note that canonical growth genes mostly exhibit type-3 association.(F) Sexually dimorphic expression pattern of canonical growth genes. Plots of RNA levels confirm type-3 association with wing size.(G) Exceptional growth genes that are associated with between-line size variation (association type 2).


Figure 3 ( C ) The ratio of compartment sizes ( P area divided by A area ) is plotted . The 14 genes were knocked down using multiple ( 2 or 3 ) RNAi lines per gene . The x - axis indicates RNAi case IDs that represent the genes knocked down and the RNAi line type ( KK , GD , or TP as TRiP ) . Detailed information is provided in Dataset EV6 . The horizontal line indicates the size ratio in the negative control cases . Wilcoxon rank sum tests evaluated the statistical significance against controls ( * : p < 0 . 05 , * * * : p < 0 . 001 , n . s . : not significant , cont . : control cases ) . ( D ) Adult wings with RNAi manipulation in the P compartment ( shaded area ) . Note that the boundary lines between A and P compartments , which are relatively straight in the control cases , bent towards the P compartment due to the reduced growth and size in the P compartment .

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

Figure 3(C) The ratio of compartment sizes (P area divided by A area) is plotted. The 14 genes were knocked down using multiple (2 or 3) RNAi lines per gene. The x-axis indicates RNAi case IDs that represent the genes knocked down and the RNAi line type (KK, GD, or TP as TRiP). Detailed information is provided in Dataset EV6. The horizontal line indicates the size ratio in the negative control cases. Wilcoxon rank sum tests evaluated the statistical significance against controls (*:p <0.05, ***:p<0.001, n.s.: not significant, cont.: control cases).(D) Adult wings with RNAi manipulation in the P compartment (shaded area). Note that the boundary lines between A and P compartments, which are relatively straight in the control cases, bent towards the P compartment due to the reduced growth and size in the P compartment.


Figure 4 ( A ) Cis - association of SNP variation with RNA expression for females and males separately . P - values for eQTLs at FDR < 20 % are plotted against the effect size of the SNPs . The top SNPs are labeled with the corresponding gene names . ( D and E ) Cis - association of SNP variation with protein expression for females and males . P - value for each pQTL is plotted against the effect size of the SNP . pQTLs at FDRs of 20 % and 30 % are depicted for females and males , respectively . The corresponding proteins are designated with distinct colors . The circle indicates that the pQTL is also eQTL for the same gene .

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

Figure 4(A) Cis-association of SNP variation with RNA expression for females and males separately. P-values for eQTLs at FDR < 20% are plotted against the effect size of the SNPs. The top SNPs are labeled with the corresponding gene names.(D and E) Cis-association of SNP variation with protein expression for females and males. P-value for each pQTL is plotted against the effect size of the SNP. pQTLs at FDRs of 20% and 30% are depicted for females and males, respectively. The corresponding proteins are designated with distinct colors. The circle indicates that the pQTL is also eQTL for the same gene.


Figure 5 ( B ) Selection of mediator RNAs and the linked eQTLs . RNAs co - associated with SNPs and wing size traits are further tested whether the SNPs are also associated with wing size traits ( absCS and relCS ) for each sex . The dotted lines indicate a significance threshold ( p = 0 . 05 ) . ( C and D ) RNAi of CG34015 in the wing P - compartment significantly reduces the size in both sexes . The images are taken from female wings . Statistical significance was evaluated by Wilcoxon test . ( E ) Genome - wide association of CG34015 expression . Manhattan plot shows a strong association of CG34015 RNA with X chromosome in both sexes . P - values are plotted against ordered SNPs along the genome . eQTLs associated with CG34015 are designated as red dots . ( F ) Zoomed plot around the CG34015 gene region on X chromosome . eQTLs linked to CG34015 are mostly located upstream of the CG34015 gene region . The red arrow indicates the transcriptional direction . ( G ) SNP variation of CG34015 - linked eQTLs . SNP states at each eQTL are depicted for each line using different colors ( grey dots : reference SNP , blue dots : alternate SNP with a negative effect on RNA level ) . Lines are ordered from left to right as CG34015 RNA levels increase . Note that the eQTLs are highly linked with each other . ( H and I ) Segregation of CG34015 RNA levels and wing size ( absCS ) by the variation of an eQTL ( X _ 16344746 _ SNP ) . P - values are shown . ( J ) Association of the eQTL ( X _ 16344746 _ SNP ) with population - wide , wing size variation among 143 DGRP lines . P - value and R2 are shown for each sex .

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

Figure 5(B) Selection of mediator RNAs and the linked eQTLs. RNAs co-associated with SNPs and wing size traits are further tested whether the SNPs are also associated with wing size traits (absCS and relCS) for each sex. The dotted lines indicate a significance threshold (p=0.05).(C and D) RNAi of CG34015 in the wing P-compartment significantly reduces the size in both sexes. The images are taken from female wings. Statistical significance was evaluated by Wilcoxon test.(E) Genome-wide association of CG34015 expression. Manhattan plot shows a strong association of CG34015 RNA with X chromosome in both sexes. P-values are plotted against ordered SNPs along the genome. eQTLs associated with CG34015 are designated as red dots.(F) Zoomed plot around the CG34015 gene region on X chromosome. eQTLs linked to CG34015 are mostly located upstream of the CG34015 gene region. The red arrow indicates the transcriptional direction.(G) SNP variation of CG34015-linked eQTLs. SNP states at each eQTL are depicted for each line using different colors (grey dots: reference SNP, blue dots: alternate SNP with a negative effect on RNA level). Lines are ordered from left to right as CG34015 RNA levels increase. Note that the eQTLs are highly linked with each other.(H and I) Segregation of CG34015 RNA levels and wing size (absCS) by the variation of an eQTL (X_16344746_SNP). P-values are shown. (J) Association of the eQTL (X_16344746_SNP) with population-wide, wing size variation among 143 DGRP lines. P-value and R2 are shown for each sex.


Figure 1IFNγ enhances macrophage host cell death after type I ( RH ) and type II ( Pru ) Toxoplasma gondii ( Tg ) infection . Graphs show cell death as measured by LDH release assay ( left ) and viability measured by XTT assay ( right ) in untreated or IFNγ - primed THP - 1 cells infected with type I or type II Tg for 24 h . LDH release assays from THP - 1 cells left untreated or primed with IFNγ , transfected with siRNA against indicated GBPs or non - targeting control ( CTRL ) and infected with indicated strains of Tg for 24 h . LDH release assay from primary monocyte - derived macrophages ( MDM ) left untreated or treated with IFNγ , transfected with siRNA against GBP1 or non - targeting control ( CTRL ) and infected with indicated strain of Tg for 24 hours . Mean ± SEM of n = 4 donors shown . LDH release assay from indicated wildtype , GBP1 knockout ( ∆ GBP1 ) or GBP1 reconstituted ( ∆ GBP1 + Tet - GBP1 ) cells infected with type I or type II Tg for 24 hours . Cells were untreated or treated with IFNγ or additionally treated with doxycycline ( Dox ) as indicated . Realtime propidium iodide ( PI ) uptake assay from indicated THP - 1 cells infected with type I or type II Tg , and untreated or treated with IFNγ and / or Dox as labelled . AU , arbitrary units . LDH release assay from wildtype THP - 1 or ∆ GBP1 cells stably reconstituted with Dox - inducible expression plasmids of the indicated mutants of GBP1 . Cells were pre - treated with IFNγ and Dox and infected with either type I or type II Tg for 24 h .

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

Figure 1IFNγ enhances macrophage host cell death after type I (RH) and type II (Pru) Toxoplasma gondii (Tg) infection. Graphs show cell death as measured by LDH release assay (left) and viability measured by XTT assay (right) in untreated or IFNγ-primed THP-1 cells infected with type I or type II Tg for 24 h.LDH release assays from THP-1 cells left untreated or primed with IFNγ, transfected with siRNA against indicated GBPs or non-targeting control (CTRL) and infected with indicated strains of Tg for 24 h.LDH release assay from primary monocyte-derived macrophages (MDM) left untreated or treated with IFNγ, transfected with siRNA against GBP1 or non-targeting control (CTRL) and infected with indicated strain of Tg for 24 hours. Mean ± SEM of n = 4 donors shown.LDH release assay from indicated wildtype, GBP1 knockout (∆GBP1) or GBP1 reconstituted (∆GBP1+Tet-GBP1) cells infected with type I or type II Tg for 24 hours. Cells were untreated or treated with IFNγ or additionally treated with doxycycline (Dox) as indicated.Realtime propidium iodide (PI) uptake assay from indicated THP-1 cells infected with type I or type II Tg, and untreated or treated with IFNγ and/or Dox as labelled. AU, arbitrary units.LDH release assay from wildtype THP-1 or ∆GBP1 cells stably reconstituted with Dox-inducible expression plasmids of the indicated mutants of GBP1. Cells were pre-treated with IFNγ and Dox and infected with either type I or type II Tg for 24 h.


Figure 2Propidium iodide ( PI ) uptake in indicated wildtype THP - 1 or ∆ CASP - 1 , ∆ CASP - 4 , ∆ ASC cells , or THP - 1 stably expressing GSDMD - targeting miRNA ( GSDMDmiR ) infected with the indicated strains of Toxoplasma gondii ( Tg ) for 24 h . Cells were untreated or treated with IFNγ as indicated . Mean area under the curve ( AUC ) ± SEM from n = 3 experiments are shown . ns , not significant , from two - way ANOVA following adjustment for multiple comparisons . Real - time apoptosis assay ( Annexin V ( AnnV ) - Glo luminescence ) from IFNγ - primed primary human MDMs transfected with non - targeting control ( siCTRL ; left ) or GBP1 siRNA ( siGBP1 ; right ) and left uninfected or infected with type I or type II Tg . Depicted is the mean ± SEM of n = 4 donors . AU , arbitrary units . Immunoblots from primary MDMs infected with the indicated Tg strains for 12 h . Cells were untreated or pre - treated with IFNγ before infection . Treatment with TNFα and cycloheximide ( CHX ) served as positive control for apoptosis . Images are representative of n = 4 biologically independent experiments . Live time - lapse imaging to visualize apoptotic cell death and measure caspase - 3 / 7 activities using a fluorescent caspase substrate . THP - 1 WT were primed with IFNγ or left untreated and infected with either type I or type II Tg . Plot in ( D ) shows cumulative caspase - 3 / 7 activity from an 18 - hour time - course ( mean ± SEM from n = 3 independent experiments ) . Representative phase - contrast images overlaid with fluorescence signal for caspase - 3 / 7 activity ( green ) images are shown in ( E ) from IFNγ - primed THP - 1 cells infected with type I Tg captured at indicated times post - infection . Scale bar 20 μm . AnnV - Glo assay from IFNγ - primed THP - 1 transfected with siRNA against indicated human caspases and infected with type I or type II Tg . Area under the curve ( AUC ) from a real - time AnnV - Glo luminescence assays similar to those in ( B ) are plotted as mean ± SEM from n = 3 independent experiments . AnnV - Glo assay from IFNγ - primed primary human MDMs transfected with siRNA against CASP8 , CASP10 and non - targeting control ( CTRL ) and infected with type I or type II Tg . Area under the curve ( AUC ) from real - time assays similar to those in ( B ) are plotted from n = 4 independent experiments . Matched shapes and color of symbols indicate donors .

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

Figure 2Propidium iodide (PI) uptake in indicated wildtype THP-1 or ∆CASP-1, ∆CASP-4, ∆ASC cells, or THP-1 stably expressing GSDMD-targeting miRNA (GSDMDmiR) infected with the indicated strains of Toxoplasma gondii (Tg) for 24 h. Cells were untreated or treated with IFNγ as indicated. Mean area under the curve (AUC) ± SEM from n = 3 experiments are shown. ns, not significant, from two-way ANOVA following adjustment for multiple comparisons.Real-time apoptosis assay (Annexin V (AnnV)-Glo luminescence) from IFNγ-primed primary human MDMs transfected with non-targeting control (siCTRL; left) or GBP1 siRNA (siGBP1; right) and left uninfected or infected with type I or type II Tg. Depicted is the mean ± SEM of n = 4 donors. AU, arbitrary units.Immunoblots from primary MDMs infected with the indicated Tg strains for 12 h. Cells were untreated or pre-treated with IFNγ before infection. Treatment with TNFα and cycloheximide (CHX) served as positive control for apoptosis. Images are representative of n = 4 biologically independent experiments.Live time-lapse imaging to visualize apoptotic cell death and measure caspase-3/7 activities using a fluorescent caspase substrate. THP-1 WT were primed with IFNγ or left untreated and infected with either type I or type II Tg. Plot in (D) shows cumulative caspase-3/7 activity from an 18-hour time-course (mean ± SEM from n = 3 independent experiments). Representative phase-contrast images overlaid with fluorescence signal for caspase-3/7 activity (green) images are shown in (E) from IFNγ-primed THP-1 cells infected with type I Tg captured at indicated times post-infection. Scale bar 20 μm.AnnV-Glo assay from IFNγ-primed THP-1 transfected with siRNA against indicated human caspases and infected with type I or type II Tg. Area under the curve (AUC) from a real-time AnnV-Glo luminescence assays similar to those in (B) are plotted as mean ± SEM from n = 3 independent experiments.AnnV-Glo assay from IFNγ-primed primary human MDMs transfected with siRNA against CASP8, CASP10 and non-targeting control (CTRL) and infected with type I or type II Tg. Area under the curve (AUC) from real-time assays similar to those in (B) are plotted from n = 4 independent experiments. Matched shapes and color of symbols indicate donors.


Figure 3AnnV - Glo assay from IFNγ - primed THP - 1 transfected with siRNA against indicated human NLR / ALR sensors and infected with type I or type II Toxoplasma gondii ( Tg ) . Area under the curve ( AUC ) from real - time assays are plotted as mean ± SEM from n = 3 independent experiments . AnnV - Glo assay from IFNγ - primed primary human MDMs transfected with siRNA against AIM2 or non - targeting control ( CTRL ) and infected with type I or type II Tg . Area under the curve ( AUC ) from real - time assays are plotted from n = 4 independent experiments . Matched shapes and color of symbols indicate donors . Representative images ( top ) from IFNγ - primed THP - 1 WT infected with type I Tg for 8 hours and stained for ASC and active caspase - 8 . Blue , Nuclei ; Red , ASC ; Green , active caspase - 8 ( IETD - FITC ) and Grey , Tg . Scale bar , 5 μm or 2 μm in the magnified images . Quantification of ASC specks ( bottom ) in Tg - infected THP - 1 WT or ∆ CASP1 treated with IFNγ or left untreated at 8 hours post infection , plotted as mean ± SEM from n = 3 independent experiment . Immunoblots from primary MDMs infected with Tg strains for indicated times . Cells were untreated or pre - treated with IFNγ before infection . Images are representative of n = 4 biologically independent experiments .

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Figure 3AnnV-Glo assay from IFNγ-primed THP-1 transfected with siRNA against indicated human NLR/ALR sensors and infected with type I or type II Toxoplasma gondii (Tg). Area under the curve (AUC) from real-time assays are plotted as mean ± SEM from n = 3 independent experiments.AnnV-Glo assay from IFNγ-primed primary human MDMs transfected with siRNA against AIM2 or non-targeting control (CTRL) and infected with type I or type II Tg. Area under the curve (AUC) from real-time assays are plotted from n = 4 independent experiments. Matched shapes and color of symbols indicate donors.Representative images (top) from IFNγ-primed THP-1 WT infected with type I Tg for 8 hours and stained for ASC and active caspase-8. Blue, Nuclei; Red, ASC; Green, active caspase-8 (IETD-FITC) and Grey, Tg. Scale bar, 5 μm or 2 μm in the magnified images. Quantification of ASC specks (bottom) in Tg-infected THP-1 WT or ∆CASP1 treated with IFNγ or left untreated at 8 hours post infection, plotted as mean ± SEM from n = 3 independent experiment.Immunoblots from primary MDMs infected with Tg strains for indicated times. Cells were untreated or pre-treated with IFNγ before infection. Images are representative of n = 4 biologically independent experiments.


Figure 4Representative images ( A ) and quantification ( B ) from IFNγ - primed THP - 1 ∆ GBP1 stably reconstituted with Tet - mCH - GBP1 , mCH - GBP1K51A , mCH - GBP1C589A or mCH - GBP1 ∆ 589 - 592 variants or monocyte derived macrophages ( MDM ) stained for GBP1 . Cells were infected with type I or type II Toxoplasma gondii ( Tg ) for 2 h . Blue , Nuclei ; Grey , Tg ; Red , mCH - GBP1 or Magenta , GBP1 . Scale bar , 5 μm . Graph in ( B ) shows mean percentage of Tg vacuoles targeted by GBP1 from n = 4 experiments .

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Figure 4Representative images (A) and quantification (B) from IFNγ-primed THP-1 ∆GBP1 stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or monocyte derived macrophages (MDM) stained for GBP1. Cells were infected with type I or type II Toxoplasma gondii (Tg) for 2 h. Blue, Nuclei; Grey, Tg; Red, mCH-GBP1 or Magenta, GBP1. Scale bar, 5 μm. Graph in (B) shows mean percentage of Tg vacuoles targeted by GBP1 from n = 4 experiments.


Figure 5LDH release assay from indicated THP - 1 cells untreated or treated with IFNγ and infected with Salmonella Typhimurium ( STm ) WT or a SPI - 1 mutant ΔinvA for 4 h at indicated multiplicities of infection ( MOI ) . Mean ± SEM of n = 3 experiments are shown . Propidium iodide ( PI ) uptake assay from IFNγ - primed primary MDMs transfected with non - targeting control siRNA ( CTRL ) or siRNA against GBP1 ( left ) or indicated lines of THP - 1 ( right ) infected with STm - GFP for 4 h . Area under the curve ( AUC ) from real - time assay plotted as mean ± SEM from n = 3 independent experiments . PI uptake assay from IFNγ - primed primary MDMs transfected with indicated siRNA or non - targeting control ( CTRL ) and infected with STm - GFP for 4 h . Area under the curve ( AUC ) from real - time assay plotted as mean ± SEM from n = 4 independent experiments . PI uptake assay from IFNγ - primed or untreated THP - 1 ΔCASP - 1 cells transfected with indicated siRNA and infected with STm - GFP for 4 h . Area under the curve ( AUC ) from real - time assay plotted as mean ± SEM from n = 3 independent experiments . Schematic on right shows overview of the pathway leading to caspase - 1 and caspase - 4 activation . LDH release assay from indicated THP - 1 cells treated with IFNγ and Dox and infected with STm - GFP for 4 h . Shown is the mean ± SEM of n = 3 experiments . Representative images from IFNγ - primed THP - 1 ∆ GBP1 cells stably reconstituted with Tet - mCH - GBP1 , mCH - GBP1K51A , mCH - GBP1C589A or mCH - GBP1 ∆ 589 - 592 variants or primary MDMs stained for GBP1 . Cells were infected with STm - GFP for 2 h . Blue , Nuclei ; Grey , STm ; Red , mCH - GBP1 or Magenta GBP1 . Scale bar , 5 μm . quantification ( G ) from IFNγ - primed THP - 1 ∆ GBP1 cells stably reconstituted with Tet - mCH - GBP1 , mCH - GBP1K51A , mCH - GBP1C589A or mCH - GBP1 ∆ 589 - 592 variants or primary MDMs stained for GBP1 . Cells were infected with STm - GFP for 2 h . Blue , Nuclei ; Grey , STm ; Red , mCH - GBP1 or Magenta GBP1 . Scale bar , 5 μm . Graph in ( G ) shows percentage of STm vacuoles targeted by GBP1 . Quantification of GBP1 recruitment to STm - GFP containing vacuoles ( SCV ) in MDMs pre - treated with IFNγ or left untreated ( UT ) . Mean ± 95 % CI of GBP1 fluorescence intensity measured on the indicated number ( n ) of SCVs for each condition from three independent experiments are shown . Representative images from THP - 1 ∆ GBP1 + Tet - mCH - GBP1 cells stably expressing YFP - CASP4C258S . Cells were infected with STm for 2 h . Blue , Nuclei & STm ( Hoechst dye ) ; Red , mCH - GBP1 ; Green , YFP - CASP4C258S . Scale bar , 5 μm . Quantification of THP - 1 ∆ GBP1 + Tet - mCH - GBP1 cells stably expressing YFP - CASP4C258S and infected with STm for 2 h . Graph shows percentage of STm vacuoles targeted by GBP1 , CASP4 or both from n = 3 experiments . n . d . not detected .

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

Figure 5LDH release assay from indicated THP-1 cells untreated or treated with IFNγ and infected with Salmonella Typhimurium (STm) WT or a SPI-1 mutant ΔinvA for 4 h at indicated multiplicities of infection (MOI). Mean ± SEM of n = 3 experiments are shown.Propidium iodide (PI) uptake assay from IFNγ-primed primary MDMs transfected with non-targeting control siRNA (CTRL) or siRNA against GBP1 (left) or indicated lines of THP-1 (right) infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments.PI uptake assay from IFNγ-primed primary MDMs transfected with indicated siRNA or non-targeting control (CTRL) and infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 4 independent experiments.PI uptake assay from IFNγ-primed or untreated THP-1 ΔCASP-1 cells transfected with indicated siRNA and infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments. Schematic on right shows overview of the pathway leading to caspase-1 and caspase-4 activation.LDH release assay from indicated THP-1 cells treated with IFNγ and Dox and infected with STm-GFP for 4 h. Shown is the mean ± SEM of n = 3 experiments.Representative images from IFNγ-primed THP-1 ∆GBP1 cells stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or primary MDMs stained for GBP1. Cells were infected with STm-GFP for 2 h. Blue, Nuclei; Grey, STm; Red, mCH-GBP1 or Magenta GBP1. Scale bar, 5 μm.quantification (G) from IFNγ-primed THP-1 ∆GBP1 cells stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or primary MDMs stained for GBP1. Cells were infected with STm-GFP for 2 h. Blue, Nuclei; Grey, STm; Red, mCH-GBP1 or Magenta GBP1. Scale bar, 5 μm. Graph in (G) shows percentage of STm vacuoles targeted by GBP1.Quantification of GBP1 recruitment to STm-GFP containing vacuoles (SCV) in MDMs pre-treated with IFNγ or left untreated (UT). Mean ± 95% CI of GBP1 fluorescence intensity measured on the indicated number (n) of SCVs for each condition from three independent experiments are shown.Representative images from THP-1 ∆GBP1+Tet-mCH-GBP1 cells stably expressing YFP-CASP4C258S. Cells were infected with STm for 2 h. Blue, Nuclei & STm (Hoechst dye); Red, mCH-GBP1; Green, YFP-CASP4C258S. Scale bar, 5 μm.Quantification of THP-1 ∆GBP1+Tet-mCH-GBP1 cells stably expressing YFP-CASP4C258S and infected with STm for 2 h. Graph shows percentage of STm vacuoles targeted by GBP1, CASP4 or both from n = 3 experiments. n.d. not detected.