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

O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O

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


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

O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O

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


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

O O O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O

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


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

O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

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


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

O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O

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


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

O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

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


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

O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

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


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

O B-EXP_ASSAY O O O O B-CELL_LINE O O O O O O B-ORGANISM O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

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


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

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

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


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

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O B-EXP_ASSAY O

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


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

O B-ORGANISM O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

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


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

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

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


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

O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

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


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

O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O B-ORGANISM O O O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY O O O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O O B-ORGANISM O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O

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


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

O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O

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


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

O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

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


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

B-CELL_LINE O O O O O O O O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

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


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

O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

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


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

O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O

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


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

O O O B-GENEPROD O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O

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


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

O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O

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


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

O B-CELL_LINE O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-CELL_LINE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O O O O

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


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

O O B-DISEASE O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O

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


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

B-CELL_LINE O O O B-ORGANISM O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

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


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

O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY O O O O O O O O O O O O O O O

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


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

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O

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


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

O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O

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


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

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


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

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


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

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O

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


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

O O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O 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-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O

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


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

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O

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


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

O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O

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


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

O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O O

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


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

O O B-DISEASE B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O O O B-CELL_LINE O B-GENEPROD O O O O O

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


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

O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-CELL_LINE O B-GENEPROD O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O

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


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

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


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

O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

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


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

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


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

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


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

O B-ORGANISM B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE I-CELL_LINE O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O

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


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

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

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


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

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


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

O B-GENEPROD O O O O O B-CELL_LINE O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O

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


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

O B-GENEPROD O O O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O

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


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

O B-GENEPROD O O O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O

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


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

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


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

O O B-EXP_ASSAY O B-CELL_TYPE O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O B-CELL_LINE O O B-GENEPROD O O O O O B-DISEASE B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-CELL_LINE O O B-GENEPROD O

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


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

O O B-EXP_ASSAY O B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O

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


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

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-ORGANISM B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O

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


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

O O O O O O O O O O O B-GENEPROD O O 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-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O

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


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

B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O

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


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

B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O

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


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

B-CELL_LINE O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-GENEPROD O

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


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

B-CELL_LINE O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O

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


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

B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O

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


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

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O

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


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

B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

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


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

B-CELL_LINE O B-GENEPROD O O O B-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O

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


U2OS ∆ ZAK were embedded in agarose subjected to cyclic compression ( 0 . 5 h ) . Lysates were analyzed by immunoblotting with indicated antibodies .

B-CELL_LINE O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O

U2OS and ∆ZAK cells were embedded in agarose and subjected to cyclic compression (0.5 h). Lysates were analyzed by immunoblotting with the indicated antibodies.


U2OS ∆ ZAK stably expressing indicated Strep - HA - tagged ZAKβ constructs were embedded in agarose subjected to cyclic compression for 5 or 30 min . Lysates were analyzed as in ( B ) .

B-CELL_LINE O B-GENEPROD O O O B-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

U2OS and ∆ZAK cells stably expressing the indicated Strep-HA-tagged ZAKβ constructs were embedded in agarose and subjected to cyclic compression for 5 or 30 min. Lysates were analyzed as in (B).


U2OS ∆ ZAK were pretreated with ZAK inhibitor ( ZAKi ) for 30 min subjected to equibiaxial ( Equi ) or uniaxial ( Uni ) cyclic stretch ( 0 . 5 h ) as indicated . Lysates were analyzed as in ( B ) .

B-CELL_LINE O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U2OS and ∆ZAK cells were pretreated with ZAK inhibitor (ZAKi) for 30 min and subjected to equibiaxial (Equi) or uniaxial (Uni) cyclic stretch (0.5 h) as indicated. Lysates were analyzed as in (B).


U2OS ∆ ZAK were transfected with indicated siRNAs subjected to equibiaxial cyclic stretch ( 5 min ) as indicated . Lysates were analyzed as in ( B ) .

B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

U2OS and ∆ZAK cells were transfected with the indicated siRNAs and subjected to equibiaxial cyclic stretch (5 min) as indicated. Lysates were analyzed as in (B).


Expression of MAP3 kinase genes in human skeletal muscle ( GTEx Portal ) . TPM , Transcripts Per Million .

B-EXP_ASSAY O O O O O B-ORGANISM B-TISSUE I-TISSUE O O O O O O O O O O O

Expression levels of MAP3 kinase genes in human skeletal muscle (GTEx Portal). TPM, Transcripts Per Million.


C2C12 were incubated in diff . medium for 3 or 6 days . Lysates were analyzed for ZAK isoform expression by immunoblotting .

B-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY O

C2C12 cells were incubated in diff. medium for 3 or 6 days. Lysates were analyzed for ZAK isoform expression by immunoblotting.


C2C12 myoblasts myotubes ( 6 d differentiation ) were pre - treated with ZAK inhibitor ( ZAKi , 0 . 5 h ) treated with 500 mM sorbitol ( 1 h ) as indicated . Lysates were analyzed by immunoblotting with indicated antibodies .

B-CELL_LINE B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O

C2C12 myoblasts and myotubes (6 d differentiation) were pre-treated with ZAK inhibitor (ZAKi, 0.5 h) and treated with 500 mM sorbitol (1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies.


Skeletal muscle ( tibialis anterior , TA ) liver from WT ZAK - / - mice were lysed analyzed for ZAK isoform expression by immunoblotting . Ponceau of indicates equal loading .

B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O O B-GENEPROD O O O B-ORGANISM O O O O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O O O O

Skeletal muscle (tibialis anterior, TA) and liver from WT and ZAK-/- mice were lysed and analyzed for ZAK isoform expression by immunoblotting. Ponceau staining of the membrane indicates equal loading.


Mouse embryonic fibroblasts ( MEF ) isolated from WT ZAK - / - mice were treated with 500 mM sorbitol ( 1 h ) . Lysates were analyzed by immunoblotting with indicated antibodies . * , unspecific band .

B-ORGANISM B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O B-GENEPROD O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O O O O O O

Mouse embryonic fibroblasts (MEF) isolated from WT and ZAK-/- mice were treated with 500 mM sorbitol (1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. *, unspecific band.


16 - 18 - week - old WT ZAK - / - mice ( n = 3 biological replicates ) were subjected to protocol in ( D ) . TA lysates were analyzed by immunoblotting with indicated antibodies .

O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O B-TISSUE O O O O B-EXP_ASSAY O O O O

16-18-week-old WT and ZAK-/- male mice (n=3 biological replicates) were subjected to the protocol in (D). TA lysates were analyzed by immunoblotting with the indicated antibodies.


ZAKβ S335 S339 phosphorylation sites upregulated in WT TA from ( D ) . Values indicate absolute phospho - peptide abundances error bars represent standard deviation ( n = 3 biological replicates ) .

B-GENEPROD O O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

ZAKβ S335 and S339 phosphorylation sites upregulated in WT TA muscles from (D). Values indicate absolute phospho-peptide abundances and error bars represent the standard deviation (n=3 biological replicates).


TA muscle in an 8 - week old WT mouse was electroporated with a ZAKβ - tdTomato construct . After 7 days muscle was harvested , sectioned longitudinally immunostained with an antibody against Z - disc marker α - actinin1 . right panel shows a higher magnification of region highlighted in yellow . Data information : scale bars , 20 μm .

B-TISSUE I-TISSUE O O O O O O O B-ORGANISM O O O O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

TA muscle in an 8-week old WT male mouse was electroporated with a ZAKβ-tdTomato construct. After 7 days the muscle was harvested, sectioned longitudinally and immunostained with an antibody against the Z-disc marker α-actinin1. The right panel shows a higher magnification of the region highlighted in yellow. Data information: scale bars, 20 μm.


C2C12 were differentiated into myotubes for 14 days transfected with indicated GFP - ZAKβ constructs . were fixed immunostained with an antibody against α - actinin1 . Data information : scale bars , 20 μm .

B-CELL_LINE O O O B-CELL_TYPE O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O

C2C12 cells were differentiated into myotubes for 14 days and transfected with the indicated GFP-ZAKβ constructs. Cells were fixed and immunostained with an antibody against α-actinin1. Data information: scale bars, 20 μm.


16 - 18 - week - old WT ZAK - / - mice were subjected to experimental protocol in ( I ) . TA muscle lysates were analyzed by immunoblotting with indicated antibodies .

O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O B-TISSUE I-TISSUE O O O O B-EXP_ASSAY O O O O

16-18-week-old WT and ZAK-/- male mice were subjected to the experimental protocol in (I). TA muscle lysates were analyzed by immunoblotting with the indicated antibodies.


H & E of soleus muscle cross sections from 8 - 22 - week - old WT ZAK - / - mice . Arrows indicate presence of centralized nuclei . Scale bars , 50 μm . of ( A ) . Values indicate of displaying centralized nuclei error bars represent standard deviation ( n = 3 biological replicates ) . * * , p < 0 . 01 * * * , p < 0 . 001 in t - with Bonferroni - Dunn correction for multiple comparison .

O O O O B-TISSUE I-TISSUE O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O 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&E staining of soleus muscle cross sections from 8- and 22-week-old WT and ZAK-/- male mice. Arrows indicate the presence of centralized nuclei. Scale bars, 50 μm. Quantification of (A). Values indicate the percentage of fibers displaying centralized nuclei and error bars represent the standard deviation (n=3 biological replicates). **, p<0.01 and ***, p<0.001 in t-test with Bonferroni-Dunn correction for multiple comparison.


from ( A ) were immunostained for type I ( red ) type IIa ( green ) using myosin isoform - specific antibodies . Scale bars , 500 μm . of ( C ) . Values indicate of positive for type I myosin error bars represent standard deviation ( n = 3 biological replicates ) . * * , p < 0 . 01 ns , not significant in t - with Bonferroni - Dunn correction for multiple comparison .

O O O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Muscles from (A) were immunostained for type I (red) and type IIa (green) fibers using myosin isoform-specific antibodies. Scale bars, 500 μm. Quantification of (C). Values indicate the percentage of fibers positive for type I myosin and error bars represent the standard deviation (n=3 biological replicates). **, p<0.01 and ns, not significant in t-test with Bonferroni-Dunn correction for multiple comparison.


Figure 4 - MAP2K1 is a predictive marker for inhibitors targeting EGFR Effect - heat maps of six drugs ( see titles of panels ) targeting EGFR . It is evident that different drugs showed different profiles but also that high MAP2K1 expression ( blue / red gradient across lines ) was consistently associated with drug resistance ( blue / yellow gradient across lines ; AUC : under curve ; see main text Appendix for details ) . See also Figure EV5 .

O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O 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-GENEPROD O O O O O O O O O O O O O O O O O O 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 - MAP2K1 is a predictive marker for inhibitors targeting EGFR Effect-size heat maps of six drugs (see titles of panels) targeting EGFR. It is evident that the different drugs showed different profiles but also that high MAP2K1 expression (blue/red gradient across cell lines) was consistently associated with drug resistance (dark blue/yellow gradient across cell lines; AUC: area under the curve; see main text and Appendix for details). See also Figure EV5.


( A ) Effect - heat maps of two drugs ( one from two different drug sensitivity screens ) targeting MEK1 / 2 show consistent association of high MERTK expression with drug resistance . colour scheme is same as in Figure 4 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

(A) Effect-size heat maps of two drugs (one from two different drug sensitivity screens) targeting MEK1/2 show consistent association of high MERTK expression with drug resistance. The colour scheme is the same as in Figure 4.


( C ) Dose - response curves of two drugs for which high MERTK ( left panels ) or ACVR2A ( right panels ) expression was predicted to confer drug resistance . For each drug , three lines predicted to be sensitive ( blue ) three lines predicted to be resistant ( yellow ) were assayed for viability . experimental data validated that lines predicted to be more sensitive to a drug indeed showed this phenotype ( data represents average of three technical replicates ; see Appendix for details ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O 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) Dose-response curves of two drugs for which high MERTK (left panels) or ACVR2A (right panels) expression was predicted to confer drug resistance. For each drug, three cell lines predicted to be sensitive (dark blue) and three cell lines predicted to be resistant (yellow) were assayed for viability. The experimental data validated that cell lines predicted to be more sensitive to a drug indeed showed this phenotype (data represents the average of three technical replicates; see Appendix for details).


( D ) Boxplots summarizing data shown in panel ( C ) using under curve ( AUC ) as a measure for drug sensitivity . High AUC values indicate drug resistance . Again , it is evident that lines predicted to be more resistant to a certain drug were in fact significantly ( p ≤ 0 . 05 , one - sided Mann - Whitney ) more resistant than lines predicted to be more sensitive .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplots summarizing the data shown in panel (C) using the area under the curve (AUC) as a measure for drug sensitivity. High AUC values indicate drug resistance. Again, it is evident that cell lines predicted to be more resistant to a certain drug were in fact significantly (p≤0.05, one-sided Mann-Whitney test) more resistant than cell lines predicted to be more sensitive.


( E ) Western blot of , visualising successful knockout by CRISPR / Cas9 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

(E) Western blot of and cells, visualising successful knockout by CRISPR/Cas9.


( F ) Dose - response curve of RDEA119 ( MEK1 / 2 inhibitor ) in . knockout is more sensitive to MEK1 / 2 inhibition than wild type . See also Figure EV6 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Dose-response curve of RDEA119 (MEK1/2 inhibitor) in and cells. The knockout is more sensitive to MEK1/2 inhibition than the wild type. See also Figure EV6.


( A ) Western Blot visualising MERTK expression in two negative ( RKO , CC07 ) two positive control lines ( CaCo - 2 ; HT55 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-CELL_LINE O B-CELL_LINE O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O

(A) Western Blot visualising MERTK expression in two negative (RKO, CC07) and two positive control cell lines (CaCo-2; HT55).


( B ) of MERTK expression in RKO ( negative control ) CaCo - 2 ( positive control ) .

O O O O B-GENEPROD O O B-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O

(B) IHC staining of MERTK expression in RKO (negative control) and CaCo-2 (positive control) cells.


( C ) of three representative TMAs from enrolled in QUASAR 2 clinical trial corresponding of signal by pathologists .

O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O

(C) IHC staining of three representative TMAs from patients enrolled in the QUASAR 2 clinical trial and the corresponding quantification of the signal by pathologists.


( D ) Kaplan - Meier plots showing worse overall , disease - free recurrence - free survival of with high / membranous expression of MERTK .

O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O B-GENEPROD O

(D) Kaplan-Meier plots showing worse overall, disease-free and recurrence-free survival of patients with high cytoplasmic/membranous expression of MERTK.


( A ) Neutrophil killing were assessed at day 6 ( section iii ) . Controls were without treatment . . A + G , Am80 - GCSF combination ; CL , chemiluminescence ; RLU , relative units ; AUC , under curve .

O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O

(A) Neutrophil bacterial killing were assessed at day 6 (section iii). Controls were without treatment. . A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.


( A ) Neutrophil morphologic differentiation were assessed at day 6 ( section ii ) . Controls were without treatment . arrows indicate neutrophilnuclear segmentation . A + G , Am80 - GCSF combination ; CL , chemiluminescence ; RLU , relative units ; AUC , under curve .

O O O B-CELL_TYPE O O 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-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O

(A) Neutrophil morphologic differentiation were assessed at day 6 (section ii). Controls were without treatment. White arrows indicate neutrophilnuclear segmentation. A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.


( A ) Neutrophil ROS production were assessed at day 6 ( iv ) . Controls were without treatment . A + G , Am80 - GCSF combination ; CL , chemiluminescence ; RLU , relative units ; AUC , under curve .

O O O B-CELL_TYPE B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O

(A) Neutrophil ROS production were assessed at day 6 (iv). Controls were without treatment. A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.


( A ) Proliferation of hematopoietic CD34 + precursors for up to 6 days ( section i ) .

O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O

(A) Proliferation analysis of hematopoietic CD34+ precursors for up to 6 days (section i).


( B ) qRT - PCR assessed RA - target gene expression after culturing CD34 + for 1 , 2 , 6 days .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O

(B) qRT-PCR assessed RA-target gene expression after culturing CD34+ cells for 1, 2, and 6 days.


( C ) Flow cytometry of CD66 - CD18 co - expression after culturing CD34 + for 6 days , using anti - human CD66 - PE CD18 - FITC antibodies ( section i ) . Isotypes were used for controls ( section i , top panel ) . CD66 - CD18 co - expression was quantified in section ii .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-EXP_ASSAY B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O

(C) Flow cytometry analysis of CD66-CD18 co-expression after culturing CD34+ cells for 6 days, using anti-human CD66-PE and CD18-FITC antibodies (section i). Isotypes were used for controls (section i, top panel). CD66-CD18 co-expression was quantified in section ii.


( D ) Fresh peripheral blood ( PB ) collected from normal human donor . Bactericidal activities of neutrophils induced by Am80 - GCSF from PB mononuclear were evaluated by ROS production ( section ii ) .

O O O O O B-TISSUE O O O O O O B-ORGANISM O O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O

(D) Fresh peripheral blood (PB) collected from normal human donor. Bactericidal activities of neutrophils induced by Am80-GCSF from PB mononuclear cells were evaluated by ROS production (section ii).


( D ) Fresh peripheral blood ( PB ) collected from normal human donor . killing was assessed in presence or absence of PBneutrophils ( section i ) .

O O O O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O

(D) Fresh peripheral blood (PB) collected from normal human donor. Bacterial killing was assessed in the presence or absence of PBneutrophils (section i).


( D ) Fresh peripheral blood ( PB ) collected from normal human donor . Bactericidal activities of neutrophils induced by Am80 - GCSF from PB mononuclear were evaluated by killing assays ( section iii ) .

O O O O O B-TISSUE O O O O O O B-ORGANISM O O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O

(D) Fresh peripheral blood (PB) collected from normal human donor. Bactericidal activities of neutrophils induced by Am80-GCSF from PB mononuclear cells were evaluated by bacterial killing assays (section iii).


( E ) Fresh PBneutrophils collected from normal human donor were tested for ROS production in presence or absence of specific antibodies . Controls were with or without IgG . Normal IgG , φ ; Ab , antibodies ; Comb Ab , combined anti - CD18 - CD11b antibodies .

O O O O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O

(E) Fresh PBneutrophils collected from normal human donor were tested for ROS production in the presence or absence of specific antibodies. Controls were with or without IgG. Normal IgG, φ; Ab, antibodies; Comb Ab, combined anti-CD18 and -CD11b antibodies.


( F ) Fresh PB mononuclear collected from normal human donors were treated for 3 days . killing ( sections i ) neutralization of killing in presence of IgG or anti - CD18 antibody were assessed at day 3 ( section ii ) .

O O O O B-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O

(F) Fresh PB mononuclear cells collected from normal human donors were treated for 3 days. Bacterial killing (sections i) and neutralization of bacterial killing in the presence of IgG or anti-CD18 antibody were assessed at day 3 (section ii).


( A ) Fresh bone marrow mononuclear were collected from acute myeloid leukemia ( AML ) . ROS production ( section ii ) were analyzed after were treated for 4 days .

O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

(A) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia (AML) patient. ROS production (section ii) were analyzed after cells were treated for 4 days.


( A ) Fresh bone marrow mononuclear were collected from acute myeloid leukemia ( AML ) . Proliferation ( section i ) were analyzed after were treated for 4 days .

O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O

(A) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia (AML) patient. Proliferation (section i) were analyzed after cells were treated for 4 days.


( B ) Fresh BM mononuclear collected from AML were treated for up to 6 days . Proliferation was assessed at day 3 in parallel ( section ii ) .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O

(B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. Proliferation was assessed at day 3 in parallel (section ii).


( B ) Fresh BM mononuclear collected from AML were treated for up to 6 days . Transcription of RA - target genes was analyzed with qRT - PCR at day 2 , 3 , 6 ( sections i ) .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

(B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. Transcription of RA-target genes was analyzed with qRT-PCR at day 2, 3, and 6 (sections i).


( B ) Fresh BM mononuclear collected from AML were treated for up to 6 days . ROS production were assessed at day 3 in parallel ( section iii ) .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

(B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. ROS production were assessed at day 3 in parallel (section iii).


( A ) Fresh BM mononuclear collected from AML were treated with 20 or 50 nM Am80 alone or combined with medium dose of GCSF ( 25 ng / ml ) for up to 9 days . Dynamic changes in proliferation ( left sections ) live on day 9 ( right sections ) were illustrated for each of specimens . Controls were without treatment .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY 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) Fresh BM mononuclear cells collected from AML patients were treated with 20 or 50 nM Am80 alone or combined with medium dose of GCSF (25 ng/ml) for up to 9 days. Dynamic changes in proliferation (left sections) and live cells on day 9 (right sections) were illustrated for each of specimens. Controls were without treatment.


( B ) Fresh PB or BM mononuclear collected from AML were treated with 20 , 100 , or 150 nM Am80 alone or combined with GCSF for up to 12 days . Dynamic changes in proliferation ( left sections ) live on day 12 ( right sections ) were illustrated for each of specimens .

O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

(B) Fresh PB or BM mononuclear cells collected from AML patients were treated with 20, 100, or 150 nM Am80 alone or combined with GCSF for up to 12 days. Dynamic changes in proliferation (left sections) and live cells on day 12 (right sections) were illustrated for each of specimens.


( C ) Fresh BM mononuclear collected from AML were treated with 150 nM Am80 alone or combined with GCSF . Proliferation ( left sections ) killing ( right sections ) were analyzed on day 12 for each of samples .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

(C) Fresh BM mononuclear cells collected from AML patients were treated with 150 nM Am80 alone or combined with GCSF. Proliferation (left sections) and bacterial killing (right sections) were analyzed on day 12 for each of samples.


( B ) Fresh BM mononuclear collected from AML were treated with 20 , 100 , or 150 nM Am80 alone or combined with GCSF for up to 12 days . ROS production was assessed at day 12 ( section ii ) .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

(B) Fresh BM mononuclear cells collected from AML patients were treated with 20, 100, or 150 nM Am80 alone or combined with GCSF for up to 12 days. ROS production was assessed at day 12 (section ii).


( D ) Fresh BM mononuclear collected from AML were treated with 150 nM Am80 alone or combined with GCSF . Morphologic differentiation was assessed at day 12 ( section iv ) . arrows indicate neutrophil segmentation .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_TYPE O O

(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Morphologic differentiation was assessed at day 12 (section iv). White arrows indicate neutrophil nuclear segmentation.


( D ) Fresh BM mononuclear collected from AML were treated with 150 nM Am80 alone or combined with GCSF . Proliferation was assessed for up to 12 days ( section i ) .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O

(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Proliferation was assessed for up to 12 days (section i).


( D ) Fresh BM mononuclear collected from AML were treated with 150 nM Am80 alone or combined with GCSF . killing was assessed at day 12 ( sections iii ) .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O

(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Bacterial killing was assessed at day 12 (sections iii).


( D ) Fresh BM mononuclear collected from AML were treated with 150 nM Am80 alone or combined with GCSF . RA - target gene expression was assessed at day 12 ( section v ) .

O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O O

(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. RA-target gene expression was assessed at day 12 (section v).


( A ) Mouse CCIN induced by cancer chemotherapy drug cyclophosphamide ( CPA ) consists of both neutrophil decrease neutrophil recovery stages . ANC , absolute neutrophil .

O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O B-CELL_TYPE O

(A) Mouse CCIN induced by cancer chemotherapy drug cyclophosphamide (CPA) consists of both neutrophil decrease and neutrophil recovery stages. ANC, absolute neutrophil count.


(D) Vetscan counting PBleukocytes.


( E ) Neutrophils induced by low dose treatment in PB ( section i ) BM ( section ii ) .

O O O B-CELL_TYPE O O O O O O B-TISSUE O O O O B-TISSUE O O O O O

(E) Neutrophils induced by low dose treatment in PB (section i) and BM (section ii).


( F ) killing by PBneutrophils was assessed at 3 16 hr post - infection in spleen ( sections i - iii ) , using blood agar of total extracellular viable .

O O O O O B-TISSUE O O O O O O O O O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O

(F) Bacterial killing by PBneutrophils was assessed at 3 and 16 hr post-infection and in spleen (sections i-iii), using blood agar analysis of total extracellular viable bacteria.


(H) Vetscan counting PBleukocytes.


( I ) Neutrophils induced by medium dose treatment in PB ( section i ) BM ( section ii ) .

(I) Neutrophils induced by medium dose treatment in PB (section i) and BM (section ii).


( J ) Similar to panel F , killing by PBneutrophils was evaluated 3 16 hr post - infection in heart .

O O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O

(J) Similar to panel F, bacterial killing by PBneutrophils was evaluated 3 and 16 hr post-infection and in heart.


(B) Vetscan counting PBleukocytes.


( C ) killing by PBneutrophils was evaluated 3 16 hr post - infection ( sections i , ii ) , using blood agar of extracellular viable .

O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-TISSUE O O O O O

(C) Bacterial killing by PBneutrophils was evaluated 3 and 16 hr post-infection (sections i, ii), using blood agar analysis of extracellular viable bacteria.


( D E ) Neutrophil recovery morphologic differentiation induced by low doses of Am80 / or GCSF in BM ( panel D ) PB ( panel E ) . arrows indicate neutrophilnuclear segmentation .

O O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-TISSUE O O O O B-TISSUE O O O O O O O B-CELL_TYPE O O

(D E) Neutrophil recovery and morphologic differentiation induced by low doses of Am80 and/or GCSF in BM (panel D) and PB (panel E). White arrows indicate neutrophilnuclear segmentation.


( B ) Survival of CCIN mice was calculated with both Kaplan - Meier plots log - rank on day 9 since CPA injection ( section i ) as well as assessed 6 days post - infection ( section ii ) . Data represent 3 independent experiments with similar results .

O O O B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O 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) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii). Data represent 3 independent experiments with similar results.


( C ) Gram Giemsa stains analyzed neutrophil phagocytosis clearance in mice surviving on day 9 . arrows indicate phagocytosed S . aureus in neutrophils .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_TYPE O

(C) Gram and Giemsa stains analyzed neutrophil phagocytosis and bacterial clearance in mice surviving on day 9. White arrows indicate phagocytosed S. aureus in neutrophils.


( E ) Survival of CCIN mice was calculated with both Kaplan - Meier plots log - rank on day 9 since CPA injection ( section i ) as well as assessed 6 days post - infection ( section ii ) . Data represent 2 independent experiments with similar results .

(E) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii). Data represent 2 independent experiments with similar results.


( G ) Survival of CCIN mice was calculated with both Kaplan - Meier plots log - rank on day 9 since CPA injection ( section i ) as well as assessed 6 days post - infection ( section ii ) .

O O O B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(G) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii).


( A B ) Neutrophil recovery morphologic differentiation were assessed in BM ( panel A ) PB ( panel B ) of CCIN mice surviving on day 9 . arrows indicate neutrophil segmentation .

O O O O B-CELL_TYPE O O O O O O B-TISSUE O O O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O B-CELL_TYPE O O

(A B) Neutrophil recovery and morphologic differentiation were assessed in BM (panel A) and PB (panel B) of CCIN mice surviving on day 9. White arrows indicate neutrophil nuclear segmentation.


( C ) PBneutrophil production segmentation were assessed in control Am80 - GCSF mice surviving on day 13 ( section i ) .

O O O B-TISSUE O O O O O O B-SMALL_MOLECULE O B-GENEPROD B-ORGANISM O O O O O O O O O

(C) PBneutrophil production and nuclear segmentation were assessed in control and Am80-GCSF mice surviving on day 13 (section i).


( C ) PBneutrophil production segmentation were assessed in control Am80 - GCSF mice surviving on day 13 ( section ii ) .

O O O B-TISSUE O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O

(C) PBneutrophil production and nuclear segmentation were assessed in control and Am80-GCSF mice surviving on day 13 (section ii).


( C ) increased spleen on day 9 in Am80 - GCSF mice dropped to similar to those in control mice on day 13 ( sections iii , iv ) .

O O O O B-TISSUE O O O O B-SMALL_MOLECULE O B-GENEPROD B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O

(C) The increased spleen weight on day 9 in Am80-GCSF mice dropped to the levels similar to those in control mice on day 13 (sections iii, iv).


( C ) increased spleen on day 9 in Am80 - GCSF mice dropped to similar to those in control mice on day 13 ( section iii ) .

O O O O B-TISSUE O O O O B-SMALL_MOLECULE O B-GENEPROD B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O

(C) The increased spleen size on day 9 in Am80-GCSF mice dropped to the levels similar to those in control mice on day 13 (section iii).


( D ) Significant loss of occurred in GCSFmice on day 9 .

(D) Significant loss of body weight occurred in GCSFmice on day 9.


( E ) Ultrastructure of neutrophils collected from mice surviving on day 11 . Neutrophil from GCSFmice showed less granules ( section i , 1 vs . 2 or 3 ) , more intracytoplasmic vacuoles ( sections i , ii ) , inner dilation ( sections i , iii ) . Pg : granules ; Sg : secondary granules .

O O O O O B-CELL_TYPE O O B-ORGANISM O O O O O B-CELL_TYPE O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O

(E) Ultrastructure analysis of neutrophils collected from mice surviving on day 11. Neutrophil from GCSFmice showed less granules (section i, images 1 vs. 2 or 3), more intracytoplasmic vacuoles (sections i, ii), and inner nuclear membrane dilation (sections i, iii). Pg: primary granules; Sg: secondary granules.


Sqr mRNA ( A , B , C ) , in cerebrum ( A , D ) , kidneys ( B , E , G ) muscle ( C , F , H ) of Coq9 + / + , Coq9R239X Coq9Q95X mice .

B-GENEPROD O O O O O O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD B-ORGANISM O

Sqr mRNA levels (A, B, C), in cerebrum (A, D), kidneys (B, E, G) and muscle (C, F, H) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


SQR ( D , E , F ) in cerebrum ( A , D ) , kidneys ( B , E , G ) muscle ( C , F , H ) of Coq9 + / + , Coq9R239X Coq9Q95X mice . Note that SQRwestern - blots were performed in isolated cerebralmitochondria due to low of this in cerebrum . In kidneys muscle western - blots were performed in tissue homogenates .

B-GENEPROD O O O O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD B-ORGANISM O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-TISSUE O O B-TISSUE I-TISSUE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

SQR protein levels (D, E, F) in cerebrum (A, D), kidneys (B, E, G) and muscle (C, F, H) of Coq9+/+, Coq9R239X and Coq9Q95X mice. Note that SQRwestern-blots were performed in isolated cerebralmitochondria due to the low levels of this protein in cerebrum. In kidneys and muscle the western-blots were performed in tissue homogenates.


SQR activity ( G , H ) in kidneys ( B , E , G ) muscle ( C , F , H ) of Coq9 + / + , Coq9R239X Coq9Q95X mice .

B-GENEPROD O O O O O O O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD B-ORGANISM O

SQR activity (G, H) in kidneys (B, E, G) and muscle (C, F, H) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


of CoQ10 in fibroblasts of controls ( C ) ( P1 - 4 ) with CoQ10 deficiency ( A ) .

O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O

Levels of CoQ10 in fibroblasts of controls (C) and patients (P1-4) with primary CoQ10 deficiency (A).


of SQR in fibroblasts of controls ( C ) ( P1 - 4 ) with CoQ10 deficiency cultured without ( vehicle ) with 5μM of CoQ10 ( + CoQ10 ) during 1 day or 7 days ( B ) .

O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O

Levels of SQR protein in fibroblasts of controls (C) and patients (P1-4) with primary CoQ10 deficiency cultured without (vehicle) and with 5μM of CoQ10 (+ CoQ10) during 1 day or 7 days (B).


Total CoQ ( CoQ9 + CoQ10 ) in kidneys ( C ) muscle ( D ) of Coq9 + / + , Coq9R239X Coq9R239X + ubiquinol - 10 ( Q10H2 ) mice .

O O O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O B-TISSUE O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-ORGANISM O

Total CoQ levels (CoQ9 + CoQ10) in kidneys (C) and muscle (D) of Coq9+/+, Coq9R239X and Coq9R239X + ubiquinol-10 (Q10H2) mice.


of SQR in kidneys ( E ) muscle ( F ) of Coq9 + / + , Coq9R239X Coq9R239X + ubiquinol - 10 ( Q10H2 ) mice .

O B-GENEPROD O B-TISSUE O O O B-TISSUE O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-ORGANISM O

Levels of SQR protein in kidneys (E) and muscle (F) of Coq9+/+, Coq9R239X and Coq9R239X + ubiquinol-10 (Q10H2) mice.


TST in cerebrum ( A ) , kidneys ( B ) muscle ( C ) of Coq9 + / + , Coq9R239X Coq9Q95X mice .

B-GENEPROD O B-TISSUE O O O O B-TISSUE O O O B-TISSUE O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD B-ORGANISM O

TST protein levels in cerebrum (A), kidneys (B) and muscle (C) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


TST activity in cerebrum ( D ) , kidneys ( E ) muscle ( F ) of Coq9 + / + , Coq9R239X Coq9Q95X mice .

B-GENEPROD O O B-TISSUE O O O O B-TISSUE O O O B-TISSUE O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD B-ORGANISM O

TST activity in cerebrum (D), kidneys (E) and muscle (F) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


ETHE1 ( SDO ) in cerebrum ( G ) , kidneys ( H ) muscle ( I ) of Coq9 + / + , Coq9R239X Coq9Q95X mice .

B-GENEPROD O O O O B-TISSUE O O O O B-TISSUE O O O B-TISSUE O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD B-ORGANISM O

ETHE1 (SDO) protein levels in cerebrum (G), kidneys (H) and muscle (I) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


SUOX in cerebrum ( J ) , kidneys ( K ) muscle ( L ) of Coq9 + / + , Coq9R239X Coq9Q95X mice .

SUOX protein levels in cerebrum (J), kidneys (K) and muscle (L) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


of sulfide in cerebrum kidneys of Coq9 + / + , Coq9R239X Coq9Q95X mice ( A ) .

O B-SMALL_MOLECULE O B-TISSUE I-TISSUE O B-GENEPROD O O O O B-GENEPROD I-GENEPROD B-ORGANISM O O O O

Quantification of sulfide levels in cerebrum and kidneys of Coq9+/+, Coq9R239X and Coq9Q95X mice (A).


Qualitative measurement of hydrogen sulfide in cerebrum kidneys of Coq9 + / + Coq9R239X mice ( B ) .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O O O

Qualitative measurement of hydrogen sulfide in cerebrum and kidneys of Coq9+/+ and Coq9R239X mice (B).


Total GSH in cytosol mitochondria of cerebrum of Coq9 + / + Coq9R239X mice ( A ) .

O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O O O

Total GSH in cytosol and mitochondria of cerebrum of Coq9+/+ and Coq9R239X mice(A).


GPx GRd activities in cerebrum of Coq9 + / + Coq9R239X mice ( B ) .

B-GENEPROD I-GENEPROD O O B-TISSUE O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O O O

Cytosolic GPx and GRd activities in cerebrum of Coq9+/+ and Coq9R239X mice (B).


of GPx4 ( C ) GRd ( D ) in cerebral homogenate of Coq9 + / + Coq9R239X mice .

O B-GENEPROD O O O B-GENEPROD O O O O B-TISSUE O O B-GENEPROD O O O B-GENEPROD B-ORGANISM O

Levels of GPx4 (C) and GRd (D) protein in cerebral homogenate of Coq9+/+ and Coq9R239X mice.


of L - Glutamate ( L - Glu ) , N - Acetyl - Glutamate ( NacGlu ) , L - Tryptophan ( L - Trp ) , 5HIAA , N - Acetyl - Tryptophan ( NALT ) , L - Tyrosine ( L - Tyr ) in cerebrum of Coq9 + / + Coq9R239X mice ( E ) .

Levels of L-Glutamate (L-Glu), N-Acetyl-Glutamate (NacGlu), L-Tryptophan (L-Trp), 5HIAA, N-Acetyl-Tryptophan (NALT), L-Tyrosine (L-Tyr) in cerebrum of Coq9+/+ and Coq9R239X mice (E).


SQR ( A ) TST ( B ) in human skin fibroblasts supplemented with H2S donor GYY4137 .

B-GENEPROD O O O B-GENEPROD O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O B-SMALL_MOLECULE O

SQR (A) and TST (B) protein levels in human skin fibroblasts supplemented with the H2S donor GYY4137.


TST level in kidneys ( C ) cerebrum ( D ) of Coq9 + / + mice supplemented with H2S donor GYY4137 .

B-GENEPROD O O B-TISSUE O O O B-TISSUE O O O O B-GENEPROD O O O B-ORGANISM O O O O B-SMALL_MOLECULE O

TST protein level in kidneys (C) and cerebrum (D) of Coq9+/+ mice supplemented with the H2S donor GYY4137.


of neurotransmitters in cerebrum of Coq9 + / + mice supplemented with H2S donor GYY4137 ( E ) .

O O O B-TISSUE O B-GENEPROD O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O

Levels of neurotransmitters in cerebrum of Coq9+/+ mice supplemented with the H2S donor GYY4137 (E).


COX activity in cerebrum , kidneys muscle ( A ) of Coq9 + / + , Coq9R239X Coq9Q95X mice .

B-GENEPROD O O B-TISSUE O B-TISSUE I-TISSUE O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD B-ORGANISM O

COX activity in cerebrum, kidneys and muscle (A) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


COX histochemistry ( B ) in gastrocnemious of Coq9 + / + , Coq9R239X Coq9Q95X mice ; scale bars : 100 μm .

B-GENEPROD O O O O O B-TISSUE O B-GENEPROD O O O O B-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O

Images COX histochemistry (B) in gastrocnemious of Coq9+/+, Coq9R239X and Coq9Q95X mice; scale bars: 100 μm.


Systolic , diastolic mean blood pressure in Coq9 + / + Coq9R239X mice ( A ) .

O O O O O O O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O O O

Systolic, diastolic and mean blood pressure in Coq9+/+ and Coq9R239X mice (A).


Heart rate in Coq9 + / + Coq9R239X mice ( B ) . * * P < 0 . 01 ; Coq9R239X mice versus Coq9 + / + mice ( t ; n = 5 for each group ) .

B-TISSUE O O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O B-GENEPROD B-ORGANISM O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O

Heart rate in Coq9+/+ and Coq9R239X mice (B). **P < 0.01; Coq9R239X mice versus Coq9+/+ mice (t test; n = 5 for each group).


A . Enrichment of lamin B1 in SGZ of DG .

O O O O B-GENEPROD I-GENEPROD O B-TISSUE O B-TISSUE O

A. Enrichment of lamin B1 in the SGZ of DG.