( B ) Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 24 ) or , as controls , Bcl - xfl / fl ( n = 9 ) and RosaCreERT2 + / Ki ( n = 6 ) mice ( males and females , aged 8 - 14 weeks , numbers also indicated in the figure legend ) were lethally γ - irradiated ( 2 x 5 . 5 Gy , 3 h apart ) and reconstituted with bone marrow from UBC - GFP mice ( referred to as GFP - Chimeras ) . After 8 weeks , reconstituted mice were treated with tamoxifen ( 200 mg / kg body weight administered in 3 daily doses oral gavage ) and monitored for up to 200 days ( termination of the experiment ) . Data are presented as % survival post - treatment with tamoxifen and statistical significance was assessed using the Mantel - Cox ( Log - rank ) test ; * * * * p < 0 . 0001 .

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

(B) Bcl-xfl/fl;RosaCreERT2+/Ki (n=24) or, as controls, Bcl-xfl/fl (n=9) and RosaCreERT2+/Ki (n=6) mice (males and females, aged 8-14 weeks, numbers also indicated in the figure legend) were lethally γ-irradiated (2 x 5.5 Gy, 3 h apart) and reconstituted with bone marrow from UBC-GFP mice (referred to as GFP-Chimeras). After 8 weeks, reconstituted mice were treated with tamoxifen (200 mg/kg body weight administered in 3 daily doses oral gavage) and monitored for up to 200 days (termination of the experiment). Data are presented as % survival post-treatment with tamoxifen and statistical significance was assessed using the Mantel-Cox (Log-rank) test; ****p<0.0001.


( C - F ) Total counts of ( C ) platelets , ( D ) red blood cells ( RBC ) , ( E ) hemoglobin ( HGB ) content and ( F ) hematocrit ( HCT ) of tamoxifen - treated Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 8 ) or , as controls , Bcl - xfl / fl ( n = 6 ) and RosaCreERT2 + / Ki ( n = 12 ) and Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimera ( n = 21 ) or , as controls , Bcl - xfl / fl ; GFP - Chimera ( n = 6 ) and RosaCreERT2 + / Ki ; GFP - Chimera ( n = 6 ) mice were determined by ADVIA . Data are presented as mean ± SEM . Each data point represents an individual mouse . Statistical significance was assessed using the Student ' s t - test ; * * * * p < 0 . 0001 . ( G ) Spleen weights were measured in sick mice ( at sacrifice ) or , for the healthy control mice , at the termination of the experiment . Data are presented as mean ± SEM . Each data point represents an individual mouse and numbers are indicated . Statistical significance was assessed using the Student ' s t - test ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Total counts of (C) platelets, (D) red blood cells (RBC), (E) hemoglobin (HGB) content and (F) hematocrit (HCT) of tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki (n=8) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=12) and Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimera (n=21) or, as controls, Bcl-xfl/fl;GFP-Chimera (n=6) and RosaCreERT2+/Ki;GFP-Chimera (n=6) mice were determined by ADVIA. Data are presented as mean ±SEM. Each data point represents an individual mouse. Statistical significance was assessed using the Student's t-test; ****p<0.0001. (G) Spleen weights were measured in sick mice (at sacrifice) or, for the healthy control mice, at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents an individual mouse and numbers are indicated. Statistical significance was assessed using the Student's t-test; ****p<0.0001.


( C - F ) Total counts of ( C ) platelets , ( D ) red blood cells ( RBC ) , ( E ) hemoglobin ( HGB ) content and ( F ) hematocrit ( HCT ) of tamoxifen - treated Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 8 ) or , as controls , Bcl - xfl / fl ( n = 6 ) and RosaCreERT2 + / Ki ( n = 12 ) and Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimera ( n = 21 ) or , as controls , Bcl - xfl / fl ; GFP - Chimera ( n = 6 ) and RosaCreERT2 + / Ki ; GFP - Chimera ( n = 6 ) mice were determined by ADVIA . Data are presented as mean ± SEM . Each data point represents an individual mouse . Statistical significance was assessed using the Student ' s t - test ; * * * * p < 0 . 0001 .

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

(C-F) Total counts of (C) platelets, (D) red blood cells (RBC), (E) hemoglobin (HGB) content and (F) hematocrit (HCT) of tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki (n=8) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=12) and Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimera (n=21) or, as controls, Bcl-xfl/fl;GFP-Chimera (n=6) and RosaCreERT2+/Ki;GFP-Chimera (n=6) mice were determined by ADVIA. Data are presented as mean ±SEM. Each data point represents an individual mouse. Statistical significance was assessed using the Student's t-test; ****p<0.0001.


( H ) Representative image of enlarged spleens from two Bcl - xfl / fl ; RosaCreERT2Ki / + mice and age - matched control Bcl - xfl / fl and RosaCreERT2 + / Ki mice ( 34 days after treatment ) .

O O O O O O O O O O O O O O O O O O O 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) Representative image of enlarged spleens from two Bcl-xfl/fl;RosaCreERT2Ki/+ mice and age-matched control Bcl-xfl/fl and RosaCreERT2+/Ki mice (34 days after treatment).


( I ) Histological analysis of H & E - stained sections of spleens of tamoxifen - treated mice of the indicated genotypes .

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) Histological analysis of H&E-stained sections of spleens of tamoxifen-treated mice of the indicated genotypes.


Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 8 ) or , as controls , Bcl - xfl / fl ( n = 6 ) and RosaCreERT2 + / Ki ( n = 12 ) mice as well as Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 21 ) , or as controls , Bcl - xfl / fl ; GFP - Chimeras ( n = 6 ) and RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) ( age 8 - 14 weeks , males and females ) were treated with tamoxifen ( 200 mg / kg / body weight administered in 3 daily doses by oral gavage ) to induce CreERT2 - mediated deletion of the floxed Bcl - x alleles . ( A ) Total white blood cell counts ( WBC ) were analyzed by ADVIA in sick mice or at the termination of the experiment ( healthy control mice ) . Data are presented as mean ± SEM . Each data point represents an individual mouse and n numbers are indicated above . Statistical significance was assessed using the Student ' s t - test . No statistically significant differences were observed .

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

Bcl-xfl/fl;RosaCreERT2+/Ki (n=8) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=12) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21), or as controls, Bcl-xfl/fl;GFP-Chimeras (n=6) and RosaCreERT2+/Ki;GFP-Chimeras (n=6) (age 8-14 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. (A) Total white blood cell counts (WBC) were analyzed by ADVIA in sick mice or at the termination of the experiment (healthy control mice). Data are presented as mean ±SEM. Each data point represents an individual mouse and n numbers are indicated above. Statistical significance was assessed using the Student's t-test. No statistically significant differences were observed.


( B ) The neutrophil / lymphocyte ratio ( NLR ) is presented as mean ± SEM for sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 21 ) or healthy control RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) at the termination of the experiment . Data are presented as mean ± SEM . Each data point represents an individual mouse . Statistical significance was assessed using the Student ' s t - test ; * p < 0 . 05 .

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

(B) The neutrophil/lymphocyte ratio (NLR) is presented as mean ±SEM for sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21) or healthy control RosaCreERT2+/Ki;GFP-Chimeras (n=6) at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents an individual mouse. Statistical significance was assessed using the Student's t-test; *p<0.05.


( C ) Histological analysis of H & E - stained sections of the sternum of sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras or healthy wild - type and RosaCreERT2 + / Ki ; GFP - Chimera control mice at the indicated time points post - treatment with tamoxifen ( dpt = days post - treatment ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histological analysis of H&E-stained sections of the sternum of sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras or healthy wild-type and RosaCreERT2+/Ki;GFP-Chimera control mice at the indicated time points post-treatment with tamoxifen (dpt=days post-treatment).


( D ) Histological analysis of H & E - stained sections of the livers of sick mice or age - matched healthy control wild - type mice or healthy RosaCreERT2 + / Ki ; GFP - Chimeras at the termination of the experiment ( dpt = days post - treatment ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histological analysis of H&E-stained sections of the livers of sick mice or age-matched healthy control wild-type mice or healthy RosaCreERT2+/Ki;GFP-Chimeras at the termination of the experiment (dpt=days post-treatment).


( E ) ALT ( left panel ) , AST ( middle panel ) and bilirubin levels ( right panel ) in the serum were determined in sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 18 ) or in healthy RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 4 ) at the termination of the experiment . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using Student ' s t - test ; * * p < 0 . 01 . nd = not detected .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ALT (left panel), AST (middle panel) and bilirubin levels (right panel) in the serum were determined in sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=18) or in healthy RosaCreERT2+/Ki;GFP-Chimeras (n=4) at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test; **p<0.01. nd=not detected.


Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 7 ) or as controls , Bcl - xfl / fl ( n = 8 ) and RosaCreERT2 + / Ki ( n = 16 ) mice as well as Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 5 ) , Bcl - xfl / fl ; GFP - Chimeras ( n = 8 ) and RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) ( age 8 - 14 weeks , males and females ) were treated with tamoxifen ( 200 mg / kg / body weight administered in 3 daily doses by oral gavage ) to induce CreERT2 - mediated deletion of the floxed Bcl - x alleles . ( A ) Kidney weights were measured in sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( at sacrifice ) or , for the healthy control mice , at the termination of the experiment . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using Student ' s t - test ; * p < 0 . 05 , * * * p < 0 . 001 .

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

Bcl-xfl/fl;RosaCreERT2+/Ki (n=7) or as controls, Bcl-xfl/fl (n=8) and RosaCreERT2+/Ki (n=16) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=5), Bcl-xfl/fl;GFP-Chimeras (n=8) and RosaCreERT2+/Ki;GFP-Chimeras (n=6) (age 8-14 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. (A) Kidney weights were measured in sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (at sacrifice) or, for the healthy control mice, at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test; *p<0.05, ***p<0.001.


Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 7 ) or as controls , Bcl - xfl / fl ( n = 8 ) and RosaCreERT2 + / Ki ( n = 16 ) mice as well as Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 5 ) , Bcl - xfl / fl ; GFP - Chimeras ( n = 8 ) and RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) ( age 8 - 14 weeks , males and females ) were treated with tamoxifen ( 200 mg / kg / body weight administered in 3 daily doses by oral gavage ) to induce CreERT2 - mediated deletion of the floxed Bcl - x alleles .

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

Bcl-xfl/fl;RosaCreERT2+/Ki (n=7) or as controls, Bcl-xfl/fl (n=8) and RosaCreERT2+/Ki (n=16) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=5), Bcl-xfl/fl;GFP-Chimeras (n=8) and RosaCreERT2+/Ki;GFP-Chimeras (n=6) (age 8-14 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles.


( B ) Picture showing both kidneys from age - matched healthy control wild - type mice ( left ) and sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( right ) .

O O O O O O O O O O O O O O O O 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) Picture showing both kidneys from age-matched healthy control wild-type mice (left) and sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (right).


( C ) Histological analysis of H & E - stained sections of the kidneys of age - matched healthy control mice or sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras as indicated , showing from left to right , shrunken scarred kidney , segmental chronic tubulo - interstitial disease , accumulation of cellular debris and amorphous material within the collecting ducts of the papilla . The higher magnification shows tubular epithelial degeneration , apoptosis and segmental secondary glomerular sclerosis . Pictures are representative of at least 5 mice for each genotype and treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histological analysis of H&E-stained sections of the kidneys of age-matched healthy control mice or sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras as indicated, showing from left to right, shrunken scarred kidney, segmental chronic tubulo-interstitial disease, accumulation of cellular debris and amorphous material within the collecting ducts of the papilla. The higher magnification shows tubular epithelial degeneration, apoptosis and segmental secondary glomerular sclerosis. Pictures are representative of at least 5 mice for each genotype and treatment.


( D ) Multiphoton analysis of fixed sections of the kidneys of age - matched healthy RosaCreERT2 + / Ki ; GFP - Chimeras ( left ) or sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( right ) . Pictures are representative of at least 3 mice for each genotype and treatment . green = second harmony , blue = short wavelength autofluorescence .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Multiphoton analysis of fixed sections of the kidneys of age-matched healthy RosaCreERT2+/Ki;GFP-Chimeras (left) or sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (right). Pictures are representative of at least 3 mice for each genotype and treatment. green=second harmony, blue=short wavelength autofluorescence.


( A ) TUNEL staining of kidney sections from control RosaCreERT2 + / Ki , Bcl - xfl / fl ; RosaCreERT2 + / Ki , RosaCreERT2 + / Ki ; GFP - Chimeras or Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras at the indicated time points post - tamoxifen treatment ( 200 mg / kg / body weight administered in 3 daily doses by oral gavage ) or γ - irradiation ( 2 x 550 Rad ) , as indicated . Slides were counterstained with hematoxylin . Arrow heads indicate examples of TUNEL + ( apoptotic ) cells . ( B ) TUNEL + cells were quantified using a personalized script for the Image J software . Kidney sections were divided into ~ 40 microscopic images and the total numbers of blue and brown ( TUNEL + ) nuclei were determined . The percentages of TUNEL + nuclei were calculated for each picture . The average value for each kidney section was then determined and plotted as a single data point . Error bars represent SEM from at least 3 independent samples for each genotype and treatment . Statistical significance was assessed using one - way ANOVA analysis with Tukey ' s multiple comparisons test ( comparing control RosaCreERT2 + / Ki ( 24 h post - tamoxifen n = 2 , 24 h post - γ - irradiation n = 9 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ( 24 h post - tamoxifen n = 6 , 24 h post - γ - irradiation n = 5 ) and control RosaCreERT2 + / Ki ; GFP - Chimeras ( 24 h post - tamoxifen n = 7 , End point n = 8 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( 24 h post - tamoxifen n = 8 , End point n = 6 ) ( * * * p < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TUNEL staining of kidney sections from control RosaCreERT2+/Ki, Bcl-xfl/fl;RosaCreERT2+/Ki, RosaCreERT2+/Ki;GFP-Chimeras or Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras at the indicated time points post-tamoxifen treatment (200 mg/kg/body weight administered in 3 daily doses by oral gavage) or γ-irradiation (2 x 550 Rad), as indicated. Slides were counterstained with hematoxylin. Arrow heads indicate examples of TUNEL+ (apoptotic) cells. (B) TUNEL+ cells were quantified using a personalized script for the Image J software. Kidney sections were divided into ~40 microscopic images and the total numbers of blue and brown (TUNEL+) nuclei were determined. The percentages of TUNEL+ nuclei were calculated for each picture. The average value for each kidney section was then determined and plotted as a single data point. Error bars represent SEM from at least 3 independent samples for each genotype and treatment. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test (comparing control RosaCreERT2+/Ki (24 h post-tamoxifen n=2, 24 h post-γ-irradiation n=9) with Bcl-xfl/fl;RosaCreERT2+/Ki (24 h post-tamoxifen n=6, 24 h post-γ-irradiation n=5) and control RosaCreERT2+/Ki;GFP-Chimeras (24 h post-tamoxifen n=7, End point n=8) with Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (24 h post-tamoxifen n=8, End point n=6) (***p<0.001).


( A ) TUNEL staining of kidney sections from control RosaCreERT2 + / Ki , Bcl - xfl / fl ; RosaCreERT2 + / Ki , RosaCreERT2 + / Ki ; GFP - Chimeras or Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras at the indicated time points post - tamoxifen treatment ( 200 mg / kg / body weight administered in 3 daily doses by oral gavage ) or γ - irradiation ( 2 x 550 Rad ) , as indicated . Slides were counterstained with hematoxylin . Arrow heads indicate examples of TUNEL + ( apoptotic ) cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TUNEL staining of kidney sections from control RosaCreERT2+/Ki, Bcl-xfl/fl;RosaCreERT2+/Ki, RosaCreERT2+/Ki;GFP-Chimeras or Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras at the indicated time points post-tamoxifen treatment (200 mg/kg/body weight administered in 3 daily doses by oral gavage) or γ-irradiation (2 x 550 Rad), as indicated. Slides were counterstained with hematoxylin. Arrow heads indicate examples of TUNEL+ (apoptotic) cells.


( A , B ) Data are presented as % survival and statistical significance was assessed using the Mantel - Cox ( Log - rank ) test comparing tamoxifen - treated Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 25 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; GFP - Chimeras ( n = 25 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim - / - ; GFP - Chimeras ( n = 18 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma + / - ; GFP - Chimeras ( n = 21 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma - / - ; GFP - Chimeras ( n = 15 ) and Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; Puma + / - ; GFP - Chimeras ( n = 22 ) . * p < 0 . 05 , * * * * p < 0 . 0001 . The survival curves for the Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B) Data are presented as % survival and statistical significance was assessed using the Mantel-Cox (Log-rank) test comparing tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=25) with Bcl-xfl/fl;RosaCreERT2+/Ki;Bim+/-;GFP-Chimeras (n=25), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim-/-;GFP-Chimeras (n=18), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma+/-;GFP-Chimeras (n=21), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma-/-;GFP-Chimeras (n=15) and Bcl-xfl/fl;RosaCreERT2+/Ki; Bim+/-;Puma+/-;GFP-Chimeras (n=22). *p<0.05, ****p<0.0001. The survival curves for the Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras


( A , B ) Data are presented as % survival and statistical significance was assessed using the Mantel - Cox ( Log - rank ) test comparing tamoxifen - treated Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 25 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; GFP - Chimeras ( n = 25 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim - / - ; GFP - Chimeras ( n = 18 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma + / - ; GFP - Chimeras ( n = 21 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma - / - ; GFP - Chimeras ( n = 15 ) and Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; Puma + / - ; GFP - Chimeras ( n = 22 ) . * p < 0 . 05 , * * * * p < 0 . 0001 . The survival curves for the Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) Histological analysis of H & E - stained sections of the kidneys of sick mice of the indicated genotypes at the indicated time points ( dpt = days post - tamoxifen treatment ) . Pictures are representative of at least 3 mice for each genotype and treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histological analysis of H&E-stained sections of the kidneys of sick mice of the indicated genotypes at the indicated time points (dpt = days post-tamoxifen treatment). Pictures are representative of at least 3 mice for each genotype and treatment.


( D - F ) ( D ) Total red blood cell ( RBC ) count , ( E ) hematocrit ( HCT ) and ( F ) hemoglobin ( HGB ) content were determined by ADVIA in the blood of sick mice of the indicated genotypes or at the termination of the experiment for healthy controls . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using Student ' s t - test comparing tamoxifen - treated RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 21 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; GFP - Chimeras ( n = 20 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim - / - ; GFP - Chimeras ( n = 12 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma + / - ; GFP - Chimeras ( n = 15 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma - / - ; GFP - Chimeras ( n = 10 ) and Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; Puma + / - ; GFP - Chimeras ( n = 17 ) ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . Data for the Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras and RosaCreERT2 + / Ki ; GFP - Chimeras

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) (D) Total red blood cell (RBC) count, (E) hematocrit (HCT) and (F) hemoglobin (HGB) content were determined by ADVIA in the blood of sick mice of the indicated genotypes or at the termination of the experiment for healthy controls. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test comparing tamoxifen-treated RosaCreERT2+/Ki;GFP-Chimeras (n=6) with Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim+/-;GFP-Chimeras (n=20), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim-/-;GFP-Chimeras (n=12), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma+/-;GFP-Chimeras (n=15), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma-/-;GFP-Chimeras (n=10) and Bcl-xfl/fl;RosaCreERT2+/Ki; Bim+/-;Puma+/-;GFP-Chimeras (n=17) ; ***p<0.001; ****p<0.0001. Data for the Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras and RosaCreERT2+/Ki;GFP-Chimeras


( D - F ) ( D ) Total red blood cell ( RBC ) count , ( E ) hematocrit ( HCT ) and ( F ) hemoglobin ( HGB ) content were determined by ADVIA in the blood of sick mice of the indicated genotypes or at the termination of the experiment for healthy controls . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using Student ' s t - test comparing tamoxifen - treated RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 21 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; GFP - Chimeras ( n = 20 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim - / - ; GFP - Chimeras ( n = 12 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma + / - ; GFP - Chimeras ( n = 15 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma - / - ; GFP - Chimeras ( n = 10 ) and Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; Puma + / - ; GFP - Chimeras ( n = 17 ) ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . Data for the Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras and RosaCreERT2 + / Ki ; GFP -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) GFP - Chimeras were treated with the BCL - XL inhibitor A1331852 ( n = 8 , 5 doses by oral gavage , 25 mg / kg body weight each dose ) . Control groups ( n = 8 each group ) include untreated GFP - Chimeras and un - irradiated C57BL / 6 - Ly5 . 1 ( wild - type ) mice treated with the BCL - XL inhibitor A1331852 or left untreated . Mice were monitored for up to 200 days post - treatment ( dpt ) . Data are presented as % survival .

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

(A) GFP-Chimeras were treated with the BCL-XL inhibitor A1331852 (n=8, 5 doses by oral gavage, 25 mg/kg body weight each dose). Control groups (n=8 each group) include untreated GFP-Chimeras and un-irradiated C57BL/6-Ly5.1 (wild-type) mice treated with the BCL-XL inhibitor A1331852 or left untreated. Mice were monitored for up to 200 days post-treatment (dpt). Data are presented as % survival.


( B ) C57BL / 6 - Ly5 . 1 ( wild - type ) mice ( females , aged 10 weeks ) were treated with Cyclophosphamide ( n = 7 , 150 mg / kg body weight , 1 dose i . v . ) or 5 - Fluorouracil ( 5 - FU , n = 7 , 100 mg / kg body weight , 1 dose i . v . ) and after 5 days additionally treated with the BCL - XL inhibitor A1331852 ( 5 doses by oral gavage , 100 mg / kg body weight each dose ) . Control groups ( n = 7 each group ) include C57BL / 6 - Ly5 . 1 ( wild - type ) mice ( females , aged 10 weeks ) treated with A1331852 alone , Cyclophosphamide alone , 5 - FU alone or left untreated . Mice were monitored for up to 150 days post - treatment ( dpt ) . Data are presented as % survival .

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

(B) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with Cyclophosphamide (n=7, 150 mg/kg body weight, 1 dose i.v.) or 5-Fluorouracil (5-FU, n=7, 100 mg/kg body weight, 1 dose i.v.) and after 5 days additionally treated with the BCL-XL inhibitor A1331852 (5 doses by oral gavage, 100 mg/kg body weight each dose). Control groups (n=7 each group) include C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) treated with A1331852 alone, Cyclophosphamide alone, 5-FU alone or left untreated. Mice were monitored for up to 150 days post-treatment (dpt). Data are presented as % survival.


( C ) C57BL / 6 - Ly5 . 1 ( wild - type ) mice ( females , aged 10 weeks ) were treated with the BCL - XL inhibitor A1331852 ( n = 7 , 5 doses by oral gavage , 25 or 100 mg / kg body weight each dose ) . Total platelet counts were determined by ADVIA at the indicated time points . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using one - way ANOVA analysis with Tukey ' s multiple comparisons test comparing untreated and drug - treated mice within each group ( * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p > 0 . 0001 ) .

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

(C) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with the BCL-XL inhibitor A1331852 (n=7, 5 doses by oral gavage, 25 or 100 mg/kg body weight each dose). Total platelet counts were determined by ADVIA at the indicated time points. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (**p<0.01, ***p<0.001, ****p>0.0001).


Total red blood cell count ( RBC ) , platelet count , hemoglobin ( HGB ) content and hematocrit ( HCT ) were determined by ADVIA as indicated in the blood of drug - treated GFP - Chimeras or control mice ( n = 8 each group ) at the termination of the experiment Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using one - way ANOVA analysis with Tukey ' s multiple comparisons test comparing untreated and drug - treated mice within each group ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p > 0 . 0001 ) .

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

Total red blood cell count (RBC), platelet count, hemoglobin (HGB) content and hematocrit (HCT) were determined by ADVIA as indicated in the blood of drug-treated GFP-Chimeras or control mice (n=8 each group) at the termination of the experiment Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (*p<0.05, **p<0.01, ***p<0.001, ****p>0.0001).


Total red blood cell count ( RBC ) , platelet count , hemoglobin ( HGB ) content and hematocrit ( HCT ) were determined by ADVIA as indicated in the blood of in drug - treated C57BL / 6 - Ly5 . 1 ( wild - type ) mice or control mice at the indicated time points ( n = 7 each group ) . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using one - way ANOVA analysis with Tukey ' s multiple comparisons test comparing untreated and drug - treated mice within each group ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p > 0 . 0001 ) .

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

Total red blood cell count (RBC), platelet count, hemoglobin (HGB) content and hematocrit (HCT) were determined by ADVIA as indicated in the blood of in drug-treated C57BL/6-Ly5.1 (wild-type) mice or control mice at the indicated time points (n=7 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (*p<0.05, **p<0.01, ***p<0.001, ****p>0.0001).


( A ) GFP - Chimeras were treated with the BCL - XL inhibitor A1331852 ( n = 8 , 5 doses by oral gavage , 25 mg / kg body weight each dose ) . Control groups ( n = 8 each group ) include untreated GFP - Chimera and un - irradiated C57BL / 6 - Ly5 . 1 ( wild - type ) mice treated with the BCL - XL inhibitor A1331852 or left untreated . Kidney ( left panel ) and spleen weights ( right panel ) were measured in drug - treated GFP - Chimeras or control mice at the termination of the experiment ( n = 8 each group ) . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using one - way ANOVA analysis with Tukey ' s multiple comparisons test . No statistically significant differences were observed .

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

(A) GFP-Chimeras were treated with the BCL-XL inhibitor A1331852 (n=8, 5 doses by oral gavage, 25 mg/kg body weight each dose). Control groups (n=8 each group) include untreated GFP-Chimera and un-irradiated C57BL/6-Ly5.1 (wild-type) mice treated with the BCL-XL inhibitor A1331852 or left untreated. Kidney (left panel) and spleen weights (right panel) were measured in drug-treated GFP-Chimeras or control mice at the termination of the experiment (n=8 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test. No statistically significant differences were observed.


( B ) C57BL / 6 - Ly5 . 1 ( wild - type ) mice ( females , aged 10 weeks ) were treated with Cyclophosphamide ( n = 7 , 150 mg / kg body weight , 1 dose i . v . ) or 5 - Fluorouracil ( 5 - FU , n = 7 , 100 mg / kg body weight , 1 dose i . v . ) and after 5 days additionally treated with the BCL - XL inhibitor A1331852 ( 5 doses by oral gavage , 100 mg / kg body weight each dose ) . Control groups ( n = 7 each group ) include C57BL / 6 - Ly5 . 1 ( wild - type ) mice ( females , aged 10 weeks ) treated with the BCL - XL inhibitor A1331852 alone , Cyclophosphamide alone , 5 - FU alone or left untreated . Kidney ( left panel ) and spleen weights ( right panel ) were measured in drug - treated mice or control mice at the termination of the experiment ( n = 7 each group ) . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using one - way ANOVA analysis with Tukey ' s multiple comparisons test . No statistically significant differences were observed .

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

(B) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with Cyclophosphamide (n=7, 150 mg/kg body weight, 1 dose i.v.) or 5-Fluorouracil (5-FU, n=7, 100 mg/kg body weight, 1 dose i.v.) and after 5 days additionally treated with the BCL-XL inhibitor A1331852 (5 doses by oral gavage, 100 mg/kg body weight each dose). Control groups (n=7 each group) include C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) treated with the BCL-XL inhibitor A1331852 alone, Cyclophosphamide alone, 5-FU alone or left untreated. Kidney (left panel) and spleen weights (right panel) were measured in drug-treated mice or control mice at the termination of the experiment (n=7 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test. No statistically significant differences were observed.


( C ) Histological analysis of H & E - stained sections of the kidneys of drug - treated GFP - Chimeras or control mice at the termination of the experiment . Pictures are representative of at least 3 mice for each treatment group .

O O O O O O O O O O O O O O O O O O O O O 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) Histological analysis of H&E-stained sections of the kidneys of drug-treated GFP-Chimeras or control mice at the termination of the experiment. Pictures are representative of at least 3 mice for each treatment group.


( D ) Histological analysis of H & E - stained sections of the kidneys of drug - treated mice or control untreated mice at the termination of the experiment . Pictures are representative of at least 3 mice for each treatment group .

O O O O O O O O O O O O O O O O O O O O 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) Histological analysis of H&E-stained sections of the kidneys of drug-treated mice or control untreated mice at the termination of the experiment. Pictures are representative of at least 3 mice for each treatment group.


C , Dose - response curves of BEAS - 2B and BEAS - 2B - KRAS cells treated for 72 hours with inhibitors of FGFR ( AZD4547 , BGJ398 ) and PLK1 ( BI2536 , BI6727 ) , alone or combination . Values of Fa and CI are shown underneath , with CI < 1 . 0 indicating synergistic effect . Data were from three independent experiments ( n = 3 ) , error bar : SD

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

C, Dose-response curves of BEAS-2B and BEAS-2B-KRAS cells treated for 72 hours with inhibitors of FGFR (AZD4547, BGJ398) and PLK1 (BI2536, BI6727), alone or combination. Values of Fa and CI are shown underneath, with CI<1.0 indicating synergistic effect. Data were from three independent experiments (n=3), error bar: SD


C , Dose - response curves of BEAS - 2B and BEAS - 2B - KRAS cells treated for 72 hours with inhibitors of FGFR ( AZD4547 , BGJ398 ) and PLK1 ( BI2536 , BI6727 ) , alone or combination . Values of Fa and CI are shown underneath , with CI < 1 . 0 indicating synergistic effect . Data were from three independent experiments ( n = 3 ) , error bar :

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


D , BEAS - 2B and BEAS - 2B - KRAS treated with the indicated drugs , alone or combination for 72 hours , were cultured in drug - free medium for additional 7 - 21 days . Surviving cells after the treatment were fixed and visualized by crystal violet staining .

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

D, BEAS-2B and BEAS-2B-KRAS treated with the indicated drugs, alone or combination for 72 hours, were cultured in drug-free medium for additional 7-21 days. Surviving cells after the treatment were fixed and visualized by crystal violet staining.


C , Clongenic assay of KRAS - mutant lung and pancreatic cells ( MIA PaCa - 2 , H2122 , H2009 , H23 , H358 and A549 ) treated with control ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or combination .

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

C, Clongenic assay of KRAS-mutant lung and pancreatic cells (MIA PaCa-2, H2122, H2009, H23, H358 and A549) treated with control (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or combination.


D , Apoptotic assay of KRAS - mutant cancer cells ( H358 , H441 , A549 , and SW620 ) and KRAS - WT lung cancer cells ( EBC - 1 ) treated with vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or in combination for 48h . Data are presented as mean ± SD ( n = 3 ) . * * * P < 0 . 001 and * * * * P < 0 . 0001 by two - way ANOVA with Tukey ' s multiple comparisons test .

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

D, Apoptotic assay of KRAS-mutant cancer cells (H358, H441, A549, and SW620) and KRAS-WT lung cancer cells (EBC-1) treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or in combination for 48h. Data are presented as mean ± SD (n=3). ***P<0.001 and ****P<0.0001 by two-way ANOVA with Tukey's multiple comparisons test.


E , KRAS mutant lung ( A549 , H358 ， H441 ) , pancreatic ( MIAPaCa - 2 ) and colon ( SW620 ) cancer cells were transfected with control siRNAs , FGFR1 - and PLK1 - specific siRNAs , alone or combination . Cell viability was determined 72 h post transfection . Data are presented as mean ± SD ( n = 3 ) . * * p < 0 . 01 , * * * * p < 0 . 0001 , and ns P ＞ 0 . 05 by two - way ANOVA with Tukey ' s multiple comparisons test . ns , no significance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, KRAS mutant lung (A549, H358，H441), pancreatic (MIAPaCa-2) and colon (SW620) cancer cells were transfected with control siRNAs, FGFR1- and PLK1-specific siRNAs, alone or combination. Cell viability was determined 72 h post transfection. Data are presented as mean ± SD (n=3). **p<0.01, ****p<0.0001, and ns P＞0.05 by two-way ANOVA with Tukey's multiple comparisons test. ns, no significance.


D , Clonogenic assay of H358 , A549 and MIA PaCa - 2 cells treated with AZD4547 , BI2536 , and NAC , alone or in combination as indicated .

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

D, Clonogenic assay of H358, A549 and MIA PaCa-2 cells treated with AZD4547, BI2536, and NAC, alone or in combination as indicated.


E , Immunoblots of H358 and A549 cells treated for 24 h with vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , in the presence or absence of NAC ( 5 mM ) .

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

E, Immunoblots of H358 and A549 cells treated for 24 h with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), in the presence or absence of NAC (5 mM).


clonogenic assay of H358 cells treated for 48 h with vehicle , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , in the presence or absence of Q - VD - OPh ( 20 µM ) . Data shown as mean ± SD ( n = 3 ) . * * * P < 0 . 001 and * * * * P < 0 . 0001 by two - way ANOVA with Tukey ' s multiple comparisons test .

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

clonogenic assay of H358 cells treated for 48 h with vehicle, AZD4547 (5 µM) and BI2536 (5 nM), in the presence or absence of Q-VD-OPh (20 µM). Data shown as mean ± SD (n=3). ***P<0.001 and ****P<0.0001 by two-way ANOVA with Tukey's multiple comparisons test.


B , C , Immunoblots ( B ) and viability assay ( C ) of H358 and A549 cells preincubated overnight with SP600125 ( 2 µM ) and SB203580 ( 1 µM ) and subsequently treated with AZD 4547 ( 5 µM ) and BI2536 ( 5 nM ) for 24 hours . Data were shown as mean ± SD ( n = 3 ) ; * * * P < 0 . 001 , * * * * P < 0 . 0001 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test .

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

B, C, Immunoblots (B) and viability assay (C) of H358 and A549 cells preincubated overnight with SP600125 (2 µM) and SB203580 (1 µM) and subsequently treated with AZD 4547 (5 µM) and BI2536 (5 nM) for 24 hours. Data were shown as mean ± SD (n=3); ***P<0.001, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.


D , E , Immunoblots ( D ) and viability assay ( E ) of H358 and A549 cells transfected with E2F1 - specific or control siRNAs followed by treatment ( 48 h post transfection ) with vehicle ( DMSO ) or combined AZD 4547 ( 5 µM ) / BI2536 ( 5 nM ) for additional 24 hours . Data were shown as mean ± SD ( n = 3 ) . * * P < 0 . 01 , * * * * P < 0 . 0001 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test .

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

D, E, Immunoblots (D) and viability assay (E) of H358 and A549 cells transfected with E2F1-specific or control siRNAs followed by treatment (48 h post transfection) with vehicle (DMSO) or combined AZD 4547 (5 µM)/BI2536(5 nM) for additional 24 hours. Data were shown as mean ± SD (n=3). **P<0.01, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.


F , H358 cells transfected with E2F1 - specific or control siRNAs for 24h were subsequently treated with vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or combination for 48h before apoptotic assay . Data were shown as mean ± SD ( n = 3 ) . * P < 0 . 05 , * * P < 0 . 01 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test .

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

F, H358 cells transfected with E2F1-specific or control siRNAs for 24h were subsequently treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or combination for 48h before apoptotic assay. Data were shown as mean ± SD (n=3). *P<0.05, **P<0.01 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.


B , A549 cells stably expressing mCHerry - GFP - LC3B were exposed to vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or in combination for 24h . Cell were then fixed and processed for immunofluorescence , scale bar = 10 µm .

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

B, A549 cells stably expressing mCHerry-GFP-LC3B were exposed to vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or in combination for 24h. Cell were then fixed and processed for immunofluorescence, scale bar=10 µm.


E , Flow cytometry - based ROS measure in H358 , A549 and MIA PaCa - 2 cells treated with vehicle ( DMSO ) , AZD4547 ( 5 µM ) and BI2536 ( 5 nM ) , alone or the combination , in the presence or absence of HCQ ( 10 µM ) for 24h . Quantification of relative ROS levels was shown to the right . Data are presented as mean ± SD ( n = 3 ) . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test .

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

E, Flow cytometry-based ROS measure in H358, A549 and MIA PaCa-2 cells treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or the combination, in the presence or absence of HCQ (10 µM) for 24h. Quantification of relative ROS levels was shown to the right. Data are presented as mean ± SD (n=3). *P<0.05, **P<0.01, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.


F , Immunoblots of H358 cells transfected with ATG5 - specific or control siRNAs for 48h and before treated for 24h with vehicle ( DMSO ) or combined AZD 4547 ( 5 µM ) / BI2536 ( 5 nM ) .

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

F, Immunoblots of H358 cells transfected with ATG5-specific or control siRNAs for 48h and before treated for 24h with vehicle (DMSO) or combined AZD 4547 (5 µM)/BI2536 (5 nM).


A , Growth curve of a KRAS - mutant lung cancer PDX ( BE564T ) treated with vehicle , HCQ ( 30 mg / kg / day ) , AZD4547 ( 10 mg / kg / day ) and BI6727 ( 5 mg / kg / day ) , alone or in combination . * P < 0 . 05 , * * * P < 0 . 001 and P ＞ 0 . 05 ( ns ) by one - way ANOVA with Tukey ' s multiple comparisons test . Data are the mean of tumor volume of each group ( 5 mice / group ) ; error bar : SD

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Growth curve of a KRAS-mutant lung cancer PDX (BE564T) treated with vehicle, HCQ (30 mg/kg/day), AZD4547 (10 mg/kg/day) and BI6727 (5 mg/kg/day), alone or in combination. *P<0.05, ***P<0.001 and P＞0.05 (ns) by one-way ANOVA with Tukey's multiple comparisons test. Data are the mean of tumor volume of each group (5 mice/group); error bar: SD


A , Growth curve of a KRAS - mutant lung cancer PDX ( BE564T ) treated with vehicle , HCQ ( 30 mg / kg / day ) , AZD4547 ( 10 mg / kg / day ) and BI6727 ( 5 mg / kg / day ) , alone or in combination . * P < 0 . 05 , * * * P < 0 . 001 and P ＞ 0 . 05 ( ns ) by one - way ANOVA with Tukey ' s multiple comparisons test . Data are the mean of tumor volume of each group ( 5 mice / group ) ; error bar :

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , E , H & E and IHC analysis ( Ki67 and Caspase - 3 ) of PDX ( BE 564T ) tumors after the treatment ( D ) . Quantification of the IHC data for the positivity of Ki - 67 and Caspase - 3 are shown in ( E ) . * P < 0 . 05 and * * P < 0 . 01 by two - way ANOVA with Tukey ' s multiple comparisons test . Data are shown as mean ± SD ( error bar ) of three independent experiments ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E, H&E and IHC analysis (Ki67 and Caspase-3) of PDX (BE 564T) tumors after the treatment (D). Quantification of the IHC data for the positivity of Ki-67 and Caspase-3 are shown in (E). *P<0.05 and **P<0.01 by two-way ANOVA with Tukey's multiple comparisons test. Data are shown as mean ± SD (error bar) of three independent experiments (n=3).


F - K , Growth curves ( F , I ) of KRAS - mutant lung cancer PDXs ( PF139 , PF563 ) treated with vehicle , HCQ ( 30 mg / kg / day ) , AZD4547 ( 10 mg / kg / day ) and BI6727 ( 5 mg / kg / day ) , alone or in combination . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by one - way ANOVA with Tukey ' s multiple comparisons test . Data are the mean of tumor volume of each group ( 4 mice / group ) ; error bar : SD Relative tumor volume ( G , J ) and weights ( H , K ) of the PDX tumors after the treatment . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and P ＞ 0 . 05 ( ns ) by unpaired two - tailed t - test . Data are the mean ± SD ( error bar ) of tumor weights of each group ( 4 mice / group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K, Growth curves (F, I) of KRAS-mutant lung cancer PDXs (PF139, PF563) treated with vehicle, HCQ (30 mg/kg/day), AZD4547 (10 mg/kg/day) and BI6727 (5 mg/kg/day), alone or in combination. *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA with Tukey's multiple comparisons test. Data are the mean of tumor volume of each group (4 mice/group); error bar: SD Relative tumor volume (G, J) and weights (H, K) of the PDX tumors after the treatment. *P<0.05, **P<0.01, ***P<0.001 and P＞0.05 (ns) by unpaired two-tailed t-test. Data are the mean ± SD (error bar) of tumor weights of each group (4 mice/group).


F , Box plots showing the percentage of Ki67 - positive cells in different treatment groups . Data are based on IHC staining of lung tissue section of LSL - KrasG12D mice . * P < 0 . 05 and P ＞ 0 . 05 ( ns ) by two - way ANOVA with Tukey ' s multiple comparisons test . The boxplots span from the first to third quartile , the whiskers extend to 1 . 5 × the interquartile range ( IQR ) . The horzontal line within the boxes represents the median of each group of 10 replicates ( n = 10 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Box plots showing the percentage of Ki67-positive cells in different treatment groups. Data are based on IHC staining of lung tissue section of LSL-KrasG12D mice. *P<0.05 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test. The boxplots span from the first to third quartile, the whiskers extend to 1.5× the interquartile range (IQR). The horzontal line within the boxes represents the median of each group of 10 replicates (n=10).


A . Immunoblot of cell extracts following depletion of WDR26 , RanBP9 , ARMC8 , and GID4 using pools of siRNAs for 72 hr in HeLa Kyoto cells . Endogenous levels of the indicated proteins were monitored by western blotting ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot of cell extracts following depletion of WDR26, RanBP9, ARMC8, and GID4 using pools of siRNAs for 72 hr in HeLa Kyoto cells. Endogenous levels of the indicated proteins were monitored by western blotting (n=3).


Western blotting of samples after ectopic overexpression of HBP1 ( B ) alone , or together with WDR26 or GID4 in HEK - 293T cells . HBP1 levels were monitored after treatment of MG132 or DMSO for 10 - 12 h ( n = 3 ) .

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

Western blotting of samples after ectopic overexpression of HBP1 (B) alone, or together with WDR26 or GID4 in HEK-293T cells. HBP1 levels were monitored after treatment of MG132 or DMSO for 10-12 h (n=3).


Western blotting of samples after ectopic overexpression of ZMYND19 ( C ) alone , or together with WDR26 or GID4 in HEK - 293T cells . ZMYND19 levels were monitored after treatment of MG132 or DMSO for 10 - 12 h ( n = 3 ) .

Western blotting of samples after ectopic overexpression of ZMYND19 (C) alone, or together with WDR26 or GID4 in HEK-293T cells. ZMYND19 levels were monitored after treatment of MG132 or DMSO for 10-12 h (n=3).


C . , D . Western blot analysis of in vitro ubiquitinated HBP1 , which was performed by mixing purified HBP1 with ubiquitin E1 , UBCH5a , and ubiquitin in the presence of the indicated hGID sub - complexes ( n = 3 ) .

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

C., D. Western blot analysis of in vitro ubiquitinated HBP1, which was performed by mixing purified HBP1 with ubiquitin E1, UBCH5a, and ubiquitin in the presence of the indicated hGID sub-complexes (n=3).


E . Immunoblots of in vitro ubiquitinated ZMYND19 , which was performed by mixing purified ZMYND19 with ubiquitin E1 , UBE2H , ubiquitin and the 6 - subunit hGID complex ( ARCM8 , RanBP9 , WDR26 , MAEA , RMND5a and TWA1 ) in the presence or absence of GID4 and a 10 - fold excess of the PGLV GID4 - specific peptide ( n = 2 )

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

E. Immunoblots of in vitro ubiquitinated ZMYND19, which was performed by mixing purified ZMYND19 with ubiquitin E1, UBE2H, ubiquitin and the 6-subunit hGID complex (ARCM8, RanBP9, WDR26, MAEA, RMND5a and TWA1) in the presence or absence of GID4 and a 10-fold excess of the PGLV GID4-specific peptide (n=2)


D . Western blotting of samples following ectopic overexpression of HBP1 either alone , or with full - length ( FL ) or WD40 - truncated WDR26 ( ΔWD40 ) in HEK - 293T cells . HBP1 levels were monitored in cells treated with MG132 or DMSO for 12 - 14 h ( n = 3 ) .

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

D. Western blotting of samples following ectopic overexpression of HBP1 either alone, or with full-length (FL) or WD40-truncated WDR26 (ΔWD40) in HEK-293T cells. HBP1 levels were monitored in cells treated with MG132 or DMSO for 12-14 h (n=3).


E . Western blot analysis of in vitro ubiquitinated HBP1 in the presence of wild type WDR26 ( WT ) or the WDR26 ( ΔWD40 ) mutant ( n = 2 ) .

O O O O O O O O O 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. Western blot analysis of in vitro ubiquitinated HBP1 in the presence of wild type WDR26 (WT) or the WDR26 (ΔWD40) mutant (n=2).


C . Transiently expressed FLAG - GID4 and HSS - tagged ARMC8 isoforms ( α or β ) in HEK293T cells . The presence of GID4 in isoform - specific ARMC8 immunoprecipitates was visualized by immunoblotting ( left panels ) . The right panel shows a western blot of transiently expressed and immunoprecipitated HSS - WDR26 from HEK - 293T cells and the presence of endogenous ARMC8 isoforms ( α or β ) was probed by immunoblotting ( n = 2 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Transiently expressed FLAG-GID4 and HSS-tagged ARMC8 isoforms (α or β) in HEK293T cells. The presence of GID4 in isoform-specific ARMC8 immunoprecipitates was visualized by immunoblotting (left panels). The right panel shows a western blot of transiently expressed and immunoprecipitated HSS-WDR26 from HEK-293T cells and the presence of endogenous ARMC8 isoforms (α or β) was probed by immunoblotting (n=2).


D . Baculoviral co - expression in Sf9 cells of the 5 - subunit hGID complex ( 5mer ; Strep - RanBP9 , His - WDR26 , FLAG - MAEA , His - RMND5a and His - TWA1 ) along with His - GID4 in the presence of ARMC8α or ARMC8β . Strep - or His - pulldowns revealed the presence of GID4 in ARMC8α , but not in ARMC8β , complexes ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Baculoviral co-expression in Sf9 cells of the 5-subunit hGID complex (5mer; Strep-RanBP9, His-WDR26, FLAG-MAEA, His-RMND5a and His-TWA1) along with His-GID4 in the presence of ARMC8α or ARMC8β. Strep- or His-pulldowns revealed the presence of GID4 in ARMC8α, but not in ARMC8β, complexes (n=3).


E . Immunoblot analysis of in vitro ubiquitinated GID4 - hGID complexes containing either ARMC8α or ARMC8β ( 6mer ; ARMC8 , RanBP9 , WDR26 , MAEA , RMND5a and TWA1 ) . Where indicated , the reaction was carried out in the presence of 20 - fold molar excess of the PGLV GID4 - binding peptide ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot analysis of in vitro ubiquitinated GID4-hGID complexes containing either ARMC8α or ARMC8β (6mer; ARMC8, RanBP9, WDR26, MAEA, RMND5a and TWA1) . Where indicated, the reaction was carried out in the presence of 20-fold molar excess of the PGLV GID4-binding peptide (n=3).


F . Western blot analysis of in vitro ubiquitinated HBP1 by the hGID 5 - subunit complex in the presence of the different ARMC8 isoforms ( α or β ) ( n = 2 ) .

O O O O O O O O O O O O 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. Western blot analysis of in vitro ubiquitinated HBP1 by the hGID 5-subunit complex in the presence of the different ARMC8 isoforms (α or β) (n=2).


Subcellular localization of dLap1mGFP and dTormGFP - E177Q in the wild - type ( w - ) or dLap1 + / ∆ 11 fat body of 3rd instar fly larvae , when expressed as UAS transgenes .

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

Subcellular localization of dLap1mGFP and dTormGFP-E177Q in the wild-type (w-) or dLap1+/∆11 fat body of 3rd instar fly larvae, when expressed as UAS transgenes.


Neutral lipid staining of the 5DO larval fat body . White arrows ( O ) indicate nuclear - localized neutral lipid staining , blue signal shows DAPI .

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

Neutral lipid staining of the 5DO larval fat body. White arrows (O) indicate nuclear-localized neutral lipid staining, blue signal shows DAPI.


GFP , Phalloidin and LipidTOX detect the expression of dTormGFP chimeras ( green ) , cell boundaries ( red ) , and LD ( red ) , respectively , in dTorKO fat body cells .

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

GFP, Phalloidin and LipidTOX detect the expression of dTormGFP chimeras (green), cell boundaries (red), and LD (red), respectively, in dTorKO fat body cells.


Bars show the fraction ( mean ± SEM ) of membrane GL and GPL lipids with each fatty acyl chain characteristic . n = 3 MS analyses of 4DO fat body samples for each genotype . MUFA , mono - unsaturated ; PUFA , poly - unsaturated . ‡ indicate a significant difference between w - and dTorKO expressing Luciferase RNAi , * indicate a significant difference compared to dTorKO expressing Luciferase RNAi . Two - way ANOVA , Dunnett ' s multiple comparison test . ‡ p < 0 . 05 and * p < 0 . 05 .

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

Bars show the fraction (mean ± SEM) of membrane GL and GPL lipids with each fatty acyl chain characteristic. n = 3 MS analyses of 4DO fat body samples for each genotype. MUFA, mono-unsaturated; PUFA, poly-unsaturated. ‡ indicate a significant difference between w- and dTorKO expressing Luciferase RNAi, * indicate a significant difference compared to dTorKO expressing Luciferase RNAi. Two-way ANOVA, Dunnett's multiple comparison test. ‡ p < 0.05 and * p < 0.05.


Control and dTorKO 5DO fat body cells expressing UAS GFP fusion proteins with the Cg - Gal4 driver , and labeled with anti - Calnexin ( red ) and a neutral lipid dye ( LipidTOX , magenta ) . Note that dTorKO cells are smaller . White arrowhead indicates NE enrichment of LipinmGFP .

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

Control and dTorKO 5DO fat body cells expressing UAS GFP fusion proteins with the Cg-Gal4 driver, and labeled with anti-Calnexin (red) and a neutral lipid dye (LipidTOX, magenta). Note that dTorKO cells are smaller. White arrowhead indicates NE enrichment of LipinmGFP.


Lipin1mGFP localization in HEK293T cells transfected with a plasmid that co - expresses CTDNEP1myc - WT or - CD ( red ) and NEP1R1V5 ( magenta ) . ( G ' ) Upper : enlargement of panel G , white arrow highlights NE enrichment . Lower : plot showing the intensity of Lipin1mGFP signal ( mean ± SD , n = 5 cells ) along a 3 µm profile that transects the NE at 0 µm .

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

Lipin1mGFP localization in HEK293T cells transfected with a plasmid that co-expresses CTDNEP1myc-WT or -CD (red) and NEP1R1V5 (magenta). (G') Upper: enlargement of panel G, white arrow highlights NE enrichment. Lower: plot showing the intensity of Lipin1mGFP signal (mean ± SD, n = 5 cells) along a 3 µm profile that transects the NE at 0 µm.


Percentage of MEFs with primarily cytosolic or nuclear Lipin1mGFP . Chi - square test . * * * * p < 0 . 0001 .

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

Percentage of MEFs with primarily cytosolic or nuclear Lipin1mGFP. Chi-square test. **** p < 0.0001.


Control ( w - ) and dTorKO fat body nuclei labeled with anti - LaminDm0 , Megator / Tpr , mAb414 , or expressing UAS Nup107GFP or UAS Nup358GFP with the Cg - Gal4 driver .

Control (w-) and dTorKO fat body nuclei labeled with anti-LaminDm0, Megator/Tpr, mAb414, or expressing UAS Nup107GFP or UAS Nup358GFP with the Cg-Gal4 driver.


mAb414 labeling of 5DO dTorKO fat body cells expressing the dTor cDNA , Luciferase RNAi , or RNAi against Nep1r1 , Ctdnep1 , or Lipin . Nuc , nucleus . The percentage of fat body cells with NE - localized mAb414 . * * and * * * * indicate that Lipin , Ctdnep1 , and Nep1r1 RNAi significantly increased the number of dTorKO cells with NE - specific mAb414 labeling compared to the Luciferase RNAi ( Luc ) . Chi - square test followed by individual post hoc Chi - square tests . * * p < 0 . 01 and * * * * p < 0 . 0001 . NE ultrastructure in 5DO dTorKO cells . Red arrows , nuclear pores ; yellow arrows , abnormal INM herniations . Nuc , nucleus ; Cyto , cytosol .

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

mAb414 labeling of 5DO dTorKO fat body cells expressing the dTor cDNA, Luciferase RNAi, or RNAi against Nep1r1, Ctdnep1, or Lipin. Nuc, nucleus. The percentage of fat body cells with NE-localized mAb414. ** and **** indicate that Lipin, Ctdnep1, and Nep1r1 RNAi significantly increased the number of dTorKO cells with NE-specific mAb414 labeling compared to the Luciferase RNAi (Luc). Chi-square test followed by individual post hoc Chi-square tests. ** p < 0.01 and **** p < 0.0001. NE ultrastructure in 5DO dTorKO cells. Red arrows, nuclear pores; yellow arrows, abnormal INM herniations. Nuc, nucleus; Cyto, cytosol.


The percentage that Lipin , Ctdnep1 , and Nep1r1 RNAi normalize the abundance of membrane GL and GPL classes in the dTorKO fat body . 0 % " normal " reflects lipid abundance in dTorKO expressing Luciferase RNAi , while 100 % " normal " reflects lipid abundance in w - expressing Luciferase RNAi . Percentage normalization was separately calculated for PI , PA , DAG , PC , PE and PS ( for dTorKO expressing Lipin , Ctdnep1 , and Nep1r1 RNAi ) . Points show the mean of all lipid class normalization per sample , bars show mean ± SEM for the group .

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

The percentage that Lipin, Ctdnep1, and Nep1r1 RNAi normalize the abundance of membrane GL and GPL classes in the dTorKO fat body. 0% "normal" reflects lipid abundance in dTorKO expressing Luciferase RNAi, while 100% "normal" reflects lipid abundance in w- expressing Luciferase RNAi. Percentage normalization was separately calculated for PI, PA, DAG, PC, PE and PS (for dTorKO expressing Lipin, Ctdnep1, and Nep1r1 RNAi). Points show the mean of all lipid class normalization per sample, bars show mean ± SEM for the group.


NE ultrastructure of 5DO fat body cells overexpressing UAS transgenes , or from dLap1 - / - animals . Yellow dotted lines highlight NE regions with subtly abnormal membrane architecture . Red arrows indicate nuclear pores . R1 , region 1 ; R2 , region2 ; Nuc , nucleus ; Cyto ,

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

NE ultrastructure of 5DO fat body cells overexpressing UAS transgenes, or from dLap1-/- animals. Yellow dotted lines highlight NE regions with subtly abnormal membrane architecture. Red arrows indicate nuclear pores. R1, region 1; R2, region2; Nuc, nucleus; Cyto, cytosol


Data from FIB - SEM imaging of fat body nuclei expressing Sec61b ( control ) , or CTDNEP1mGFP . ( J - L ) 3D reconstructions , ( M ) distance between the INM and ONM at NPC in wild - type cells versus CTDNEP1mGFP - induced structures . Kolmogorov - Smirnov non - parametric T - Test . * * * * p < 0 .

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

Data from FIB-SEM imaging of fat body nuclei expressing Sec61b (control), or CTDNEP1mGFP. (J - L) 3D reconstructions, (M) distance between the INM and ONM at NPC in wild-type cells versus CTDNEP1mGFP-induced structures. Kolmogorov-Smirnov non-parametric T-Test. **** p < 0.0001


mAb414 localization in 5DO fat body cells expressing the indicated UAS transgenes or from a dLap1 - / - animal . * * * and * * * * indicate significant difference compared to control mGFPSec61b expressing cells . Chi - square test followed by individual post hoc Chi - square tests . * * * p < 0 . 001 and * * * * p < 0 . 0001 .

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

mAb414 localization in 5DO fat body cells expressing the indicated UAS transgenes or from a dLap1-/- animal. *** and **** indicate significant difference compared to control mGFPSec61b expressing cells. Chi-square test followed by individual post hoc Chi-square tests. *** p < 0.001 and **** p < 0.0001.


NE ultrastructure of 5DO fat body cells from a ( I ) control animal , ( J ) homozygous Nup35BG01311 and ( K ) homozygous Nup35MB02683 animals . Nuc , nucleus ; Cyto , cytosol .

NE ultrastructure of 5DO fat body cells from a (I) control animal, (J) homozygous Nup35BG01311 and (K) homozygous Nup35MB02683 animals. Nuc, nucleus; Cyto, cytosol.


( H Anti - FruM signals in 3 - 6h WTCS males are stronger than those in 4 - 6d WTCS males ( H ) n = 7 for each . Data information : Scale bars , 100 μm .

O O O O O O O O O O O O O O O O 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 Anti-FruM signals in 3-6h WTCS males are stronger than those in 4-6d WTCS males (H) n = 7 for each. Data information: Scale bars, 100 μm.


( J and Anti - GFP signals are grossly similar in 3 - 6h and 4 - 6d UAS - myrGFP ; fruGAL4 males ( J ) , n = 6 for each . Data information : Scale bars , 100 μm .

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

(J and Anti-GFP signals are grossly similar in 3-6h and 4-6d UAS-myrGFP; fruGAL4 males (J), n = 6 for each. Data information: Scale bars, 100 μm.


( A ) Wing extension indices ( zero ) of isolated 3 - 6h juvenile w1118 / Y ; UAS - dTrpA1 / + ; fruGAL4 / + , Met27 / Y ; UAS - dTrpA1 / + ; fruGAL4 / + and Gce2 . 5k / Y ; UAS - dTrpA1 / + ; fruGAL4 / + males at 22 and 27 ℃ . n = 20 for each . ( B and C ) Wing extension indices ( B ) and latencies ( C ) of isolated 4 - 6d adult w1118 / Y ; UAS - dTrpA1 / + ; fruGAL4 / + , Met27 / Y ; UAS - dTrpA1 / + ; fruGAL4 / + and Gce2 . 5k / Y ; UAS - dTrpA1 / + ; fruGAL4 / + males at 22 and 27 ℃ . n = 20 for each . n . s . , not significant , * p < 0 . 05 , * * * p < 0 . 001 , Mann - Whitney U test for ( B ) and unpaired t test for ( C ) . NA , not applicable for latency analysis . Data information : Error bars indicate SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wing extension indices (zero) of isolated 3-6h juvenile w1118/Y; UAS-dTrpA1/+; fruGAL4/+, Met27/Y; UAS-dTrpA1/+; fruGAL4/+ and Gce2.5k/Y; UAS-dTrpA1/+; fruGAL4/+ males at 22 and 27℃. n = 20 for each. (B and C) Wing extension indices (B) and latencies (C) of isolated 4-6d adult w1118/Y; UAS-dTrpA1/+; fruGAL4/+, Met27/Y; UAS-dTrpA1/+; fruGAL4/+ and Gce2.5k/Y; UAS-dTrpA1/+; fruGAL4/+ males at 22 and 27℃. n = 20 for each. n.s., not significant, *p < 0.05, ***p < 0.001, Mann-Whitney U test for (B) and unpaired t test for (C). NA, not applicable for latency analysis. Data information: Error bars indicate SEM.


( E and F ) Tracking indices of 3 - 6h and 4 - 6d w1118 / Y ; UAS - dTrpA1 / + ; fruGAL4 / + , Met27 / Y ; UAS - dTrpA1 / + ; fruGAL4 / + and Gce2 . 5k / Y ; UAS - dTrpA1 / + ; fruGAL4 / + males toward moving dots at 22 and 27 ℃ . n = 24 for each in ( E ) . n = 22 , 20 , 24 , 24 , 24 and 24 from left to right in ( F ) . n . s . , not significant , * * * p < 0 . 001 , Mann - Whitney U test . Data information : Error bars indicate SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and F) Tracking indices of 3-6h and 4-6d w1118/Y; UAS-dTrpA1/+; fruGAL4/+, Met27/Y; UAS-dTrpA1/+; fruGAL4/+ and Gce2.5k/Y; UAS-dTrpA1/+; fruGAL4/+ males toward moving dots at 22 and 27℃. n = 24 for each in (E). n = 22, 20, 24, 24, 24 and 24 from left to right in (F). n.s., not significant, ***p < 0.001, Mann-Whitney U test. Data information: Error bars indicate SEM.


( A and B ) qPCR experiments show that Met ( A ) and Gce ( B ) mRNA levels are significantly knocked down in actin - GAL4 > UAS - Met : : Gce - IR adult flies . n = 9 for each . * * * p < 0 . 001 , unpaired t test . Data information : Error bars indicate SEM .

(A and B) qPCR experiments show that Met (A) and Gce (B) mRNA levels are significantly knocked down in actin-GAL4>UAS-Met::Gce-IR adult flies. n = 9 for each. ***p<0.001, unpaired t test. Data information: Error bars indicate SEM.


Courtship behaviors of 3 - 6h juvenile and 4 - 6d adult males are not significantly affected by knocking down JH receptors Met and Gce ( UAS - Met : : Gce - IR ) pan - neuronally or specifically in neurons driven by 57C10 - GAL4 ( C ) , fruGAL4 ( D ) N = 22 for each in ( C ) , n = 24 for each in ( D ) n . s . , not significant , Mann - Whitney U test . Data information : Error bars indicate SEM .

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

Courtship behaviors of 3-6h juvenile and 4-6d adult males are not significantly affected by knocking down JH receptors Met and Gce (UAS-Met::Gce-IR) pan-neuronally or specifically in neurons driven by 57C10-GAL4 (C), fruGAL4 (D) N = 22 for each in (C), n = 24 for each in (D) n.s., not significant, Mann-Whitney U test. Data information: Error bars indicate SEM.


Courtship behaviors of 3 - 6h juvenile and 4 - 6d adult males are not significantly affected by knocking down JH receptors Met and Gce ( UAS - Met : : Gce - IR ) pan - neuronally or specifically in R41A01 - GAL4 ( E ) , NPF - GAL4 ( F ) , TH - GAL4 ( G ) and R71G01 - GAL4 ( H ) respectively . n = 24 , 24 , 21 , and 22 from left to right in ( E ) , n = 24 , 24 , 24 , and 23 from left to right in ( F ) , n = 24 , 24 , 20 and 24 from left to right in ( G ) and n = 24 , 24 , 23 and 24 from left to right in ( H ) . n . s . , not significant , Mann - Whitney U test . Data information : Error bars indicate SEM .

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

Courtship behaviors of 3-6h juvenile and 4-6d adult males are not significantly affected by knocking down JH receptors Met and Gce (UAS-Met::Gce-IR) pan-neuronally or specifically in R41A01-GAL4 (E), NPF-GAL4 (F), TH-GAL4 (G) and R71G01-GAL4 (H) respectively. n = 24, 24, 21, and 22 from left to right in (E), n = 24, 24, 24, and 23 from left to right in (F), n = 24, 24, 20 and 24 from left to right in (G) and n = 24, 24, 23 and 24 from left to right in (H). n.s., not significant, Mann-Whitney U test. Data information: Error bars indicate SEM.


( A - D ) Expression pattern of R41A01 - GAL4 ( pCd , A ) , NPF - GAL4 ( B ) , TH - GAL4 ( C ) , and R71G01 - GAL4 ( P1 , D ) in adult male brains . Representative of three samples for each . Data information : Scale bars , 100 μm .

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

(A-D) Expression pattern of R41A01-GAL4 (pCd, A), NPF-GAL4 (B), TH-GAL4 (C), and R71G01-GAL4 (P1, D) in adult male brains. Representative of three samples for each. Data information: Scale bars, 100 μm.


( I - L ) Courtship behaviors of 4 - 6d adult males toward virgin females are similarly intensive and not significantly influenced by activating pCd ( I ) , NPF ( J ) , TH ( K ) or P1 ( L ) neurons at 29 ℃ . n = 24 for each in ( I ) , n = 24 , 24 , 23 , and 23 from left to right in ( J ) , n = 24 , 24 , 24 , and 23 from left to right in ( K ) , and n = 24 for each in ( L ) . n . s . , not significant , * * p < 0 . 01 , * * * p < 0 . 001 , Mann - Whitney U test . Data information : Error bars indicate SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-L) Courtship behaviors of 4-6d adult males toward virgin females are similarly intensive and not significantly influenced by activating pCd (I), NPF (J), TH (K) or P1 (L) neurons at 29℃. n = 24 for each in (I), n = 24, 24, 23, and 23 from left to right in (J), n = 24, 24, 24, and 23 from left to right in (K), and n = 24 for each in (L). n.s., not significant, **p < 0.01, ***p < 0.001, Mann-Whitney U test. Data information: Error bars indicate SEM.


( A and B ) Anti - GFP signals are grossly similar in 3 - 6h and 4 - 6d UAS - myrGFP / + ; dsxGAL4 / + males ( A ) , which are quantified in ( B ) . n = 6 and 8 , respectively . n . s . , not significant , Mann - Whitney U test . Data information : Scale bars , 50 μm . Error bars indicate SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) Anti-GFP signals are grossly similar in 3-6h and 4-6d UAS-myrGFP/+; dsxGAL4/+ males (A), which are quantified in (B). n = 6 and 8, respectively. n.s., not significant, Mann-Whitney U test. Data information: Scale bars, 50 μm. Error bars indicate SEM.


( C and D ) Anti - DsxM signals in 3 - 6h WTCS males are stronger than those in 4 - 6d WTCS males ( C ) , which are quantified in ( D ) . n = 6 for each . * p < 0 . 05 , Mann - Whitney U test . Data information : Scale bars , 50 μm . Error bars indicate SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and D) Anti-DsxM signals in 3-6h WTCS males are stronger than those in 4-6d WTCS males (C), which are quantified in (D). n = 6 for each. *p < 0.05, Mann-Whitney U test. Data information: Scale bars, 50 μm. Error bars indicate SEM.


( E - L ) Synaptic transmission levels are visualized by syb - GRASP signals ( GFP ) . ( E and F ) syb - GRASP signals from NPF neurons to dsx neurons are only observed in 4 - 6d adult males but not in 3 - 6h or 1d males ( E ) , which are quantified in ( F ) . The genotype is LexAop - nSyb - spGFP1 - 10 , UAS - CD4 - spGFP11 / + ; NPF - lexA / dsxGal4 . n = 12 , 7 and 5 from left to right . ( G and H ) syb - GRASP signals from dsx neurons to NPF neurons in 4 - 6d adult males are much stronger than those in 3 - 6h and 1d males . syb - GRASP signals in ( G ) are quantified in ( H ) . The genotype is UAS - nSyb - spGFP1 - 10 , LexAop - CD4 - spGFP11 / + ; NPF - lexA / dsxGal4 . n = 13 , 7 and 6 from left to right . ( I and J ) syb - GRASP signals from TH neurons to dsx neurons increase with age from 3 - 6h juvenile males to 4 - 6d adult males ( I ) , which are quantified in ( J ) . The genotype is LexAop - nSyb - spGFP1 - 10 , UAS - CD4 - spGFP11 / TH - LexA ; dsxGal4 / + . n = 12 , 5 and 6 , respectively . ( K and L ) syb - GRASP signals from dsx neurons to TH neurons gradually increase from juvenile males to adult males . syb - GRASP signals in ( K ) are quantified in ( L ) . The genotype is UAS - nSyb - spGFP1 - 10 , LexAop - CD4 - spGFP11 / TH - LexA ; dsxGal4 / + . n = 14 , 6 and 6 , respectively . White arrowheads indicate syb - GRASP signals in the same position in brains of juvenile males and adult males . n . s . , not significant , * * p < 0 . 01 , * * * p < 0 . 001 , Mann - Whitney U test . Data information : Scale bars , 50 μm . Error bars indicate SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-L) Synaptic transmission levels are visualized by syb-GRASP signals (GFP). (E and F) syb-GRASP signals from NPF neurons to dsx neurons are only observed in 4-6d adult males but not in 3-6h or 1d males (E), which are quantified in (F). The genotype is LexAop-nSyb-spGFP1-10, UAS-CD4-spGFP11/+; NPF-lexA/dsxGal4. n = 12, 7 and 5 from left to right. (G and H) syb-GRASP signals from dsx neurons to NPF neurons in 4-6d adult males are much stronger than those in 3-6h and 1d males. syb-GRASP signals in (G) are quantified in (H). The genotype is UAS-nSyb-spGFP1-10, LexAop-CD4-spGFP11/+; NPF-lexA/dsxGal4. n = 13, 7 and 6 from left to right. (I and J) syb-GRASP signals from TH neurons to dsx neurons increase with age from 3-6h juvenile males to 4-6d adult males (I), which are quantified in (J). The genotype is LexAop-nSyb-spGFP1-10, UAS-CD4-spGFP11/TH-LexA; dsxGal4/+. n = 12, 5 and 6, respectively. (K and L) syb-GRASP signals from dsx neurons to TH neurons gradually increase from juvenile males to adult males. syb-GRASP signals in (K) are quantified in (L). The genotype is UAS-nSyb-spGFP1-10, LexAop-CD4-spGFP11/TH-LexA; dsxGal4/+. n = 14, 6 and 6, respectively. White arrowheads indicate syb-GRASP signals in the same position in brains of juvenile males and adult males. n.s., not significant, **p < 0.01, ***p < 0.001, Mann-Whitney U test. Data information: Scale bars, 50 μm. Error bars indicate SEM.


( A - D ) Activation of NPF neurons with dTrpA1 induced Ca2 + rise in pC1 dsx neurons in 4 - 6d males but not in 3 - 6h males . ( A and B ) Ca2 + imaging of pC1 neurons from 22 ˚ C to 27 ˚ C and 30 ˚ C in 3 - 6h ( A ) and 4 - 6d ( B ) males . ( C ) Time - course of fluorescent changes in pC1 neurons upon activation of NPF neurons in 3 - 6h and 4 - 6d males ( UAS - GCaMP6m / LexAop - dTrpA1 ; NPF - lexA / dsxGAL4 ) , and in 4 - 6d control males ( UAS - GCaMP6m / + ; dsxGAL4 / + ) without NPF neuronal activation , which are quantified in ( D ) . n = 8 for 4 - 6d control males , 15 for 3 - 6h and 8 for 4 - 6d males with dTrpA1 . n . s . , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , unpaired t test . Data information : Scale bars , 10 μm . Error bars indicate SEM .

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

(A-D) Activation of NPF neurons with dTrpA1 induced Ca2+ rise in pC1 dsx neurons in 4-6d males but not in 3-6h males. (A and B) Ca2+ imaging of pC1 neurons from 22˚C to 27˚C and 30˚C in 3-6h (A) and 4-6d (B) males. (C) Time-course of fluorescent changes in pC1 neurons upon activation of NPF neurons in 3-6h and 4-6d males (UAS-GCaMP6m/LexAop-dTrpA1; NPF-lexA/dsxGAL4), and in 4-6d control males (UAS-GCaMP6m/+; dsxGAL4/+) without NPF neuronal activation, which are quantified in (D). n = 8 for 4-6d control males, 15 for 3-6h and 8 for 4-6d males with dTrpA1. n.s., not significant, *p < 0.05, **p < 0.01, ***p<0.001, unpaired t test. Data information: Scale bars, 10 μm. Error bars indicate SEM.


( E - H ) Activation of TH neurons with dTrpA1 triggered Ca2 + increase in pC1 dsx neurons in both 3 - 6h and 4 - 6d males . ( E and F ) Ca2 + imaging of pC1 neurons from 22 ˚ C to 27 ˚ C and 30 ˚ C in 3 - 6h ( E ) and 4 - 6d ( F ) males . ( G ) Time - course of fluorescent changes in pC1 neurons upon activation of TH neurons in 3 - 6h and 4 - 6d males ( UAS - dTrpA1 / LexAop - GCaMP6s ; dsxLexA / TH - GAL4 ) , and in 4 - 6d control males ( LexAop - GCaMP6s / + ; dsxLexA / + ) without activating TH neurons , which are quantified in ( H ) . n = 8 for each . n . s . , not significant , * p < 0 . 05 , Mann - Whitney U test . Data information : Scale bars , 10 μm . Error bars indicate SEM .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) Activation of TH neurons with dTrpA1 triggered Ca2+ increase in pC1 dsx neurons in both 3-6h and 4-6d males. (E and F) Ca2+ imaging of pC1 neurons from 22˚C to 27˚C and 30˚C in 3-6h (E) and 4-6d (F) males. (G) Time-course of fluorescent changes in pC1 neurons upon activation of TH neurons in 3-6h and 4-6d males (UAS-dTrpA1/LexAop-GCaMP6s; dsxLexA/TH-GAL4), and in 4-6d control males (LexAop-GCaMP6s/+; dsxLexA/+) without activating TH neurons, which are quantified in (H). n = 8 for each. n.s., not significant, *p < 0.05, Mann-Whitney U test. Data information: Scale bars, 10 μm. Error bars indicate SEM.


A Z score for AUC and IC50 of 37 different drugs ( shown along the horizontal axis ) identified as hits in 13 of 17 samples screened . A drug hit is defined as z score of less than - 2 for both AUC and IC50 . Each dot in a column represents a sample screened for that drug . The size of the dot corresponds to the IC50 z score for that sample ( the larger the dot , the smaller the IC50 ) . Dots below the black horizontal line represent sample with AUC z score of less than - 2 . Dots are color coded for drug hit types . All color dots below the black line represent a hit for the corresponding drug .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Z score for AUC and IC50 of 37 different drugs (shown along the horizontal axis) identified as hits in 13 of 17 samples screened. A drug hit is defined as z score of less than -2 for both AUC and IC50. Each dot in a column represents a sample screened for that drug. The size of the dot corresponds to the IC50 z score for that sample (the larger the dot, the smaller the IC50). Dots below the black horizontal line represent sample with AUC z score of less than -2. Dots are color coded for drug hit types. All color dots below the black line represent a hit for the corresponding drug.


Event - free survival ( EFS ) and percentage of human CD45 + leukocytes in peripheral blood in acute lymphoblastic leukemia ( ALL ) orthotopic models .

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Event-free survival (EFS) and percentage of human CD45+ leukocytes in peripheral blood in acute lymphoblastic leukemia (ALL) orthotopic models.


EFS and tumor volume in non - CNS subcutaneous PDX models which demonstrated objective response in 1 or more treatments .

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

EFS and tumor volume in non-CNS subcutaneous PDX models which demonstrated objective response in 1 or more treatments.


L EFS in a CNS orthotopic model in which drug sensitivity was observed . EFS is time of inoculation of tumor cells to event ( defined by neurologic symptoms or weight loss ) .

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

L EFS in a CNS orthotopic model in which drug sensitivity was observed. EFS is time of inoculation of tumor cells to event (defined by neurologic symptoms or weight loss).


D Treatment response by therapy tier . 14 of 29 patients with molecular - based therapeutic options received the treatment .

D Treatment response by therapy tier. 14 of 29 patients with molecular-based therapeutic options received the treatment.


( a ) Immunoblot analysis of caspase - 1 and IL - 1β in lysates ( Lys ) and supernatants ( Sup ) of peritoneal macrophages obtained from Map1lc3b - / - mice and Becn1 + / - mice and the corresponding wild - type littermate mice , then left unstimulated ( - ) or stimulated with LPS alone ( LPS ) or LPS followed by 30 min of ATP treatment ( L + A ) . p10 , mature form of caspase - 1 . Right margin , molecular size in kilodaltons ( kDa ) .

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

(a) Immunoblot analysis of caspase-1 and IL-1β in lysates (Lys) and supernatants (Sup) of peritoneal macrophages obtained from Map1lc3b-/- mice and Becn1+/- mice and the corresponding wild-type littermate mice, then left unstimulated (-) or stimulated with LPS alone (LPS) or LPS followed by 30 min of ATP treatment (L + A). p10, mature form of caspase-1. Right margin, molecular size in kilodaltons (kDa).


( b ) Enzyme - linked immunosorbent assay ( ELISA ) of cytokine secretion by peritoneal macrophages stimulated with LPS , followed by treatment for 1 h with ATP .

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

(b) Enzyme-linked immunosorbent assay (ELISA) of cytokine secretion by peritoneal macrophages stimulated with LPS, followed by treatment for 1 h with ATP.


( c ) Immunoblot analysis of caspase - 1 and IL - 1β in lysates ( left ) and ELISA of cytokines in supernatants ( right ) of Map1lc3b + / + and Map1lc3b - / - BMDMs incubated with LPS , followed by 30 min ( immunoblot ) or 1 h ( ELISA ) of ATP treatment .

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

(c) Immunoblot analysis of caspase-1 and IL-1β in lysates (left) and ELISA of cytokines in supernatants (right) of Map1lc3b+/+ and Map1lc3b-/- BMDMs incubated with LPS, followed by 30 min (immunoblot) or 1 h (ELISA) of ATP treatment.