( G ) DFCP1 - GFP cells were transfected with control siRNA or Bim siRNA . The numbers of GFP puncta were assessed . Data are shown as mean ± SD . ∗ p < 0 . 05 . Images represent DFCP1 - GFP vesicles in cells transfected with control siRNA and Bim siRNA . Arrows mark DFCP1 - GFP puncta .

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

(G) DFCP1-GFP cells were transfected with control siRNA or Bim siRNA. The numbers of GFP puncta were assessed. Data are shown as mean ± SD.∗p < 0.05.Images represent DFCP1-GFP vesicles in cells transfected with control siRNA and Bim siRNA. Arrows mark DFCP1-GFP puncta.


( H ) HeLa cells were treated with control siRNA or Bim siRNA . Blots were probed with the indicated antibodies . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 .

O O O O O O O O O O O O O O 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) HeLa cells were treated with control siRNA or Bim siRNA. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01.


( I ) Bim ( EL ) / control siRNA , Bim ( EL ) / Bim siRNA , Bim ( EL ) EE / control siRNA , Bim ( EL ) EE / Bim siRNA , vector / control siRNA , and vector / Bim siRNA were transfected into HeLa cells . Blots were probed with the indicated antibodies . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bim(EL)/control siRNA, Bim(EL)/Bim siRNA, Bim(EL)EE/control siRNA, Bim(EL)EE/Bim siRNA, vector/control siRNA, and vector/Bim siRNA were transfected into HeLa cells. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01.


( J ) HeLa cells were transfected with control plasmid or Bim ( EL ) EE . Blots were probed with the indicated antibodies . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 .

O O O O O O O O O O O O O O O O O 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) HeLa cells were transfected with control plasmid or Bim(EL)EE. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01.


( K ) Bax / Bak DKO MEFs were transfected with control plasmid / GFP - LC3 , wild - type Bim ( EL ) / GFP - LC3 ( 3 : 1 ) , or Bim ( EL ) EE / GFP - LC3 . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 .

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

(K) Bax/Bak DKO MEFs were transfected with control plasmid /GFP-LC3, wild-type Bim(EL)/GFP-LC3 (3:1), or Bim (EL) EE/GFP-LC3. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01.


( L ) Two independent control vector ( pcDNA3 ) or Bim ( EL ) EE stably expressing HeLa cell clones were transfected with GFP - LC3 respectively . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ ∗ 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

(L) Two independent control vector (pcDNA3) or Bim(EL)EE stably expressing HeLa cell clones were transfected with GFP-LC3 respectively. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001.


( M ) HeLa cells were transfected with control plasmid / GFP - PX or Bim ( EL ) EE / GFP - PX ( 3 : 1 ) . The percentages of cells with GFP - vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . Fluorescent microscope images show GFP - PX vesicles .

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

(M) HeLa cells were transfected with control plasmid/GFP-PX or Bim(EL)EE/GFP-PX (3:1). The percentages of cells with GFP-vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01. Fluorescent microscope images show GFP-PX vesicles.


( A ) Lysates from spleen - derived T cells of wild - type ( Bim + / + ) and Bim - knockout ( Bim − / − ) mice were immunoblotted and probed with indicated antibodies . Statistics were from three knockout and control mice . Data are shown as mean ± SD . ∗ ∗ ∗ 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

(A) Lysates from spleen-derived T cells of wild-type (Bim+/+) and Bim-knockout (Bim−/−) mice were immunoblotted and probed with indicated antibodies. Statistics were from three knockout and control mice. Data are shown as mean ± SD. ∗∗∗p < 0.001.


( B ) T cells from Bim + / + and Bim − / − mice were cultured with / without Baf for 4 hr preceding harvest , immunoblotted , and probed with the indicated antibodies .

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

(B) T cells from Bim+/+ and Bim−/− mice were cultured with/without Baf for 4 hr preceding harvest, immunoblotted, and probed with the indicated antibodies.


( C ) Spleen - derived T cells from Bim + / + or Bim − / − mice were stained with rabbit anti - LC3 antibody . Representative confocal images and quantification from triplicates ( n = 100 - 110 ) are shown . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 0001 .

(C) Spleen-derived T cells from Bim+/+ or Bim−/− mice were stained with rabbit anti-LC3 antibody. Representative confocal images and quantification from triplicates (n = 100-110) are shown. Data are shown as mean ± SD. ∗∗∗p < 0.0001.


( D ) Bim + / + and Bim − / − mouse liver lysates were immunoblotted and probed with the indicated antibodies ( n = 3 ) . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O 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) Bim+/+ and Bim−/− mouse liver lysates were immunoblotted and probed with the indicated antibodies (n = 3). Data are shown as mean ± SD. ∗∗p < 0.01.


( E ) Bim + / + and Bim − / − mouse liver lysates were immunoblotted and probed with the indicated antibodies ( n = 3 ) . Data are shown as mean ± SD . ∗ ∗ ∗ 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

(E) Bim+/+ and Bim−/− mouse liver lysates were immunoblotted and probed with the indicated antibodies (n = 3). Data are shown as mean ± SD. ∗∗∗p < 0.001.


( F ) RNA from Bim wild - type or Bim - knockout mouse livers was analyzed by qRT - PCR for p62 / beta - actin mRNA . The mean ± SD from three mice per group is shown . NS , not significant .

(F) RNA from Bim wild-type or Bim-knockout mouse livers was analyzed by qRT-PCR for p62/beta-actin mRNA. The mean ± SD from three mice per group is shown. NS, not significant.


( A ) HeLa cells were treated with control siRNA , Bim siRNA + control siRNA , control siRNA + Beclin 1 siRNA , or Bim siRNA + Beclin 1 siRNA . Blots were probed as indicated . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA. Blots were probed as indicated. Data are shown as mean ± SD. ∗∗p < 0.01.


( B ) HeLa cells were treated with control siRNA , Bim siRNA + control siRNA , control siRNA + Beclin 1 siRNA , or Bim siRNA + Beclin 1 siRNA . After 24 hr , cells were transfected with GFP - PX for 24 hr . GFP - PX vesicles were assessed with a confocal microscope . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . Arrows label cells with increased number / size of GFP - PX vesicles .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA. After 24 hr, cells were transfected with GFP-PX for 24 hr. GFP-PX vesicles were assessed with a confocal microscope. Data are shown as mean ± SD. ∗∗p < 0.01. Arrows label cells with increased number/size of GFP-PX vesicles.


( C ) GFP - mRFP - LC3 HeLa cells were treated with control , Bim siRNA , or Bcl - 2 siRNA and then analyzed with Cellomics microscope . Data are shown as mean ± SD . ∗ ∗ ∗ 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

(C) GFP-mRFP-LC3 HeLa cells were treated with control, Bim siRNA, or Bcl-2 siRNA and then analyzed with Cellomics microscope. Data are shown as mean ± SD. ∗∗∗p < 0.001.


( D ) GFP - mRFP - LC3 stable HeLa cells were treated with control siRNA , Bim siRNA + control siRNA , control siRNA + Beclin 1 siRNA , or Bim siRNA + Beclin 1 siRNA and then analyzed with Cellomics microscope and matching confocal images are shown . Data are shown as mean ± SD . ∗ ∗ ∗ 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

(D) GFP-mRFP-LC3 stable HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA and then analyzed with Cellomics microscope and matching confocal images are shown. Data are shown as mean ± SD. ∗∗∗p < 0.001.


( A ) Bim ( EL ) EE / empty vector ( IP negative control ) or Bim ( EL ) EE / Beclin 1 - Flag ( in duplicate ) were transfected into HeLa cells . After 20 hr , one set of cells with Bim ( EL ) EE / Beclin 1 - Flag were starved in HBSS for 2 hr . Anti - Flag antibody ( M2 ) was used for immunoprecipitation , and blots were probed as indicated . Data are shown as mean ± SD . ∗ ∗ ∗ 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

(A) Bim(EL)EE/empty vector (IP negative control) or Bim(EL)EE/Beclin 1-Flag (in duplicate) were transfected into HeLa cells. After 20 hr, one set of cells with Bim(EL)EE/Beclin 1-Flag were starved in HBSS for 2 hr. Anti-Flag antibody (M2) was used for immunoprecipitation, and blots were probed as indicated. Data are shown as mean ± SD. ∗∗∗p < 0.001.


( B ) Panel i : HeLa cells were treated with control siRNA or Bim siRNA . After 48 hr , one set of transfections were starved in HBSS for 2 hr . Blots were probed as indicated . LC3 - II / tubulin ratio of siRNA - transfected cells is set as 1 ( n = 3 ) . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 ; NS , not significant . Panel ii : HeLa cells were treated with control siRNA or Bim siRNA . After 48 hr , one set of transfections were treated with Baf for 2 hr ; one set of transfections were starved and treated with Baf for 2 hr . Blots were probed as indicated and analyzed as in ( Bi ) . ∗ ∗ p < 0 . 01 ; NS , not significant .

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

(B) Panel i: HeLa cells were treated with control siRNA or Bim siRNA. After 48 hr, one set of transfections were starved in HBSS for 2 hr. Blots were probed as indicated. LC3-II/tubulin ratio of siRNA-transfected cells is set as 1 (n = 3). Data are shown as mean ± SD. ∗∗∗p < 0.001; NS, not significant. Panel ii: HeLa cells were treated with control siRNA or Bim siRNA. After 48 hr, one set of transfections were treated with Baf for 2 hr; one set of transfections were starved and treated with Baf for 2 hr. Blots were probed as indicated and analyzed as in (Bi). ∗∗p < 0.01; NS, not significant.


( C ) HeLa cells were transfected with control plasmid / GFP - LC3 ( 3 : 1 ) , or Bim ( EL ) EE / GFP - LC3 ( 3 : 1 ) . After 20 hr , one set of transfected cells were starved in HBSS for 2 hr and then fixed . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . NS , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were transfected with control plasmid /GFP-LC3 (3:1), or Bim(EL)EE/GFP-LC3 (3:1). After 20 hr, one set of transfected cells were starved in HBSS for 2 hr and then fixed. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01. NS, not significant.


( D ) BimS - Beclin 1 binding is not sensitive to HBSS starvation . BimEL - EE ( EL ) / vector ( IP negative control ) , BimEL - EE ( EL ) / Beclin 1 - Flag ( two replicates ) , BimL - EE ( L ) / Beclin 1 - Flag ( two replicates ) or BimS - EE ( S ) / Beclin 1 - Flag ( two replicates ) were transfected into HeLa cells . After 20 hr , one of EL - Beclin 1 - Flag transfections , one of L - Beclin - Flag transfections and one of S - Beclin 1 - Flag transfections were subjected to 2 hr HBSS starvation . Anti - Flag ( M2 ) was used for immunoprecipitation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BimS-Beclin 1 binding is not sensitive to HBSS starvation. BimEL-EE (EL)/vector (IP negative control), BimEL-EE (EL)/Beclin 1-Flag (two replicates), BimL-EE (L)/Beclin 1-Flag (two replicates) or BimS-EE (S)/Beclin 1-Flag (two replicates) were transfected into HeLa cells. After 20 hr, one of EL-Beclin 1-Flag transfections, one of L-Beclin-Flag transfections and one of S-Beclin 1-Flag transfections were subjected to 2 hr HBSS starvation. Anti-Flag (M2) was used for immunoprecipitation.


( E ) HBSS starvation increases Bim T116 phosphorylation . Bim ( EL ) EE or BimEE ( EL ) - T116A were transfected into HeLa cells . After 20 hr , one set of Bim ( EL ) EE transfections and one of Bim ( EL ) EE - T116A were starved for 2 hr in HBSS . Blots were probed with the indicated antibodies .

(E) HBSS starvation increases Bim T116 phosphorylation. Bim(EL)EE or BimEE(EL)-T116A were transfected into HeLa cells. After 20 hr, one set of Bim(EL)EE transfections and one of Bim(EL)EE-T116A were starved for 2 hr in HBSS. Blots were probed with the indicated antibodies.


( F ) Bim T116 phosphorylation disables Bim - Beclin 1 interaction . Bim ( EL ) EE / vector ( IP negative control ) , Bim ( EL ) EE / Beclin 1 - Flag ( in duplicate ) , or Bim ( EL ) EE - T116E ( phospho - mimic , designated as T116E here ) / Beclin - Flag ( in duplicate ) were transfected into HeLa cells . After 20 hr , one set of Bim ( EL ) EE / Beclin 1 - Flag transfections and one of Bim ( EL ) EE - T116E / Beclin 1 - Flag transfections were starved for 2 hr in HBSS ( Starv ) . Anti - Flag ( M2 ) was used for immunoprecipitation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bim T116 phosphorylation disables Bim-Beclin 1 interaction. Bim(EL)EE /vector (IP negative control), Bim(EL)EE/Beclin 1-Flag (in duplicate), or Bim(EL)EE-T116E (phospho-mimic, designated as T116E here)/Beclin-Flag (in duplicate) were transfected into HeLa cells. After 20 hr, one set of Bim(EL)EE/Beclin 1-Flag transfections and one of Bim(EL)EE-T116E/Beclin 1-Flag transfections were starved for 2 hr in HBSS (Starv). Anti-Flag (M2) was used for immunoprecipitation.


( G ) HeLa cells were transfected with control plasmid / GFP - LC3 , Bim ( EL ) - EE / GFP - LC3 ( 3 : 1 ) , or Bim ( EL ) - EE T116E / GFP - LC3 ( 3 : 1 ) . After 20 hr , one set of transfected cells were starved in HBSS for 2 hr and the cells were then fixed . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 ; NS , not significant .

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

(G) HeLa cells were transfected with control plasmid /GFP-LC3, Bim(EL)-EE/GFP-LC3 (3:1), or Bim(EL)-EE T116E/GFP-LC3 (3:1). After 20 hr, one set of transfected cells were starved in HBSS for 2 hr and the cells were then fixed. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01; NS, not significant.


( A ) Bim - 147 does not bind to Beclin 1 . Beclin 1 - Flag / vector ( IP negative control ) , Beclin 1 - Flag / Myc - BimEL - 1 - 147aa ( Myc - 147 ) , or Beclin 1 - Flag / Myc - BimEL - ΔBH3 ( Myc - ΔBH3 ) were transfected into HeLa cells . Anti - Myc ( Rabbit ) was used for immunoprecipitation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bim-147 does not bind to Beclin 1. Beclin 1-Flag/vector (IP negative control), Beclin 1-Flag/Myc-BimEL-1-147aa (Myc-147), or Beclin 1-Flag/Myc-BimEL-ΔBH3 (Myc-ΔBH3) were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation.


( B ) Bim - 147 is competent for LC8 binding . LC8 / vector ( IP negative control ) , LC8 / Myc - BimEL - 1 - 147aa ( Myc - 147 ) , or LC8 / Myc - BimEL - ΔBH3 ( Myc - ΔBH3 ) were transfected into HeLa cells . Anti - Myc ( Rabbit ) was used for immunoprecipitation .

(B) Bim-147 is competent for LC8 binding. LC8/vector (IP negative control), LC8/Myc-BimEL-1-147aa (Myc-147), or LC8/Myc-BimEL-ΔBH3 (Myc-ΔBH3) were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation.


( C ) Mutating LC8 binding consensus sites within Bim disrupts the Bim - LC8 interaction . Bim ( EL ) EE / vector ( IP negative control ) , Bim ( EL ) EE / Myc - LC8 , Bim ( EL ) EE - S109A , S113A , T114A ( AAA ) / Myc - LC8 , or Bim ( EL ) EE - T116E / Myc - LC8 were transfected into HeLa cells . Anti - Myc ( Rabbit ) was used for immunoprecipitation . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

(C) Mutating LC8 binding consensus sites within Bim disrupts the Bim-LC8 interaction. Bim(EL)EE/vector (IP negative control), Bim(EL)EE/Myc-LC8, Bim(EL)EE-S109A, S113A, T114A (AAA)/ Myc-LC8, or Bim(EL)EE-T116E/ Myc-LC8 were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation. Data are shown as mean ± SD. ∗∗∗p < 0.001.


( D ) Mutating LC8 binding consensus sites within Bim disrupts the Bim - Beclin 1 interaction . Bim ( EL ) EE / vector ( IP negative control ) , Bim ( EL ) EE / Beclin 1 - Flag , Bim ( EL ) EE - S109A , S113A , T114A ( AAA ) / Beclin 1 - Flag , or Bim ( EL ) EE - T116E / Beclin 1 - Flag were transfected into HeLa cells . Anti - Flag ( M2 ) was used for immunoprecipitation . Data are shown as mean ± SD . ∗ ∗ ∗ 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

(D) Mutating LC8 binding consensus sites within Bim disrupts the Bim-Beclin 1 interaction. Bim(EL)EE/vector (IP negative control), Bim(EL)EE/Beclin 1-Flag, Bim(EL)EE-S109A, S113A, T114A (AAA)/Beclin 1-Flag, or Bim(EL)EE-T116E/Beclin 1-Flag were transfected into HeLa cells. Anti-Flag (M2) was used for immunoprecipitation. Data are shown as mean ± SD. ∗∗∗p < 0.001.


( E ) Bim - S does not bind to LC8 . BimEL - EE ( EL ) / vector ( IP negative control ) , BimEL - EE ( EL ) / Myc - LC8 , BimL - EE ( L ) / Myc - LC8 , or BimS - EE ( S ) / Myc - LC8 were transfected into HeLa cells . Anti - Myc ( Rabbit ) was used for immunoprecipitation .

(E) Bim-S does not bind to LC8. BimEL-EE (EL)/vector (IP negative control), BimEL-EE (EL)/Myc-LC8, BimL-EE (L)/Myc-LC8, or BimS-EE (S)/Myc-LC8 were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation.


( G ) LC8 enhances Bim - Beclin 1 interaction in vitro . His - Bim , His - LC8 and GST - Beclin 1 were expressed in BL21 ( DE3 ) E . coli and purified . Two micrograms of His - Bim was combined with 2 μg GST without or with 1 μg His - LC8 ( as controls ) ; 2 μg His - Bim was combined with 2 μg GST - Beclin 1 without or with 1 μg His - LC8 . The mixtures were incubated in buffer A for 3 hr . Glutathione beads were then used to pull down GST or GST - Beclin 1 . The pulldown products were detected with anti - Bim and anti - Beclin 1 .

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

(G) LC8 enhances Bim-Beclin 1 interaction in vitro. His-Bim, His-LC8 and GST-Beclin 1 were expressed in BL21(DE3) E. coli and purified. Two micrograms of His-Bim was combined with 2 μg GST without or with 1 μg His-LC8 (as controls); 2 μg His-Bim was combined with 2 μg GST-Beclin 1 without or with 1 μg His-LC8. The mixtures were incubated in buffer A for 3 hr. Glutathione beads were then used to pull down GST or GST-Beclin 1. The pulldown products were detected with anti-Bim and anti-Beclin 1.


( H ) LC8 siRNA knockdown reduces the Bim - Beclin 1 interaction . Control siRNA or LC8 siRNA were transfected into HeLa cells . After 48 hr , HA - Bim ( EL ) EE / vector ( IP negative control ) or HA - Bim ( EL ) EE / Beclin 1 - Flag was transfected into control siRNA transfected HeLa cells ; HA - Bim ( EL ) EE / Beclin 1 - Flag was transfected into LC8 siRNA - transfected HeLa cells . Anti - Flag ( M2 ) was used for immunoprecipitation . Note that in the presence of BimEE , endogenous LC8 was pulled down by Beclin 1 when cells were treated with control siRNA . Data are shown as mean ± SD . ∗ ∗ ∗ 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

(H) LC8 siRNA knockdown reduces the Bim-Beclin 1 interaction. Control siRNA or LC8 siRNA were transfected into HeLa cells. After 48 hr, HA-Bim(EL)EE/vector (IP negative control) or HA-Bim(EL)EE/Beclin 1-Flag was transfected into control siRNA transfected HeLa cells; HA-Bim(EL)EE/Beclin 1-Flag was transfected into LC8 siRNA-transfected HeLa cells. Anti-Flag (M2) was used for immunoprecipitation. Note that in the presence of BimEE, endogenous LC8 was pulled down by Beclin 1 when cells were treated with control siRNA. Data are shown as mean ± SD. ∗∗∗p < 0.001.


( A ) HeLa cells were transfected with vector ( vec ) / GFP - LC3 , BimEL - EE ( EL ) / GFP - LC3 , BimL - EE ( L ) / GFP - LC3 , BimS - EE ( S ) / GFP - LC3 , or BimEL - EE - AAA ( AAA ) / GFP - LC3 ( 3 : 1 ) . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ p < 0 . 01 ; NS , not significant . Note that BimEL - EE - S109A , S113A , T114A is designated as BimEL - EE - AAA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were transfected with vector (vec) /GFP-LC3, BimEL-EE (EL)/GFP-LC3, BimL-EE (L)/GFP-LC3, BimS-EE (S)/GFP-LC3, or BimEL-EE-AAA (AAA)/GFP-LC3 (3:1). The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001; ∗∗p < 0.01; NS, not significant. Note that BimEL-EE-S109A, S113A, T114A is designated as BimEL-EE-AAA.


( B ) Bim bridges the Beclin 1 - LC8 interaction . Dynein light chain1 ( Myc - LC8 ) / empty vectors ( IP negative control ) , Myc - LC8 / Beclin 1 - Flag / empty vector , Myc - LC8 / Beclin 1 - Flag / BimEL - EE ( EL ) , Myc - LC8 / Beclin 1 - Flag / BimL - EE ( L ) , Myc - LC8 / Beclin 1 - Flag / BimS - EE ( S ) , and Myc - LC8 / Beclin 1 - Flag / BimEL - EE - S109A , S113A , T114A ( AAA ) were transfected into HeLa cells . Anti - Flag ( M2 ) was used for immunoprecipitation .

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(B) Bim bridges the Beclin 1-LC8 interaction. Dynein light chain1 (Myc-LC8)/empty vectors (IP negative control), Myc-LC8/Beclin 1-Flag/empty vector, Myc-LC8/Beclin 1-Flag/BimEL-EE (EL), Myc-LC8/Beclin 1-Flag/BimL-EE (L), Myc-LC8/Beclin 1-Flag/BimS-EE (S), and Myc-LC8/Beclin 1-Flag/BimEL-EE-S109A, S113A, T114A (AAA) were transfected into HeLa cells. Anti-Flag (M2) was used for immunoprecipitation.


( C ) Starvation reduces the ability of Bim to bridge the Beclin 1 - LC8 interaction . Myc - LC8 / empty vectors ( IP negative control ) , Myc - LC8 / Beclin 1 - Myc / empty vector , or Myc - LC8 / Beclin 1 - Flag / HA - BimEL - EE ( HA - Bim ) ( two replicates ) were transfected into HeLa cells . After 20 hr , one set of cells with Myc - LC8 / Beclin 1 - Flag / HA - HA - Bim was starved in HBSS for 2 hr . Anti - Flag ( M2 ) antibody was used for immunoprecipitation .

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(C) Starvation reduces the ability of Bim to bridge the Beclin 1-LC8 interaction. Myc-LC8/empty vectors (IP negative control), Myc-LC8/Beclin 1-Myc/empty vector, or Myc-LC8/Beclin 1-Flag/HA-BimEL-EE (HA-Bim) (two replicates) were transfected into HeLa cells. After 20 hr, one set of cells with Myc-LC8/Beclin 1-Flag/HA-HA-Bim was starved in HBSS for 2 hr. Anti-Flag (M2) antibody was used for immunoprecipitation.


( D ) HeLa cells were cultured with DMEM with 10 % serum ( Control ) or starved ( Starve ) in HBSS for 2 hr . The fixed cells were stained with Beclin 1 and tubulin antibodies and analyzed by confocal microscopy . White boxes show the areas where Beclin 1 is enriched . Yellow boxes show enlarged areas .

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(D) HeLa cells were cultured with DMEM with 10% serum (Control) or starved (Starve) in HBSS for 2 hr. The fixed cells were stained with Beclin 1 and tubulin antibodies and analyzed by confocal microscopy. White boxes show the areas where Beclin 1 is enriched. Yellow boxes show enlarged areas.


( E ) HeLa cells were treated with control siRNA or LC8 siRNA . After 24 hr , cells were split . Vector / GFP - LC3 or Bim ( EL ) EE / GFP - LC3 ( 3 : 1 ) were transfected into the control siRNA - transfected or LC8 siRNA - transfected cells . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 ; NS , not significant .

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(E) HeLa cells were treated with control siRNA or LC8 siRNA. After 24 hr, cells were split. Vector/GFP-LC3 or Bim(EL)EE/GFP-LC3 (3:1) were transfected into the control siRNA-transfected or LC8 siRNA-transfected cells. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01; NS, not significant.


( F ) HeLa cells were treated with control siRNA , Bim siRNA , or LC8 siRNA for 48 hr . Cells were then fixed in 37 ° C , 4 % PFA for 10 min . Cells were stained with Beclin 1 and tubulin antibodies and analyzed by confocal microscopy . White boxes / triangle show areas where Beclin 1 is enriched . Yellow boxes show enlarged areas . Colocalizations were quantified from images in 12 - 15 cells with Volocity program ( Colocalization coefficient Mx ) . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 .

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(F) HeLa cells were treated with control siRNA, Bim siRNA, or LC8 siRNA for 48 hr. Cells were then fixed in 37°C, 4% PFA for 10 min. Cells were stained with Beclin 1 and tubulin antibodies and analyzed by confocal microscopy. White boxes/triangle show areas where Beclin 1 is enriched. Yellow boxes show enlarged areas. Colocalizations were quantified from images in 12-15 cells with Volocity program (Colocalization coefficient Mx). Data are shown as mean ± SD. ∗∗∗p < 0.001.


( B ) Immunoblot of WT , cGAS KO , and MAVS KO THP1 cells Data information : Panel ( B ) is representative of two independent biological experiments .

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(B) Immunoblot of WT, cGAS KO, and MAVS KO THP1 cells Data information: Panel (B) is representative of two independent biological experiments.


( C ) THP1 cells of the indicated genotypes were mock infected or infected with VZV for 24 and 48 hours using the transwell assay described in ( A ) . Infected cells were quantified by surface staining for VZV - gE / gI and flow cytometry analysis . Data information : Panel ( C ) , show pooled data from four repeats , where each data point represents an independent biological experiment ( n = 4 ± SEM ) . Statistical analysis in panels ( C ) , was paired t - tests WT : wild type , KO : knockout .

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(C) THP1 cells of the indicated genotypes were mock infected or infected with VZV for 24 and 48 hours using the transwell assay described in (A). Infected cells were quantified by surface staining for VZV-gE/gI and flow cytometry analysis. Data information: Panel (C), show pooled data from four repeats, where each data point represents an independent biological experiment (n=4 ± SEM). Statistical analysis in panels (C), was paired t-tests WT: wild type, KO: knockout.


( D ) RT - qPCR analysis of VZV ORF63 ( immediate early ( IE ) gene ) , ORF54 ( early ( E ) gene ) , and ORF40 ( late ( L ) gene ) transcripts in cells infected as in ( C ) . Graphs show expression relative to GAPDH . Data information : Panel ( D ) show pooled data from three repeats , where each data point represents an independent biological experiment ( n = 3 ± SEM ) . Statistical analysis in panels ( D ) , was paired t - tests hpi : hours post

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(D) RT-qPCR analysis of VZV ORF63 (immediate early (IE) gene), ORF54 (early (E) gene), and ORF40 (late (L) gene) transcripts in cells infected as in (C). Graphs show expression relative to GAPDH. Data information: Panel (D) show pooled data from three repeats, where each data point represents an independent biological experiment (n=3 ± SEM). Statistical analysis in panels (D), was paired t-tests hpi: hours post infection


( E ) RT - qPCR analysis of IFNB1 , IFI44 , and IFIT1 expression in cells infected as in ( C ) . Graphs show expression relative to GAPDH . Data information : Panel ( E ) show pooled data from three repeats , where each data point represents an independent biological experiment ( n = 3 ± SEM ) . Statistical analysis in panel ( E ) paired ratio t - tests . hpi : hours post

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(E) RT-qPCR analysis of IFNB1, IFI44, and IFIT1 expression in cells infected as in (C). Graphs show expression relative to GAPDH. Data information: Panel (E) show pooled data from three repeats, where each data point represents an independent biological experiment (n=3 ± SEM). Statistical analysis in panel (E) paired ratio t-tests. hpi: hours post infection


( F ) Activity of Lucia luciferase ( secreted under an IRF3 - dependent promotor ) was determined in supernatants of cells infected as in ( C ) by QUANTI - Luc assay . Data information : Panel , ( F ) , show pooled data from four repeats , where each data point represents an independent biological experiment ( n = 4 ± SEM ) . Statistical analysis in panels ( F ) , was paired t - tests hpi : hours post infection , WT : wild type , KO : knockout .

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(F) Activity of Lucia luciferase (secreted under an IRF3-dependent promotor) was determined in supernatants of cells infected as in (C) by QUANTI-Luc assay. Data information: Panel , (F), show pooled data from four repeats, where each data point represents an independent biological experiment (n=4 ± SEM). Statistical analysis in panels (F), was paired t-tests hpi: hours post infection, WT: wild type, KO: knockout.


( G ) Concentrations of CXCL10 in supernatants of cells infected as in ( C ) were determined by ELISA . Data information : Panel ( G ) show pooled data from four repeats , where each data point represents an independent biological experiment ( n = 4 ± SEM ) . Statistical analysis in panels ( G ) was paired t - tests hpi : hours post

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(G) Concentrations of CXCL10 in supernatants of cells infected as in (C) were determined by ELISA. Data information: Panel (G) show pooled data from four repeats, where each data point represents an independent biological experiment (n=4 ± SEM). Statistical analysis in panels (G) was paired t-tests hpi: hours post infection


( A ) HEK293T cells were transfected with pRL - TK , p125 - F - Luc , expression plasmids for human cGAS and human STING , as well as expression plasmids for individual ORFs . The next day luciferase activity was determined . For each ORF a z - value was calculated and ORFs sorted by descending values . ( B ) The experiment shown in panel ( A ) was repeated three times and for each ORF in each experiment a rank was determined according to z - value ( highest z - value = rank 1 ) . Ranks are displayed as a heatmap . The first row shows results displayed in panel ( A ) . Data information : Panel ( A ) is representative of three independent biological experiments , where each data point represents a technical replicate ( mean ± SD ) . The three independent experiments are summarised in panel ( B ) , where the data from ( A ) forms the first row .

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(A) HEK293T cells were transfected with pRL-TK, p125-F-Luc, expression plasmids for human cGAS and human STING, as well as expression plasmids for individual ORFs. The next day luciferase activity was determined. For each ORF a z-value was calculated and ORFs sorted by descending values. (B) The experiment shown in panel (A) was repeated three times and for each ORF in each experiment a rank was determined according to z-value (highest z-value = rank 1). Ranks are displayed as a heatmap. The first row shows results displayed in panel (A). Data information: Panel (A) is representative of three independent biological experiments, where each data point represents a technical replicate (mean ± SD). The three independent experiments are summarised in panel (B), where the data from (A) forms the first row.


( C ) HEK293T cells were transfected as in ( A , B ) with expression constructs for VZV ORF9 or empty vector . In parallel , reporter expression was stimulated by co - transfection of the RIG - I - CARD plasmid instead of hcGAS and hSTING . Data information Panel ( C ) is representing two independent biological experiments , where each data point represents a technical replicate ( mean ± SD ) .

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(C) HEK293T cells were transfected as in (A,B) with expression constructs for VZV ORF9 or empty vector. In parallel, reporter expression was stimulated by co-transfection of the RIG-I-CARD plasmid instead of hcGAS and hSTING. Data information Panel (C) is representing two independent biological experiments, where each data point represents a technical replicate (mean ± SD).


( D ) Western blot analysis of PMA - differentiated THP1 monocytes stably transduced with either V5 - tagged VZV ORF9 or GFP using the indicated antibodies . Data information : Panels ( D ) are representing two independent biological experiments . In ( E ) each data point represents a technical replicate .

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(D) Western blot analysis of PMA-differentiated THP1 monocytes stably transduced with either V5-tagged VZV ORF9 or GFP using the indicated antibodies. Data information: Panels (D) are representing two independent biological experiments. In (E) each data point represents a technical replicate.


( E ) THP1 monocytes stably transduced with either VZV ORF9 or GFP were PMA - differentiated and transfected with indicated doses of dsDNA . Expression of IFNB1 and IFNL1 was assessed by RT - qPCR . Graphs show expression relative to GAPDH . Data information : Panels ( E ) are representing two independent biological experiments . In ( E ) each data point represents a technical replicate .

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(E) THP1 monocytes stably transduced with either VZV ORF9 or GFP were PMA-differentiated and transfected with indicated doses of dsDNA. Expression of IFNB1 and IFNL1 was assessed by RT-qPCR. Graphs show expression relative to GAPDH. Data information: Panels (E) are representing two independent biological experiments. In (E) each data point represents a technical replicate.


( F ) THP1 monocytes stably transduced with either VZV ORF9 or GFP were treated with the STING agonist diABZi ( 0 . 005 μM ) . Expression of IFNB1 , IFIT1 and IFI44 was assessed by RT - qPCR . Graphs show expression relative to GAPDH . Data information : Panel ( F ) shows pooled data from three and two repeats , respectively , where each data point represents an independent biological experiment ( horizontal bars show means ) . Statistical analysis in ( F ) were paired t - tests .

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

(F) THP1 monocytes stably transduced with either VZV ORF9 or GFP were treated with the STING agonist diABZi (0.005 μM). Expression of IFNB1, IFIT1 and IFI44 was assessed by RT-qPCR. Graphs show expression relative to GAPDH. Data information: Panel (F) shows pooled data from three and two repeats, respectively, where each data point represents an independent biological experiment (horizontal bars show means). Statistical analysis in (F) were paired t-tests.


( I ) PMA - differentiated THP1 cells were co - cultured with VZV - ORF9ecDHFR - infected MeWo cells for 1h in the presence of TMP , washed , and incubated for another 24h in the presence of TMP . Cells were then washed extensively and fresh medium was added that was supplemented or not with TMP . Expression levels of IFNB1 , IFIT1 , and IFI44 were measured by RT - qPCR after a further 24h incubation . Graphs show expression relative to GAPDH . Mean fold changes are indicated . Data information : Panel ( I ) shows pooled data from three repeats , where each data point represents an independent biological experiment . Statistical analysis in ( I ) were paired t - tests .

O O 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 B-CONTROLLED_VAR 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

(I) PMA-differentiated THP1 cells were co-cultured with VZV-ORF9ecDHFR-infected MeWo cells for 1h in the presence of TMP, washed, and incubated for another 24h in the presence of TMP. Cells were then washed extensively and fresh medium was added that was supplemented or not with TMP. Expression levels of IFNB1, IFIT1, and IFI44 were measured by RT-qPCR after a further 24h incubation. Graphs show expression relative to GAPDH. Mean fold changes are indicated. Data information: Panel (I) shows pooled data from three repeats, where each data point represents an independent biological experiment. Statistical analysis in (I) were paired t-tests.


( J ) Cells infected and treated as in ( I ) with the addition of uninfected cells were analysed by western blotting using the indicated antibodies . Data information : Panel ( J ) is representative of three independent experiments .

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(J) Cells infected and treated as in (I) with the addition of uninfected cells were analysed by western blotting using the indicated antibodies. Data information: Panel (J) is representative of three independent experiments.


( A ) HEK293T cells were transfected with expression plasmids for ORF9 - V5 , human cGAS - FLAG , and human STING - HA . The next day , cells were lysed , and overexpressed proteins were immunoprecipitated with α - V5 , α - FLAG , α - HA , or IgG isotype control antibody . Input , unbound and IP fractions were subjected to immunoblotting using the indicated antibodies . Data information : Panels ( A ) are representative of two independent experiments .

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(A) HEK293T cells were transfected with expression plasmids for ORF9-V5, human cGAS-FLAG, and human STING-HA. The next day, cells were lysed, and overexpressed proteins were immunoprecipitated with α-V5, α-FLAG, α-HA, or IgG isotype control antibody. Input, unbound and IP fractions were subjected to immunoblotting using the indicated antibodies. Data information: Panels (A) are representative of two independent experiments.


( B ) THP1 monocytes stably transduced with either VZV FLAG - ORF9 or FLAG - GFP were PMA - differentiated overnight . The next day , cells were lysed and ectopically expressed proteins were immunoprecipitated using α - FLAG antibody . Input , unbound and IP fractions were subjected to immunoblotting using the indicated antibodies . Data information : Panels ( B ) , are representative of three independent experiments .

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(B) THP1 monocytes stably transduced with either VZV FLAG-ORF9 or FLAG-GFP were PMA-differentiated overnight. The next day, cells were lysed and ectopically expressed proteins were immunoprecipitated using α-FLAG antibody. Input, unbound and IP fractions were subjected to immunoblotting using the indicated antibodies. Data information: Panels (B), are representative of three independent experiments.


( C ) HaCaT cells and PMA - differentiated THP1 cells were infected with WT VZV or VZVORF9 - V5 through co - culture with infected MeWo cells for 48 hours . Cells were lysed and ORF9 was immunoprecipitated using α - V5 antibody . Input and IP fractions were subjected to immunoblotting using the indicated antibodies . Data information : Panel ( C ) is representative of two ( HaCaT ) and three ( THP1 ) independent experiments .

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(C) HaCaT cells and PMA-differentiated THP1 cells were infected with WT VZV or VZVORF9-V5 through co-culture with infected MeWo cells for 48 hours. Cells were lysed and ORF9 was immunoprecipitated using α-V5 antibody. Input and IP fractions were subjected to immunoblotting using the indicated antibodies. Data information: Panel (C) is representative of two (HaCaT) and three (THP1) independent experiments.


( D ) HEK293T cells were seeded onto glass coverslips and were transfected with human cGAS - V5 , FLAG - ORF9 , or both together . The next day , cells were fixed , permeabilised and stained using α - V5 - FITC , rabbit - α - FLAG , and goat - α - rabbit - AF647 antibodies , and DAPI . Mounted coverslips were imaged using confocal microscopy . Scale bars : 15 µm . Data information : Panels ( D ) , are representative of three independent experiments .

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(D) HEK293T cells were seeded onto glass coverslips and were transfected with human cGAS-V5, FLAG-ORF9, or both together. The next day, cells were fixed, permeabilised and stained using α-V5-FITC, rabbit-α-FLAG, and goat-α-rabbit-AF647 antibodies, and DAPI. Mounted coverslips were imaged using confocal microscopy. Scale bars: 15 µm. Data information: Panels (D), are representative of three independent experiments.


( F ) Far western analysis of cGAS - ORF9 interaction . Recombinant FLAG - ORF9 and FLAG - GFP protein were run on an SDS - PAGE gel and transferred to a membrane . After renaturation of proteins , the membrane was incubated with recombinant human cGAS as a probe , which was detected using α - cGAS antibody . After stripping , the proteins on the membrane were detected using α - FLAG antibody . Data information : Panels ( F ) are representative of three independent experiments .

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(F) Far western analysis of cGAS-ORF9 interaction. Recombinant FLAG-ORF9 and FLAG-GFP protein were run on an SDS-PAGE gel and transferred to a membrane. After renaturation of proteins, the membrane was incubated with recombinant human cGAS as a probe, which was detected using α-cGAS antibody. After stripping, the proteins on the membrane were detected using α-FLAG antibody. Data information: Panels (F) are representative of three independent experiments.


( G ) Recombinant human cGAS - HA was mixed with recombinant FLAG - ORF9 or FLAG - GFP . Proteins were immunoprecipitated using α - HA and α - FLAG antibodies . Input , unbound and IP fractions were analysed by immunoblotting . Data information : Panels ( G ) are representative of two independent experiments .

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(G) Recombinant human cGAS-HA was mixed with recombinant FLAG-ORF9 or FLAG-GFP. Proteins were immunoprecipitated using α-HA and α-FLAG antibodies. Input, unbound and IP fractions were analysed by immunoblotting. Data information: Panels (G) are representative of two independent experiments.


( B ) HEK293T cells were transfected with expression plasmids for human cGAS and ORF9 truncation mutants . The next day cells were lysed and ORF9 proteins were immunoprecipitated using α - FLAG antibody . IP fractions were subjected to immunoblotting using the indicated antibodies . Data information : Results shown in panels ( B ) are representative of two independent experiments .

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(B) HEK293T cells were transfected with expression plasmids for human cGAS and ORF9 truncation mutants. The next day cells were lysed and ORF9 proteins were immunoprecipitated using α-FLAG antibody. IP fractions were subjected to immunoblotting using the indicated antibodies. Data information: Results shown in panels (B) are representative of two independent experiments.


( D ) Recombinant cGAS , FLAG - ORF9 , FLAG - GFP , and biotinylated VACV70mer dsDNA were incubated in the indicated combinations . Recombinant proteins were precipitated using streptavidin beads . Fractions were analysed by immunoblotting . Data information : Results shown are representative of two independent experiments .

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(D) Recombinant cGAS, FLAG-ORF9, FLAG-GFP, and biotinylated VACV70mer dsDNA were incubated in the indicated combinations. Recombinant proteins were precipitated using streptavidin beads. Fractions were analysed by immunoblotting. Data information: Results shown are representative of two independent experiments.


( E ) HEK293T cells were transfected with expression plasmids for FLAG - GFP or FLAG - ORF9 . The next day cells were lysed , and the lysate was spiked with biotinylated VACV70mer dsDNA . DNA was precipitated using streptavidin beads and fractions were subjected to immunoblotting using the indicated antibodies . Data information : Results shown are representative of two independent experiments .

O O O O O O O O O O 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

(E) HEK293T cells were transfected with expression plasmids for FLAG-GFP or FLAG-ORF9. The next day cells were lysed, and the lysate was spiked with biotinylated VACV70mer dsDNA. DNA was precipitated using streptavidin beads and fractions were subjected to immunoblotting using the indicated antibodies. Data information: Results shown are representative of two independent experiments.


( F ) VACV70mer dsDNA was incubated with indicated proteins and analysed by agarose gel EMSA . Triangles indicate concentrations of ORF9 ( 0 . 7 , 1 . 4 , 2 . 9 , 5 . 7 , 8 . 6 µM ) , cGAS ( 0 . 6 , 1 . 1 , 2 . 2 , 4 . 5 , 6 . 7 µM ) , and ORF9 ( as before ) in the presence of 4 . 5 µM cGAS . Data information : Results shown are representative of two independent experiments .

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

(F) VACV70mer dsDNA was incubated with indicated proteins and analysed by agarose gel EMSA. Triangles indicate concentrations of ORF9 (0.7, 1.4, 2.9, 5.7, 8.6 µM), cGAS (0.6, 1.1, 2.2, 4.5, 6.7 µM), and ORF9 (as before) in the presence of 4.5 µM cGAS. Data information: Results shown are representative of two independent experiments.


( C ) HEK293T cells were transfected with expression plasmids for FLAG - GFP or FLAG - ORF9 as indicated . The next day cells were lysed , and the lysate was spiked with biotinylated VACV70mer dsDNA . DNA was precipitated using streptavidin beads and proteins were analysed by immunoblotting using the indicated antibodies . Data information : Data shown in panels ( C ) are representative of two independent experiments .

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

(C) HEK293T cells were transfected with expression plasmids for FLAG-GFP or FLAG-ORF9 as indicated. The next day cells were lysed, and the lysate was spiked with biotinylated VACV70mer dsDNA. DNA was precipitated using streptavidin beads and proteins were analysed by immunoblotting using the indicated antibodies. Data information: Data shown in panels (C) are representative of two independent experiments.


( D ) HEK293T cells were transfected with expression plasmids for human cGAS and FLAG - GFP or FLAG - ORF9 . The next day cells were lysed , and proteins were immunoprecipitated using α - FLAG antibody and analysed by immunoblotting using the indicated antibodies . Data information : Data shown in panels ( D ) are representative of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293T cells were transfected with expression plasmids for human cGAS and FLAG-GFP or FLAG-ORF9. The next day cells were lysed, and proteins were immunoprecipitated using α-FLAG antibody and analysed by immunoblotting using the indicated antibodies. Data information: Data shown in panels (D) are representative of two independent experiments.


( E ) THP1 cells stably transduced with GFP , ORF9 , or ORF9 - DM were transfected with the indicated amounts of E . coli dsDNA . The next day , IFNB1 expression was assessed by RT - qPCR . Data information : Panel ( E ) shows pooled data from two repeats , where each data point represents an independent biological experiment ( n = 2 + / - range ) .

O O 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

(E) THP1 cells stably transduced with GFP, ORF9, or ORF9-DM were transfected with the indicated amounts of E.coli dsDNA. The next day, IFNB1 expression was assessed by RT-qPCR. Data information: Panel (E) shows pooled data from two repeats, where each data point represents an independent biological experiment (n=2 +/- range).


( F ) In vitro cGAS activity assay . Recombinant hcGAS was incubated with ATP , GTP , and radioactive α32P - ATP at 37 ° C with addition of dsDNA and other proteins as indicated ( triangles represent concentrations of 0 . 5 , 1 , 2 , 4 , 6 µM ) . ORF9 - DM was tested only at 0 . 5 , 1 , 2 µM . Reactions were treated with calf - intestinal phosphatase and products were analysed by thin - layer chromatography and phosphorimaging . ( G ) Signal intensities from ( F ) were determined by densitometry analysis and normalized to the average BSA signal . Data information : Panel ( F ) is representative of three independent experiments . Panel ( G ) shows pooled data from three repeats , where each data point represents an independent biological experiment ( n = 3 + / - 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 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

(F) In vitro cGAS activity assay. Recombinant hcGAS was incubated with ATP, GTP, and radioactive α32P-ATP at 37°C with addition of dsDNA and other proteins as indicated (triangles represent concentrations of 0.5, 1, 2, 4, 6 µM). ORF9-DM was tested only at 0.5, 1, 2 µM. Reactions were treated with calf-intestinal phosphatase and products were analysed by thin-layer chromatography and phosphorimaging. (G) Signal intensities from (F) were determined by densitometry analysis and normalized to the average BSA signal. Data information: Panel (F) is representative of three independent experiments. Panel (G) shows pooled data from three repeats, where each data point represents an independent biological experiment (n=3 +/- SD).


B , C Representative Iba1 stainings of retinal flat mounts detecting microglia / macrophages in laser spots 3 days after laser coagulation in C57BL6 / J controls ( B ) and Ifnar1 - / - ( C ) mice . Scale bar : 20 µm . D Quantification of amoeboid - shaped mononuclear phagocytes in laser spots . Values show mean ± SD . ( n = 11 - 12 retinas ; unpaired Student ' s t - test : * * * p = 0 . 0004 ) . E Quantification of immune cell morphology in laser spots using a grid image analysis system . Values show mean ± SD . ( n = 42 - 62 cells ; unpaired 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative Iba1 stainings of retinal flat mounts detecting microglia/macrophages in laser spots 3 days after laser coagulation in C57BL6/J controls (B) and Ifnar1-/- (C) mice. Scale bar: 20 µm.D Quantification of amoeboid-shaped mononuclear phagocytes in laser spots. Values show mean ± SD. (n = 11-12 retinas; unpaired Student's t-test: ***p = 0.0004).E Quantification of immune cell morphology in laser spots using a grid image analysis system. Values show mean ± SD. (n = 42-62 cells; unpaired Student's t-test: ***p < 0.0001).


F - K Representative fundus fluorescein angiography images of C57BL6 / J ( F - H ) and Ifnar1 - / - ( I - K ) mice 3 , 7 and 14 days after laser - induced damage . L Quantification of vascular leakage by analyzing pixel intensities at 3 , 7 and 14 days after laser - induced retinal damage in C57BL6 / J controls versus Ifnar1 - / - mice . Values show mean ± SD . ( n = 14 - 22 eyes ; One - way ANOVA followed by Tukey ' s post - 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

F-K Representative fundus fluorescein angiography images of C57BL6/J (F-H) and Ifnar1-/- (I-K) mice 3, 7 and 14 days after laser-induced damage.L Quantification of vascular leakage by analyzing pixel intensities at 3, 7 and 14 days after laser-induced retinal damage in C57BL6/J controls versus Ifnar1-/- mice. Values show mean ± SD. (n = 14-22 eyes; One-way ANOVA followed by Tukey's post-test: ***p < 0.0001).


M - P Representative images of lectin stained choroidal flat mounts 7 and 14 days after laser coagulation in C57BL6 / J control mice ( M , N ) and Ifnar1 - / - animals ( O , P ) . Dashed lines indicate CNV areas and the asterisk marks the central optic nerve head . Scale bar : 200 µm . Q Quantification of lectin - stained CNV areas with ImageJ software . Bars show mean ± SD . ( n = 4 - 11 RPE / choroidal flat mounts ; One - way ANOVA followed by Tukey ' s post - test : * p = 0 . 0281 ) .

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

M-P Representative images of lectin stained choroidal flat mounts 7 and 14 days after laser coagulation in C57BL6/J control mice (M, N) and Ifnar1-/- animals (O, P). Dashed lines indicate CNV areas and the asterisk marks the central optic nerve head. Scale bar: 200 µm.Q Quantification of lectin-stained CNV areas with ImageJ software. Bars show mean ± SD. (n = 4-11 RPE/choroidal flat mounts; One-way ANOVA followed by Tukey's post-test: *p = 0.0281).


B , C Representative Iba1 stainings of retinal flat mounts detecting microglia / macrophages in laser spots 3 days after laser coagulation in control mice ( B ) or IFN - b treated animals ( C ) . Scale bar : 20 µm . D Quantification of amoeboid - shaped mononuclear phagocytes in laser spots . Values show mean ± SD . ( n = 7 - 10 retinas ; unpaired Student ' s t - test : * * * p < 0 . 0001 ) . E Quantification of immune cell morphology in laser spots using a grid image analysis system . Values show mean ± SD . ( n = 41 - 62 cells ; unpaired 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative Iba1 stainings of retinal flat mounts detecting microglia/macrophages in laser spots 3 days after laser coagulation in control mice (B) or IFN-b treated animals (C).Scale bar: 20 µm.D Quantification of amoeboid-shaped mononuclear phagocytes in laser spots. Values show mean ± SD. (n = 7-10 retinas; unpaired Student's t-test: ***p < 0.0001).E Quantification of immune cell morphology in laser spots using a grid image analysis system. Values show mean ± SD. (n = 41-62 cells; unpaired Student's t-test: ***p < 0.0001).


F - K Representative fundus fluorescein angiography images of control mice ( F - H ) or IFN - b treated animals ( I - K ) 3 , 7 and 14 days after laser - induced damage . L Quantification of vascular leakage by analyzing pixel intensities at 3 , 7 and 14 days after laser - induced retinal damage in control mice and IFN - b treated animals . Values show mean ± SD . ( n = 15 - 22 eyes ; One - way ANOVA followed by Tukey ' s post - test : * * * p = 0 . 0004 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative fundus fluorescein angiography images of control mice (F-H) or IFN-b treated animals (I-K) 3, 7 and 14 days after laser-induced damage.L Quantification of vascular leakage by analyzing pixel intensities at 3, 7 and 14 days after laser-induced retinal damage in control mice and IFN-b treated animals. Values show mean ± SD. (n = 15-22 eyes; One-way ANOVA followed by Tukey's post-test: ***p = 0.0004).


M - P Representative images of lectin stained choroidal flat mounts 7 and 14 days after laser coagulation in control mice ( M , N ) and IFN - b treated animals ( O , P ) . Dashed lines indicate CNV areas and the asterisk marks the central optic nerve head . Scale bar : 200 µmQ Quantification of lectin - stained CNV areas with ImageJ software . Bars show mean ± SD . ( n = 7 - 12 RPE / choroidal flat mounts ; One - way ANOVA followed by Tukey ' s post - test : * * p = 0 . 0038 ) .

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

M-P Representative images of lectin stained choroidal flat mounts 7 and 14 days after laser coagulation in control mice (M, N) and IFN-b treated animals (O, P). Dashed lines indicate CNV areas and the asterisk marks the central optic nerve head. Scale bar: 200 µmQ Quantification of lectin-stained CNV areas with ImageJ software. Bars show mean ± SD. (n = 7-12 RPE/choroidal flat mounts; One-way ANOVA followed by Tukey's post-test: **p = 0.0038).


B - D Representative Iba1 staining results of retinal flat mounts detecting microglia / macrophages in laser spot 3 days after laser coagulation in Cx3cr1CreER ( B ) , Ifnar1fl / fl ( C ) and Cx3cr1CreER : Ifnar1fl / fl ( D ) mice . Scale bar : 20 µm . E Quantification of amoeboid - shaped mononuclear phagocytes in laser spots . Values show mean ± SD . ( n = 17 - 29 retinas ; unpaired Student ' s t - test : * * * p = 0 . 0003 , * * p = 0 . 0044 ) . F Quantification of immune cell morphology in laser spots using a grid image analysis system . Values show mean ± SD . ( n = 44 - 52 cells ; unpaired 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B-D Representative Iba1 staining results of retinal flat mounts detecting microglia/macrophages in laser spot 3 days after laser coagulation in Cx3cr1CreER (B), Ifnar1fl/fl (C) and Cx3cr1CreER:Ifnar1fl/fl (D) mice. Scale bar: 20 µm.E Quantification of amoeboid-shaped mononuclear phagocytes in laser spots. Values show mean ± SD. (n = 17-29 retinas; unpaired Student's t-test: ***p = 0.0003, **p = 0.0044).F Quantification of immune cell morphology in laser spots using a grid image analysis system. Values show mean ± SD. (n = 44-52 cells; unpaired Student's t-test: ***p < 0.0001).


G - O Representative fundus fluorescein angiography images of Cx3cr1CreER ( G - I ) , Ifnar1fl / fl ( J - L ) and Cx3cr1CreER : Ifnar1fl / fl ( M - O ) mice 3 , 7 and 14 days after laser - induced damage . P Quantification of vascular leakage by analyzing pixel intensities at 3 , 7 and 14 days after laser - induced retinal damage in Cx3cr1CreER , Ifnar1fl / fl and Cx3cr1CreER : Ifnar1fl / fl mice . Values show mean ± SD . ( n = 5 - 12 eyes ; One - way ANOVA followed by Tukey ' s post - test : * * p = 0 . 0032 , * p = 0 . 0247 ) .

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

G-O Representative fundus fluorescein angiography images of Cx3cr1CreER (G-I), Ifnar1fl/fl (J-L) and Cx3cr1CreER:Ifnar1fl/fl (M-O) mice 3, 7 and 14 days after laser-induced damage.P Quantification of vascular leakage by analyzing pixel intensities at 3, 7 and 14 days after laser-induced retinal damage in Cx3cr1CreER, Ifnar1fl/fl and Cx3cr1CreER:Ifnar1fl/fl mice. Values show mean ± SD. (n = 5-12 eyes; One-way ANOVA followed by Tukey's post-test: **p = 0.0032, *p = 0.0247).


Q - V Representative images of lectin stained choroidal flat mounts 7 and 14 days after laser coagulation in Cx3cr1CreER ( Q , R ) , Ifnar1fl / fl ( S , T ) and Cx3cr1CreER : Ifnar1fl / fl ( U , V ) mice . Dashed lines indicate CNV areas and the asterisk marks the central optic nerve head . Scale bar : 200 µm . W Quantification of lectin - stained CNV areas with ImageJ software . Bars show mean ± SD . ( n = 4 - 11 RPE / choroidal flat mounts ; One - way ANOVA followed by Tukey ' s post - test : * p = 0 . 043 , * * * p = 0 . 0007 ) .

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

Q-V Representative images of lectin stained choroidal flat mounts 7 and 14 days after laser coagulation in Cx3cr1CreER (Q, R), Ifnar1fl/fl (S, T) and Cx3cr1CreER:Ifnar1fl/fl (U, V) mice. Dashed lines indicate CNV areas and the asterisk marks the central optic nerve head. Scale bar: 200 µm.W Quantification of lectin-stained CNV areas with ImageJ software. Bars show mean ± SD. (n = 4-11 RPE/choroidal flat mounts; One-way ANOVA followed by Tukey's post-test: *p = 0.043, ***p = 0.0007).


Coq9Q95X mice at 21 postnatal days showing the loss of corporal hair .

Coq9Q95X mice at 21 postnatal days showing the loss of corporal hair.


Representative Western blot images of COQ9protein in kidney homogenate from Coq9 + / + ( n = 4 ) and Coq9Q95Xmice ( n = 4 ) at 3 months of age . Antibody sc - 271892 was used to map the C - terminal region of the COQ9protein and antibody ab - 104189 was used to map the internal sequence of the COQ9protein .

Representative Western blot images of COQ9protein in kidney homogenate from Coq9+/+ (n = 4) and Coq9Q95Xmice (n = 4) at 3 months of age. Antibody sc-271892 was used to map the C-terminal region of the COQ9protein and antibody ab-104189 was used to map the internal sequence of the COQ9protein.


High - resolution LC - MS / MS proteomic analysis of kidneymitochondria from Coq9 + / + ( n = 3 ) and Coq9Q95Xmice ( n = 3 ) at 3 months of age . None of the six peptides of the COQ9 protein identified in Coq9 + / + mice was detected in Coq9Q95Xmice .

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

High-resolution LC-MS/MS proteomic analysis of kidneymitochondria from Coq9+/+ (n = 3) and Coq9Q95Xmice (n = 3) at 3 months of age. None of the six peptides of the COQ9 protein identified in Coq9+/+mice was detected in Coq9Q95Xmice.


A - F CoQ9 levels in tissue homogenates from brain ( A ) , cerebellum ( B ) , heart ( C ) , kidney ( D ) , extensor ( E ) and triceps surae ( F ) of male and female Coq9 + / + and Coq9Q95Xmice at 6 and 12 months of age . Data are expressed as mean ± SD . Statistical analysis was performed on 6 - month - old Coq9 + / + mice versus 6 - month - old Coq9Q95Xmice and 12 - month - old Coq9 + / + mice versus 12 - month - old Coq9Q95Xmice . * * P < 0 . 01 ; * * * P < 0 . 001 . Student ' s t - test ( n = 8 for each group ) .

A-F CoQ9 levels in tissue homogenates from brain (A), cerebellum (B), heart (C), kidney (D), extensor (E) and triceps surae (F) of male and female Coq9+/+ and Coq9Q95Xmice at 6 and 12 months of age. Data are expressed as mean ± SD. Statistical analysis was performed on 6-month-old Coq9+/+mice versus 6-month-old Coq9Q95Xmice and 12-month-old Coq9+/+mice versus 12-month-old Coq9Q95Xmice. **P < 0.01; ***P < 0.001. Student's t-test (n = 8 for each group).


A - F Residual CoQ9 levels in tissue homogenates from brain ( A ) , cerebellum ( B ) , heart ( C ) , kidney ( D ) , liver ( E ) and skeletal muscle ( F ) of Coq9 + / + , Coq9Q95X and Coq9R239X mice at 1 , 3 and 5 months of age . Data are expressed as mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239Xmice versus Coq9 + / + mice . # P < 0 . 05 ; # # P < 0 . 01 ; # # # P < 0 . 001 ; Coq9Q95X versus Coq9R239Xmice ( one - way ANOVA with a Tukey ' s post hoc test ; n = 8 for each group ; numbers above columns indicate P - values of the one - way ANOVA test ) .

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

A-F Residual CoQ9 levels in tissue homogenates from brain (A), cerebellum (B), heart (C), kidney (D), liver (E) and skeletal muscle (F) of Coq9+/+, Coq9Q95X and Coq9R239X mice at 1, 3 and 5 months of age. Data are expressed as mean ± SD. **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. #P < 0.05; ##P < 0.01; ###P < 0.001; Coq9Q95X versus Coq9R239Xmice (one-way ANOVA with a Tukey's post hoc test; n = 8 for each group; numbers above columns indicate P-values of the one-way ANOVA test).


A - E mRNA expression levels of Coq9 ( A ) , Coq7 ( B ) , Adck3 ( C ) , Coq5 ( D ) and Coq6 ( E ) on cerebrum of Coq9 + / + , Coq9Q95X and Coq9Q95X mice at 3 months of age . * * P < 0 . 01 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239Xmice versus Coq9 + / + mice . # # # P < 0 . 001 ; Coq9Q95X versus Coq9R239Xmice . F - J mRNA expression levels of Coq9 ( F ) , Coq7 ( G ) , Adck3 ( H ) , Coq5 ( I ) and Coq6 ( J ) on kidney of Coq9 + / + , Coq9Q95X and Coq9Q95Xmice at 3 months of age . * * * P < 0 . 001 ; Coq9Q95X and Coq9R239Xmice versus Coq9 + / + mice . # P < 0 . 05 ; # # # P < 0 . 001 ; Coq9Q95X versus Coq9R239Xmice . K - O mRNA expression levels of Coq9 ( K ) , Coq7 ( L ) , Adck3 ( M ) , Coq5 ( N ) and Coq6 ( O ) on triceps surae of Coq9 + / + , Coq9Q95X and Coq9Q95Xmice at 3 months of age . * P < 0 . 05 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239Xmice versus Coq9 + / + mice . # P < 0 . 05 ; Coq9Q95X versus Coq9R239Xmice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E mRNA expression levels of Coq9 (A), Coq7 (B), Adck3 (C), Coq5 (D) and Coq6 (E) on cerebrum of Coq9+/+, Coq9Q95X and Coq9Q95X mice at 3 months of age. **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. ###P < 0.001; Coq9Q95X versus Coq9R239Xmice.F-J mRNA expression levels of Coq9 (F), Coq7 (G), Adck3 (H), Coq5 (I) and Coq6 (J) on kidney of Coq9+/+, Coq9Q95X and Coq9Q95Xmice at 3 months of age. ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. #P < 0.05; ###P < 0.001; Coq9Q95X versus Coq9R239Xmice.K-O mRNA expression levels of Coq9 (K), Coq7 (L), Adck3 (M), Coq5 (N) and Coq6 (O) on triceps surae of Coq9+/+, Coq9Q95X and Coq9Q95Xmice at 3 months of age. *P < 0.05; ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. #P < 0.05; Coq9Q95X versus Coq9R239Xmice.


A - D Representative Western blot and quantitation of Western blot bands of COQ7 ( A ) , ADCK3 ( B ) , COQ5 ( C ) and COQ6 ( D ) , and VDAC1 as a loading control in the kidneys of 3 - month - old mice . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239X mice versus Coq9 + / + mice . # # P < 0 . 01 ; # # # P < 0 . 001 ; Coq9Q95X versus Coq9R239X mice . One - way ANOVA with a Tukey ' s post hoc test . E - H Representative Western blot and quantitation of Western blot bands of COQ7 ( E ) , ADCK3 ( F ) , COQ5 ( G ) and COQ6 ( H ) , and VDAC1 as a loading control in skeletal muscle of 3 - month - old mice . * * P < 0 . 01 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239X mice versus Coq9 + / + mice . # P < 0 . 05 ; # # P < 0 . 01 ; Coq9Q95X versus Coq9R239X mice . Data information : All values are presented as mean ± SD . One - way ANOVA with a Tukey ' s post hoc test . Numbers above columns indicate P - values of the one - way ANOVA test . Coq9 + / + mice n = 4 ; Coq9Q95X and Coq9R239Xmice n = 5 .

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A-D Representative Western blot and quantitation of Western blot bands of COQ7 (A), ADCK3 (B), COQ5 (C) and COQ6 (D), and VDAC1 as a loading control in the kidneys of 3-month-old mice. *P < 0.05; **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239X mice versus Coq9+/+mice. ##P < 0.01; ###P < 0.001; Coq9Q95X versus Coq9R239X mice. One-way ANOVA with a Tukey's post hoc test.E-H Representative Western blot and quantitation of Western blot bands of COQ7 (E), ADCK3 (F), COQ5 (G) and COQ6 (H), and VDAC1 as a loading control in skeletal muscle of 3-month-old mice. **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239X mice versus Coq9+/+mice. #P < 0.05; ##P < 0.01; Coq9Q95X versus Coq9R239X mice.Data information: All values are presented as mean ± SD. One-way ANOVA with a Tukey's post hoc test. Numbers above columns indicate P-values of the one-way ANOVA test. Coq9+/+mice n = 4; Coq9Q95X and Coq9R239Xmice n = 5.


A - C Mitochondrial CoQ9 levels from cerebrum ( A ) , kidney ( B ) and skeletal muscle ( C ) of Coq9 + / + and Coq9Q95X males and females . n = 8 for each group .

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A-C Mitochondrial CoQ9 levels from cerebrum (A), kidney (B) and skeletal muscle (C) of Coq9+/+ and Coq9Q95X males and females. n = 8 for each group.


D - F CI + CIII activity in cerebrum ( D ) , kidney ( E ) and skeletal muscle ( F ) of male and female Coq9 + / + and Coq9Q95X mice . n = 6 for each group . G - I CII + CIII activity in cerebrum ( G ) , kidney ( H ) and skeletal muscle ( I ) of male and female Coq9 + / + and Coq9Q95X mice . n = 6 for each group .

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D-F CI+CIII activity in cerebrum (D), kidney (E) and skeletal muscle (F) of male and female Coq9+/+ and Coq9Q95X mice. n = 6 for each group.G-I CII+CIII activity in cerebrum (G), kidney (H) and skeletal muscle (I) of male and female Coq9+/+ and Coq9Q95X mice. n = 6 for each group.


J - L Blue - native gel electrophoresis ( BNGE ) followed by immunoblotting analysis of mitochondrial supercomplexes from Coq9 + / + ( n = 3 ) and Coq9Q95X mice ( n = 4 ) at 3 months of age .

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J-L Blue-native gel electrophoresis (BNGE) followed by immunoblotting analysis of mitochondrial supercomplexes from Coq9+/+ (n = 3) and Coq9Q95X mice (n = 4) at 3 months of age.


A - D Measurement of phosphorylating respiration ( represented as State 3o , in the presence of ADP and substrates ) in kidney ( A ) and skeletal muscle ( C ) from male and female Coq9 + / + , Coq9Q95X and Coq9R239X mice at 3 months of age . Representative O2 consumption graphic in kidney ( B ) and skeletal muscle ( D ) from female Coq9 + / + , Coq9Q95X and Coq9R239X mice . Data information : All values are presented as mean ± SD . ( A , C ) * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Coq9Q95X and Coq9R239Xmice versus Coq9 + / + mice . # P < 0 . 05 ; Coq9Q95X versus Coq9R239Xmice . One - way ANOVA with a Tukey ' s post hoc test . Numbers above columns indicate P - values of the one - way ANOVA test ( n = 3 for each group ) .

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A-D Measurement of phosphorylating respiration (represented as State 3o, in the presence of ADP and substrates) in kidney (A) and skeletal muscle (C) from male and female Coq9+/+, Coq9Q95X and Coq9R239X mice at 3 months of age. Representative O2 consumption graphic in kidney (B) and skeletal muscle (D) from female Coq9+/+, Coq9Q95X and Coq9R239X mice.Data information: All values are presented as mean ± SD. (A, C) *P < 0.05; **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. #P < 0.05; Coq9Q95X versus Coq9R239Xmice. One-way ANOVA with a Tukey's post hoc test. Numbers above columns indicate P-values of the one-way ANOVA test (n = 3 for each group).


A - H Complex II ( SDH ) and complex IV ( COX ) histochemistry of triceps surae showing a decreased stain in 18 - month - old Coq9Q95X female mice ( D , H ) in contrast to normal SDH and COX activity in 6 - and 18 - month - old Coq9 + / + ( A , C , E , G ) , as well as 6 - month - old Coq9Q95X female mice ( B , F ) . I - L Gomori trichrome stain ( TGM ) of triceps surae showed no differences between 6 - and 18 - month - old Coq9 + / + and Coq9Q95X female mice . M - P Hematoxylin and eosin ( H & amp ; amp ; E ) stains of triceps surae did not reveal any structural abnormality . Data information : Scale bars : 100 μm . n = 3 for each group . Complex IV , cytochrome c oxidase ( COX ) ; complex II , succinate dehydrogenase ( SDH ) .

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A-H Complex II (SDH) and complex IV (COX) histochemistry of triceps surae showing a decreased stain in 18-month-old Coq9Q95X female mice (D, H) in contrast to normal SDH and COX activity in 6- and 18-month-old Coq9+/+ (A, C, E, G), as well as 6-month-old Coq9Q95X female mice (B, F).I-L Gomori trichrome stain (TGM) of triceps surae showed no differences between 6- and 18-month-old Coq9+/+ and Coq9Q95X female mice.M-P Hematoxylin and eosin (H&amp;E) stains of triceps surae did not reveal any structural abnormality.Data information: Scale bars: 100 μm. n = 3 for each group. Complex IV, cytochrome c oxidase (COX); complex II, succinate dehydrogenase (SDH).


A - C Voluntary wheel running test . Distance traveled on the wheel and average speed during the use of the wheel were decreased in female Coq9Q95X mice at 6 months of age ( B , C ) .

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A-C Voluntary wheel running test. Distance traveled on the wheel and average speed during the use of the wheel were decreased in female Coq9Q95X mice at 6 months of age (B, C).


D Hanging wire test . Coq9Q95X female mice obtained less reaches score in the ' fall and reaches ' method .

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D Hanging wire test. Coq9Q95X female mice obtained less reaches score in the 'fall and reaches' method.


E Open - field test . Coq9Q95X mice showed a reduction in the average distance traveled in Coq9Q95X female mice at 6 months of age .

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E Open-field test. Coq9Q95X mice showed a reduction in the average distance traveled in Coq9Q95X female mice at 6 months of age.


F Grip test : Muscle strength was not affected in Coq9Q95X mice at 6 months of age .

F Grip test: Muscle strength was not affected in Coq9Q95X mice at 6 months of age.


A , B KidneyCoQ9 levels in Coq9 + / + , Coq9Q95X and Coq9R239Xmice treated with 2 , 4 - diHB ( + 2 , 4 - diHB ) compared with the non - treated littermate ( vehicle ) . Statistical analysis was performed on + 2 , 4 - diHBCoq9 + / + , Coq9Q95X and Coq9R239Xmice versus vehicle Coq9 + / + , Coq9Q95X and Coq9R239Xmice , respectively ( n = 3 for each group ) .

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A, B KidneyCoQ9 levels in Coq9+/+, Coq9Q95X and Coq9R239Xmice treated with 2,4-diHB (+2,4-diHB) compared with the non-treated littermate (vehicle). Statistical analysis was performed on +2,4-diHBCoq9+/+, Coq9Q95X and Coq9R239Xmice versus vehicle Coq9+/+, Coq9Q95X and Coq9R239Xmice, respectively (n = 3 for each group).


C , D CoQ10 levels in COQ9R244X skinfibroblasts treated with 2 , 4 - diHB ( + 2 , 4 - diHB , blue bars ) compared with the non - treated controls ( vehicle , yellow bars ) . Statistical analysis was performed on + 2 , 4 - diHBCOQ9R244X versus vehicle COQ9R244X ( n = 4 for each group ) .

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C, D CoQ10 levels in COQ9R244X skinfibroblasts treated with 2,4-diHB (+2,4-diHB, blue bars) compared with the non-treated controls (vehicle, yellow bars). Statistical analysis was performed on +2,4-diHBCOQ9R244X versus vehicle COQ9R244X (n = 4 for each group).


( A ) Cell count - based comparison of cellular proliferation of T - ALL cells after treatment with dexamethasone ( Dexa ) . Individual and mean values of three biological replicates ± SD analyzed by two - way ANOVA are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(A) Cell count-based comparison of cellular proliferation of T-ALL cells after treatment with dexamethasone (Dexa). Individual and mean values of three biological replicates ± SD analyzed by two-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( B ) Flow cytometric analysis of cell death by AnnexinV staining in T - ALL cell lines upon treatment with indicated Dexa concentrations . Cell death is represented by the number of AnnexinV positive ( AnnexinV + ) , apoptotic cells as a percentage ( % ) of total cells . Mean values of n ≥ 4 biological replicates ± SD are shown . Data information : ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(B) Flow cytometric analysis of cell death by AnnexinV staining in T-ALL cell lines upon treatment with indicated Dexa concentrations. Cell death is represented by the number of AnnexinV positive (AnnexinV+), apoptotic cells as a percentage (%) of total cells. Mean values of n≥4 biological replicates ± SD are shown. Data information: ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( C ) Expression levels of human GRα and LRH - 1 mRNA in human T - ALL cell lines were determined by probe - based real - time quantitative PCR , calculated as relative expression compared to beta - actin and used to determine the GR : LRH - 1 ratio ( right ) . Individual and mean values of four biological replicates ± SD are shown and statistically significant differences between CEM - C7 and GC - resistant cell lines were determined by one - way ANOVA .

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(C) Expression levels of human GRα and LRH-1 mRNA in human T-ALL cell lines were determined by probe-based real-time quantitative PCR, calculated as relative expression compared to beta-actin and used to determine the GR:LRH-1 ratio (right). Individual and mean values of four biological replicates ± SD are shown and statistically significant differences between CEM-C7 and GC-resistant cell lines were determined by one-way ANOVA.


( D ) Immunoblots of GR , BimEL , BimL and BimS and Tubulin from T - ALL cells treated for 24 h with 20 µM 3d2 or 30 µM Dexa . Representative results from n = 2 biological replicates are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by Ø was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(D) Immunoblots of GR, BimEL, BimL and BimS and Tubulin from T-ALL cells treated for 24 h with 20 µM 3d2 or 30 µM Dexa. Representative results from n=2 biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by Ø was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( E ) Immunoblots of GR , PARP ( nuclear loading control ) and Tubulin ( cytoplasmatic loading control ) from cytoplasmic ( CP ) and nucleoplasmic ( NP ) lysates of T - ALL cells treated with Dexa . Note : Nuclear extracts were more highly concentrated ( approx . 6 - fold ) than cytosolic fractions . Representative results from two biological replicates are shown .

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(E) Immunoblots of GR, PARP (nuclear loading control) and Tubulin (cytoplasmatic loading control) from cytoplasmic (CP) and nucleoplasmic (NP) lysates of T-ALL cells treated with Dexa. Note: Nuclear extracts were more highly concentrated (approx. 6-fold) than cytosolic fractions. Representative results from two biological replicates are shown.


( F ) GR activity in human T - ALL cells transfected with a control luciferase reporter plasmid ( pGL3 ) ( F ) GR responsive ( GRE ) luciferase reporter and treated with 3d2 and / or Dexa . β - galactosidase ( bGal ) was co - transfected as an internal transfection control . Luciferase reporter activity was normalized to bGal activity and calculated as relative to the DMSO - treated pGL3 control . Individual and mean values analyzed by two - way ANOVA of three biological replicates ± SD are shown for Jurkat and CEM - C7 whereas technical triplicates ± SD of a representative experiment ( n = 3 biological replicates ) are shown for MOLT - 4 . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(F) GR activity in human T-ALL cells transfected with a control luciferase reporter plasmid (pGL3) (F) GR responsive (GRE) luciferase reporter and treated with 3d2 and/or Dexa. β-galactosidase (bGal) was co-transfected as an internal transfection control. Luciferase reporter activity was normalized to bGal activity and calculated as relative to the DMSO-treated pGL3 control. Individual and mean values analyzed by two-way ANOVA of three biological replicates ± SD are shown for Jurkat and CEM-C7 whereas technical triplicates ± SD of a representative experiment (n=3 biological replicates) are shown for MOLT-4. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( G ) LRH - 1 activity in human T - ALL cells transfected with a control luciferase reporter plasmid ( pGL3 ) and ( G ) LRH - 1 responsive ( 5xRE ) luciferase reporter and treated with 3d2 β - galactosidase ( bGal ) was co - transfected as an internal transfection control . Luciferase reporter activity was normalized to bGal activity and calculated as relative to the DMSO - treated pGL3 control . Individual and mean values analyzed by two - way ANOVA of three biological replicates ± SD are shown for Jurkat and CEM - C7 whereas technical triplicates ± SD of a representative experiment ( n = 3 biological replicates ) are shown for MOLT - 4 . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(G) LRH-1 activity in human T-ALL cells transfected with a control luciferase reporter plasmid (pGL3) and (G) LRH-1 responsive (5xRE) luciferase reporter and treated with 3d2 β-galactosidase (bGal) was co-transfected as an internal transfection control. Luciferase reporter activity was normalized to bGal activity and calculated as relative to the DMSO-treated pGL3 control. Individual and mean values analyzed by two-way ANOVA of three biological replicates ± SD are shown for Jurkat and CEM-C7 whereas technical triplicates ± SD of a representative experiment (n=3 biological replicates) are shown for MOLT-4. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( H ) Flow cytometry - based quantification of BiFC in Jurkat cells transiently co - transfected with YFP1 - LRH - 1 and GR - YFP2 and treated for indicated time periods with Dexa . YFP + cells are shown as a percentage ( % ) of viable cells . Individual and mean values ± SD of three biological replicates analyzed by one - way ANOVA are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by 0h ) was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(H) Flow cytometry-based quantification of BiFC in Jurkat cells transiently co-transfected with YFP1-LRH-1 and GR-YFP2 and treated for indicated time periods with Dexa. YFP+ cells are shown as a percentage (%) of viable cells. Individual and mean values ± SD of three biological replicates analyzed by one-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0h) was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( A ) Daily cell counting - based comparison of cellular proliferation of T - ALL cells after ( A ) treatment with 3d2 Individual and mean values of technical triplicates of one representative experiment ( n = 3 biological replicates ) ± SD anlyzed by two - way ANOVA are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; was used as a solvent control . ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(A) Daily cell counting-based comparison of cellular proliferation of T-ALL cells after (A) treatment with 3d2 Individual and mean values of technical triplicates of one representative experiment (n=3 biological replicates) ± SD anlyzed by two-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, *** p < 0.001.


( B ) Daily cell counting - based comparison of cellular proliferation of T - ALL cells after ( B ) small hairpin RNA - mediated LRH - 1 knockdown ( shLRH - 1 ) . Individual and mean values of technical triplicates of one representative experiment ( n = 3 biological replicates ) ± SD anlyzed by two - way ANOVA are shown . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(B) Daily cell counting-based comparison of cellular proliferation of T-ALL cells after (B) small hairpin RNA-mediated LRH-1 knockdown (shLRH-1). Individual and mean values of technical triplicates of one representative experiment (n=3 biological replicates) ± SD anlyzed by two-way ANOVA are shown. Data information: ns = not significant, * p < 0.05, *** p < 0.001.


( C ) Cell cycle distribution of T - ALL cells treated for 24 h with indicated concentrations of 3d2 as measured by propidium iodide flow cytometry . Representative results from n = 3 biological replicates are shown . Stacked bars represent the mean of technical triplicates ± SD . Data information : ns = not significant , * 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 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

(C) Cell cycle distribution of T-ALL cells treated for 24 h with indicated concentrations of 3d2 as measured by propidium iodide flow cytometry. Representative results from n=3 biological replicates are shown. Stacked bars represent the mean of technical triplicates ± SD. Data information: ns = not significant, * p < 0.05, *** p < 0.001.


( D ) Simultaneous flow cytometric analysis of cell cycle distribution by EdU / DAPI staining and cell death by AnnexinV staining in MOLT - 4 cells upon treatment with 3d2 . Dead cells are represented by the number of AnnexinV positive ( AnnexinV + ) , apoptotic cells as a percentage ( % ) of total cells and cell cycle phase distribution as % of viable , AnnexinV negative ( AnnexinV - ) cells . Representative results from three biological replicates are shown . Bars represent the mean of technical triplicates ± SD and differences in AnnexinV + cells were analyzed by one - way ANOVA . Data information : ns = not significant , * 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 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

(D) Simultaneous flow cytometric analysis of cell cycle distribution by EdU/DAPI staining and cell death by AnnexinV staining in MOLT-4 cells upon treatment with 3d2. Dead cells are represented by the number of AnnexinV positive (AnnexinV+), apoptotic cells as a percentage (%) of total cells and cell cycle phase distribution as % of viable, AnnexinV negative (AnnexinV-) cells. Representative results from three biological replicates are shown. Bars represent the mean of technical triplicates ± SD and differences in AnnexinV+ cells were analyzed by one-way ANOVA. Data information: ns = not significant, * p < 0.05, *** p < 0.001.