Figure 3 ( A ) Cell viability ( TO - PRO - 3 FACS staining ) of GT1 - 7 neuronal cells transfected for 24 hours with either wild type or indicated mutant constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA , PG or GR frame . ( B ) Immunoblotting against the GFP , endogenous C9ORF72 or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to the GFP in the GA frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA . ( C ) Immunoblotting against the HA tag , endogenous C9ORF72 or GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA tag in the PG frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with Bafilomycin A1 for 15 hours . ( D ) Immunoblotting against the HA tag , endogenous C9ORF72 or GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA tag in the GR frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with Bafilomycin A1 for 15 hours . ( E ) Cell viability ( TO - PRO - 3 FACS staining ) of GT1 - 7 neuronal cells co - transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA and wild - type or mutants constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA , PG or GR frame .

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

Figure 3(A) Cell viability (TO-PRO-3 FACS staining) of GT1-7 neuronal cells transfected for 24 hours with either wild type or indicated mutant constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA, PG or GR frame.(B) Immunoblotting against the GFP, endogenous C9ORF72 or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to the GFP in the GA frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA.(C) Immunoblotting against the HA tag, endogenous C9ORF72 or GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA tag in the PG frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with Bafilomycin A1 for 15 hours.(D) Immunoblotting against the HA tag, endogenous C9ORF72 or GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA tag in the GR frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with Bafilomycin A1 for 15 hours.(E) Cell viability (TO-PRO-3 FACS staining) of GT1-7 neuronal cells co-transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA and wild-type or mutants constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA, PG or GR frame.


Figure 4 ( A ) Left panel , immunoblotting against the HA tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with a construct expressing 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA in the GA frame and treated with 10 µM of the indicated compound for 15 hours . Right panel , quantification of polyGA expression relative to the GAPDH . ( B ) Left panel , immunoblotting against the HA tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with a construct expressing 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA tag in the PG frame and treated with 10 µM of the indicated compound for 15 hours . Right panel , quantification of polyPG expression relative to the GAPDH . ( C ) Cell viability ( TO - PRO - 3 FACS staining ) of GT1 - 7 neuronal cells treated with 1 , 3 or 10 µM of promethazine and co - transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA and a construct expressing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA or PG frame .

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

Figure 4(A) Left panel, immunoblotting against the HA tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with a construct expressing 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA in the GA frame and treated with 10 µM of the indicated compound for 15 hours. Right panel, quantification of polyGA expression relative to the GAPDH.(B) Left panel, immunoblotting against the HA tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with a construct expressing 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA tag in the PG frame and treated with 10 µM of the indicated compound for 15 hours. Right panel, quantification of polyPG expression relative to the GAPDH.(C) Cell viability (TO-PRO-3 FACS staining) of GT1-7 neuronal cells treated with 1, 3 or 10 µM of promethazine and co-transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA and a construct expressing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA or PG frame.


Figure 1 ( A ) Bcl - xL was cotransfected into HeLa cells with empty vector ( IP negative control ) , Beclin 1 - Flag , Beclin 1 - Flag / HA - Bim ( EL ) , Beclin 1 - Flag / HA - Puma , Beclin 1 - Flag / HA - Noxa , or Beclin 1 - Flag / Bad . Anti - Flag ( M2 ) was used for immunoprecipitation , and proteins were detected with anti - Bcl - xL , anti - HA , or anti - Bad , anti - Flag ( Rabbit ) . ( B ) HA - Bim ( wild - type ) was cotransfected into Bax / Bak double - knockout ( DKO ) mouse embryonic fibroblasts ( MEFs ) with empty vector ( IP negative control ) or Beclin 1 - Flag . Immunoprecipitations were performed as in ( A ) . ( C ) Beclin 1 - Flag was cotransfected into Bax / Bak DKO MEFs with empty vector ( IP negative control ) or HA - Bim ( wild - type ) . Anti - HA was used for immunoprecipitation . ∗ , antibody heavy chain . ( D ) HeLa cell lysates were subjected to GFP antibody ( negative control ) and anti - Bim antibody immunoprecipitation . Proteins were detected with anti - Beclin 1 and anti - Bim . ∗ ∗ , antibody heavy chain ; ∗ , antibody light chain . ( E ) Bcl - xL was cotransfected into HeLa cells with empty vector ( IP negative control ) , wild - type HA - Bim ( EL ) , or HA - Bim L152E F159E [ Bim ( EL ) EE ] . Anti - Bim was used for immunoprecipitation . ∗ , antibody light chain . ( F ) HeLa cells were transfected with vector , wild - type HA - Bim , or HA - BimEE . Lysates were probed with the indicated antibodies . HeLa cells treated with staurosporine ( STS ) for 5 hr were a positive control . Note that wild - type Bim causes apoptosis , which reduces its levels compared to BimEE . ( G ) Vector , wild - type Bim ( EL ) , or Bim ( EL ) L152E F159E ( BimEE ) was transfected into HeLa cells . Transfection efficiency was > 90 % . After 20 hr , cell viability was determined by measurement pf ATP levels . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 . ( H ) HeLa cells were transfected with vector / GFP ( 3 : 1 ) , wild - type HA - Bim / GFP ( 3 : 1 ) , or HA - BimEE / GFP ( 3 : 1 ) ( to ensure that Bim - transfected cells also contain GFP ) . After 16 hr , cells were stained with Annexin V - APC and analyzed by FACS . The percentage of APC and GFP double - positive cells / GFP - positive cells are shown . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 . ( I ) Bim ( EL ) / Bcl - xL were cotransfected into HeLa cells with empty vector ( IP negative control ) or Beclin 1 - Flag ; Bim ( EL ) - EE / Bcl - xL were also cotransfected into HeLa cells with Beclin 1 - Flag . Anti - Flag ( M2 ) was used for immunoprecipitation . Data are shown as mean ± SD . NS , not significant . ( J ) BimEL - EE ( EL ) / vector ( IP negative control ) , BimEL - EE ( EL ) / Beclin 1 - Flag , BimL - EE ( L ) / Beclin 1 - Flag , and BimS - EE ( S ) / Beclin 1 - Flag were transfected into HeLa cells . Anti - Flag was used for immunoprecipitation . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 ; NS , not significant .

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

Figure 1(A) Bcl-xL was cotransfected into HeLa cells with empty vector (IP negative control), Beclin 1-Flag, Beclin 1-Flag/HA-Bim(EL), Beclin 1-Flag/HA-Puma, Beclin 1-Flag/HA-Noxa, or Beclin 1-Flag/Bad. Anti-Flag (M2) was used for immunoprecipitation, and proteins were detected with anti-Bcl-xL, anti-HA, or anti-Bad, anti-Flag (Rabbit).(B) HA-Bim (wild-type) was cotransfected into Bax/Bak double-knockout (DKO) mouse embryonic fibroblasts (MEFs) with empty vector (IP negative control) or Beclin 1-Flag. Immunoprecipitations were performed as in (A).(C) Beclin 1-Flag was cotransfected into Bax/Bak DKO MEFs with empty vector (IP negative control) or HA-Bim (wild-type). Anti-HA was used for immunoprecipitation. ∗, antibody heavy chain.(D) HeLa cell lysates were subjected to GFP antibody (negative control) and anti-Bim antibody immunoprecipitation. Proteins were detected with anti-Beclin 1 and anti-Bim. ∗∗, antibody heavy chain; ∗, antibody light chain.(E) Bcl-xL was cotransfected into HeLa cells with empty vector (IP negative control), wild-type HA-Bim(EL), or HA-Bim L152E F159E [Bim(EL)EE]. Anti-Bim was used for immunoprecipitation. ∗, antibody light chain.(F) HeLa cells were transfected with vector, wild-type HA-Bim, or HA-BimEE. Lysates were probed with the indicated antibodies. HeLa cells treated with staurosporine (STS) for 5 hr were a positive control. Note that wild-type Bim causes apoptosis, which reduces its levels compared to BimEE.(G) Vector, wild-type Bim(EL), or Bim(EL) L152E F159E (BimEE) was transfected into HeLa cells. Transfection efficiency was >90%. After 20 hr, cell viability was determined by measurement pf ATP levels. Data are shown as mean ± SD. ∗∗∗p < 0.001.(H) HeLa cells were transfected with vector/GFP (3:1), wild-type HA-Bim/GFP (3:1), or HA-BimEE/GFP (3:1) (to ensure that Bim-transfected cells also contain GFP). After 16 hr, cells were stained with Annexin V-APC and analyzed by FACS. The percentage of APC and GFP double-positive cells/GFP-positive cells are shown. Data are shown as mean ± SD. ∗∗∗p < 0.001.(I) Bim(EL)/Bcl-xL were cotransfected into HeLa cells with empty vector (IP negative control) or Beclin 1-Flag; Bim(EL)-EE/Bcl-xL were also cotransfected into HeLa cells with Beclin 1-Flag. Anti-Flag (M2) was used for immunoprecipitation. Data are shown as mean ± SD. NS, not significant.(J) BimEL-EE (EL)/vector (IP negative control), BimEL-EE (EL)/Beclin 1-Flag, BimL-EE (L)/Beclin 1-Flag, and BimS-EE (S)/Beclin 1-Flag were transfected into HeLa cells. Anti-Flag was used for immunoprecipitation. Data are shown as mean ± SD. ∗∗∗p < 0.001; NS, not significant.


Figure 2 ( A ) Bim − / − and Bim + / + MEFs were cultured in 6 - well dishes . Cells were treated with vehicle or Bafilomycin A1 ( Baf ) for 4 hr . Blots were probed with the indicated antibodies . LC3 - II / tubulin in wild - type control MEFs is set as 1 . The relative value of LC3 - II / tubulin in Bim - knockout MEFs is shown ( n = 5 ) . Data are mean ± SD . ∗ ∗ ∗ p < 0 . 001 . ( B ) HeLa cells were transfected with control siRNA and Bim siRNA . After 48 hr , cells were treated with vehicle or Baf for 4 hr . Blots were probed with the indicated antibodies and analyzed as described in ( A ) . ∗ ∗ ∗ p < 0 . 001 . ( C ) Bim − / − and Bim + / + MEFs were transfected with GFP - LC3 . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . ( D ) HeLa cells were transfected with control plasmid ( pMKO ) / GFP - LC3 or pMKO - BimshRNA / GFP - LC3 ( to ensure that GFP - LC3 - positive cells also contain BimshRNA or control plasmid , the ratio of pMKO or pMKO - BimshRNA / GFP - LC3 is 3 : 1 ) . The percentages of cells with GFP - LC3 vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 . ( E ) HeLa cells were transfected with control plasmid ( pMKO ) or pMKO - BimshRNA . Blots were probed with the indicated antibodies . Data are shown as mean ± SD . ∗ ∗ ∗ p < 0 . 001 . ( F ) HeLa cells were transfected with control plasmid ( pMKO ) / GFP - PX or pMKO - BimshRNA / GFP - PX ( 3 : 1 ) . The percentages of cells with GFP - vesicles were assessed . Data are shown as mean ± SD . ∗ ∗ p < 0 . 01 . ( 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 . ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Bim−/− and Bim+/+ MEFs were cultured in 6-well dishes. Cells were treated with vehicle or Bafilomycin A1 (Baf) for 4 hr. Blots were probed with the indicated antibodies. LC3-II/tubulin in wild-type control MEFs is set as 1. The relative value of LC3-II/tubulin in Bim-knockout MEFs is shown (n = 5). Data are mean ± SD. ∗∗∗p < 0.001.(B) HeLa cells were transfected with control siRNA and Bim siRNA. After 48 hr, cells were treated with vehicle or Baf for 4 hr. Blots were probed with the indicated antibodies and analyzed as described in (A). ∗∗∗p < 0.001.(C) Bim−/− and Bim+/+ MEFs were transfected with GFP-LC3. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01.(D) HeLa cells were transfected with control plasmid (pMKO)/GFP-LC3 or pMKO-BimshRNA/GFP-LC3 (to ensure that GFP-LC3-positive cells also contain BimshRNA or control plasmid, the ratio of pMKO or pMKO-BimshRNA/GFP-LC3 is 3:1). The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001.(E) HeLa cells were transfected with control plasmid (pMKO) or pMKO-BimshRNA. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗∗p < 0.001.(F) HeLa cells were transfected with control plasmid (pMKO)/GFP-PX or pMKO-BimshRNA/GFP-PX (3:1). The percentages of cells with GFP-vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01.(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.(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.(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.(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.


Figure 3 ( 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 . ( 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 . ( 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 .

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

Figure 3(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.(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.(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.


Figure 4 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.(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.


Figure 5 ( 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 . ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.(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.(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.(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.


Figure 6 ( 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 . ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.(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.(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.(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.


Figure 7 ( 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 . ( 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 . ( 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 .

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

Figure 7(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.(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.(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.


Figure 1 ( 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 . ( 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 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 . ( 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

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

Figure 1(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.(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 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.(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


Figure 2 ( 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 ) . ( 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 . ( 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 . ( 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 .

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

Figure 2(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).(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.(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.(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.


Figure 3 ( 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 . ( 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 . ( 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 .

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

Figure 3(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.(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.(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.


Figure 4 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.(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.


Figure 6 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.(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).


Figure 1B , 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B, 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).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).


Figure 2B , 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B, 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).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).


Figure 3B - 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B-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).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).


Figure 1Coq9Q95X 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 . 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 B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Coq9Q95X 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.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.


Figure 2A - 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 ) .

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

Figure 2A-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).


Figure 3A - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-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).


Figure 4A - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-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.


Figure 5A - 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 .

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

Figure 5A-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.


Figure 6A - 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 . 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 .

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

Figure 6A-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.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.