E Spine densities were comparable between experimental groups at 3dpT [ iML p = 0 . 1512 , oML p = 0 . 3841 ] . 13dpT β - catex3 iDCX neurons showed significantly increased spine density in the inner molecular layer [ iML p < 0 . 0001 , oML p = 0 . 0402 ] ( 3dpT : control : n = 20 cells from 3 animals , β - catex3 iDCX : n = 20 cells from 4 animals ; 13dpT control : n = 20 cells from 5 animals , β - catex3 iDCX : n = 20 cells from 7 animals ) . Representative images showing dendritic segments in the inner molecular layer of the DG at 13dpT . Scale bars = 1µm .

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

E Spine densities were comparable between experimental groups at 3dpT [iML p=0.1512, oML p=0.3841]. 13dpT β-catex3 iDCX neurons showed significantly increased spine density in the inner molecular layer [iML p<0.0001 , oML p=0.0402] (3dpT: control: n= 20 cells from 3 animals, β-catex3 iDCX: n= 20 cells from 4 animals; 13dpT control: n= 20 cells from 5 animals, β-catex3 iDCX: n= 20 cells from 7 animals). Representative images showing dendritic segments in the inner molecular layer of the DG at 13dpT. Scale bars= 1µm.


F Representative images of recombined ( GFP + ) neurons co - expressing Prox1 ( grey ) as a marker for neuronal fate and the stage specific markers DCX ( red ) for immature neurons and Calbindin ( red ) for mature neurons . Arrows and arrowheads indicate marker negative and marker positive cells , respectively . Scale bar = 10µm . G Fraction of DCX expressing neurons is slightly reduced in β - catex3 iDCX at 3dpT [ p = 0 . 0338 ] , while fraction of Calbindin expressing cells is slightly increased in β - catex3 iDCX at 3dpT [ p = 0 . 0345 ] . At 13dpT no difference is visible in stage specific marker expression [ DCX p = 0 . 8564 , Calbindin p = 0 . 0841 ] ( 3dpT : control : n = 12 animals , β - catex3 iDCX : n = 12 animals , 13dpT : control : n = 10 animals , β - catex3 iDCX : n = 14 animals ) .

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

F Representative images of recombined (GFP+) neurons co-expressing Prox1 (grey) as a marker for neuronal fate and the stage specific markers DCX (red) for immature neurons and Calbindin (red) for mature neurons. Arrows and arrowheads indicate marker negative and marker positive cells, respectively. Scale bar = 10µm. G Fraction of DCX expressing neurons is slightly reduced in β-catex3 iDCX at 3dpT [p=0.0338], while fraction of Calbindin expressing cells is slightly increased in β-catex3 iDCX at 3dpT [p=0.0345]. At 13dpT no difference is visible in stage specific marker expression [DCX p=0.8564, Calbindin p=0.0841] (3dpT: control: n= 12 animals, β-catex3 iDCX: n= 12 animals, 13dpT: control: n= 10 animals, β-catex3 iDCX: n= 14 animals).


B Expression of DCX and Calbindin in BrdU + neurons was comparable between experimental groups at 3dpT and 13dpT ( control : n = 5 animals , β - catex3 iDCX : n = 5 animals ) .

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

B Expression of DCX and Calbindin in BrdU+ neurons was comparable between experimental groups at 3dpT and 13dpT (control: n= 5 animals, β-catex3 iDCX: n= 5 animals).


C Representative reconstructions of control and β - catex3 iDCX neurons expressing RFP at 3dpT . Scale bar = 20µm .

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

C Representative reconstructions of control and β-catex3 iDCX neurons expressing RFP at 3dpT. Scale bar= 20µm.


D Quantification of dendritic length [ p = 0 . 0003 ] , branch points [ p = 0 . 0433 ] , and Sholl analysis [ p < 0 . 0001 ] showed a higher dendritic complexity of β - catex3 iDCX neurons at 3dpT ( control : n = 22 cells from 4 animals , β - catex3 iDCX : n = 19 cells from 5 animals ) .

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

D Quantification of dendritic length [p=0.0003], branch points [p=0.0433], and Sholl analysis [p<0.0001] showed a higher dendritic complexity of β-catex3 iDCX neurons at 3dpT (control: n= 22 cells from 4 animals, β-catex3 iDCX: n= 19 cells from 5 animals).


E Representative reconstructions of control and β - catex3 iDCX neurons expressing RFP at 13dpT . Scale bar = 20µm .

E Representative reconstructions of control and β-catex3 iDCX neurons expressing RFP at 13dpT. Scale bar= 20µm.


F Quantification of dendritic length [ p < 0 . 0001 ] , and branch points [ p = 0 . 0495 ] , and Sholl analysis [ p < 0 . 0001 ] showed a lower dendritic complexity of β - catex3 iDCX neurons at 13dpT ( control : n = 20 cells from 4 animals , β - catex3 iDCX : n = 19 cells from 5 animals ) .

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

F Quantification of dendritic length [p<0.0001], and branch points [p=0.0495], and Sholl analysis [p<0.0001] showed a lower dendritic complexity of β-catex3 iDCX neurons at 13dpT (control: n= 20 cells from 4 animals, β-catex3 iDCX: n= 19 cells from 5 animals).


A Representative overview images and quantification of the β - Galactosidase reporter signal in the DG of 8 - week old , 24 - week old and 36 - week old BATGAL mice . Note the decrease in reporter signal with increasing age ( n = 4 animals per age , 200 - 300 cells per animal ) [ 8 weeks vs 24 weeks p = 0 . 0268 , 8 weeks vs 36 weeks p = 0 . 0268 ] . Scale bar = 100µm .

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

A Representative overview images and quantification of the β-Galactosidase reporter signal in the DG of 8-week old, 24-week old and 36-week old BATGAL mice. Note the decrease in reporter signal with increasing age (n= 4 animals per age, 200-300 cells per animal) [8 weeks vs 24 weeks p=0.0268, 8 weeks vs 36 weeks p=0.0268]. Scale bar= 100µm.


B Representative images of BrdU + cells ( red ) expressing DCX ( grey ) or β - Galactosidase ( green ) at 14 days post BrdU injection ( dpBrdU ) , 28dpBrdU and 42dpBrdu in 8 - week old and 24 - week old BATGAL mice . Scale bar = 10µm . C Percentage of BrdU + cells expressing DCX in 8 - week old and 24 - week old BATGAL mice ( 14dpi : n = 3 animals , 28dpi : n = 3 animals , 42dpi : n = 3 animals ) . Note the prolonged expression of DCX in 24 - week old mice [ 28dpBrdU p = 0 . 0155 ] . D Percentage of BrdU + cells expressing the β - Galactosidase reporter in 8 - week old and 24 - week old BATGAL mice ( 14dpi : n = 3 animals , 28dpi : n = 3 animals , 42dpi : n = 3 animals ) . Note the reduced reporter expression in 24 - week old mice [ 28dpBrdU p = 0 . 0117 , 42dpBrdU p = 0 . 0034 ] .

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

B Representative images of BrdU+ cells (red) expressing DCX (grey) or β-Galactosidase (green) at 14 days post BrdU injection (dpBrdU), 28dpBrdU and 42dpBrdu in 8-week old and 24-week old BATGAL mice. Scale bar= 10µm. C Percentage of BrdU+ cells expressing DCX in 8-week old and 24-week old BATGAL mice (14dpi: n= 3 animals, 28dpi: n= 3 animals, 42dpi: n= 3 animals). Note the prolonged expression of DCX in 24-week old mice [28dpBrdU p=0.0155]. D Percentage of BrdU+ cells expressing the β-Galactosidase reporter in 8-week old and 24-week old BATGAL mice (14dpi: n= 3 animals, 28dpi: n= 3 animals, 42dpi: n= 3 animals). Note the reduced reporter expression in 24-week old mice [28dpBrdU p=0.0117, 42dpBrdU p=0.0034].


B Representative reconstructions of control and β - catex3 iDCX neurons in 24 - week old mice at 3dpT . Scale bar = 20µm .

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

B Representative reconstructions of control and β-catex3 iDCX neurons in 24-week old mice at 3dpT. Scale bar = 20µm.


C Dendritic length [ p < 0 . 0001 ] and number of branch points [ p < 0 . 0001 ] was increased in 24 - week - old β - catex3 iDCX compared to 24 - week - old controls at 3dpT ( control : n = 21 cells from 5 animals , β - catex3 iDCX : n = 20 cells from 6 animals ) . At 13dpT dendritic length [ p = 0 . 5788 ] and number of branch points [ p = 0 . 4978 ] were comparable ( control : n = 21 cells from 5 animals , β - catex3 iDCX : n = 20 cells from 8 animals ) .

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

C Dendritic length [p<0.0001] and number of branch points [p<0.0001] was increased in 24-week-old β-catex3 iDCX compared to 24-week-old controls at 3dpT (control: n= 21 cells from 5 animals, β-catex3 iDCX: n= 20 cells from 6 animals). At 13dpT dendritic length [p=0.5788] and number of branch points [p=0.4978] were comparable (control: n= 21 cells from 5 animals, β-catex3 iDCX: n= 20 cells from 8 animals).


D Representative images showing dendritic segment of the inner molecular layer of the DG in 24 - week old mice at 13dpT . Quantification of the number of spines showed increased spine density in inner molecular and outer molecular layer in 24 - week old β - catex3 iDCX mice at 13dpT [ iML p = 0 . 0047 , oML p < 0 . 0001 ] ( control n = 19 cells from 4 animals , β - catex3 iDCX : n = 20 cells from 4 animals ) . Scale bars = 1µm .

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

D Representative images showing dendritic segment of the inner molecular layer of the DG in 24-week old mice at 13dpT. Quantification of the number of spines showed increased spine density in inner molecular and outer molecular layer in 24-week old β-catex3 iDCX mice at 13dpT [iML p=0.0047, oML p<0.0001] (control n=19 cells from 4 animals, β-catex3 iDCX: n= 20 cells from 4 animals). Scale bars = 1µm.


E Representative images of recombined ( GFP + , green ) neurons in 24 - week old animals at 13dpT co - expressing Prox1 ( grey ) as a marker for neuronal fate and the stage specific markers DCX ( red ) for immature neurons and Calbindin ( red ) for mature neurons . Arrows and arrowheads indicate marker negative and marker positive cells , respectively . Scale bar = 10µm . F Quantification of marker expression . The fraction of Calbindin expressing cells was increased in the recombined population in β - catex3 iDCX at 3dpT [ p = 0 . 0081 ] and 13dpT [ p = 0 . 0259 ] ( 3dpT : control : n = 4 animals , β - catex3 iDCX : n = 9 animals , 13dpT : control : n = 8 animals , β - catex3 iDCX : n = 10 animals ) .

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

E Representative images of recombined (GFP+, green) neurons in 24-week old animals at 13dpT co-expressing Prox1 (grey) as a marker for neuronal fate and the stage specific markers DCX (red) for immature neurons and Calbindin (red) for mature neurons. Arrows and arrowheads indicate marker negative and marker positive cells, respectively. Scale bar= 10µm. F Quantification of marker expression. The fraction of Calbindin expressing cells was increased in the recombined population in β-catex3 iDCX at 3dpT [p=0.0081] and 13dpT [p=0.0259] (3dpT: control: n= 4 animals, β-catex3 iDCX: n= 9 animals, 13dpT: control: n= 8 animals, β-catex3 iDCX: n= 10 animals).


B Representative images of recombined ( GFP + , green ) BrdU + ( grey ) neurons in 24 - week old control and β - catex3 iDCX animals at 13dpT expressing the stage specific markers DCX ( red ) for immature neurons and Calbindin ( red ) for mature neurons . Arrows and arrowheads indicate marker negative and marker positive cells , respectively . Scale bar = 10µm . C Quantification of DCX and Calbindin expression in BrdU + recombined cells . At 13dpT , labeled neurons in β - catex3 iDCX animals show a shift towards a more mature marker profile [ DCX p = 0 . 0238 , Calbindin p = 0 . 0714 ] ( 3dpT : control : n = 4 animals , β - catex3 iDCX : n = 8 animals ; 13dpT control : n = 5 animals , β - catex3 iDCX : n = 5 animals ) .

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

B Representative images of recombined (GFP+, green) BrdU+ (grey) neurons in 24-week old control and β-catex3 iDCX animals at 13dpT expressing the stage specific markers DCX (red) for immature neurons and Calbindin (red) for mature neurons. Arrows and arrowheads indicate marker negative and marker positive cells, respectively. Scale bar= 10µm. C Quantification of DCX and Calbindin expression in BrdU+ recombined cells. At 13dpT, labeled neurons in β-catex3 iDCX animals show a shift towards a more mature marker profile [DCX p=0.0238, Calbindin p=0.0714] (3dpT: control: n= 4 animals, β-catex3 iDCX: n= 8 animals; 13dpT control: n= 5 animals, β-catex3 iDCX: n= 5 animals).


D Representative reconstructions of RFP - birthdated control and β - catex3 iDCX neurons in 24 - week old mice at 3dpT . Scale bar = 20µm .

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

D Representative reconstructions of RFP-birthdated control and β-catex3 iDCX neurons in 24-week old mice at 3dpT. Scale bar= 20µm.


E Quantification of dendritic length [ p = 0 . 0007 ] , branch points [ p = 0 . 3909 ] , and Sholl analysis [ p = 0 . 0001 ] showed increased complexity of birthdated β - catex3 iDCX cells at 3dpT ( control : n = 20 cells from 4 animals , β - catex3 iDCX : n = 19 cells from 5 animals ) .

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

E Quantification of dendritic length [p=0.0007], branch points [p=0.3909], and Sholl analysis [p=0.0001] showed increased complexity of birthdated β-catex3 iDCX cells at 3dpT (control: n= 20 cells from 4 animals, β-catex3 iDCX: n= 19 cells from 5 animals).


F Representative reconstructions of RFP - birthdated control and β - catex3 iDCX neurons in 24 - week old mice at 13dpT . Scale bar = 20µm .

F Representative reconstructions of RFP-birthdated control and β-catex3 iDCX neurons in 24-week old mice at 13dpT. Scale bar= 20µm.


G Quantification of dendritic length [ p = 0 . 1857 ] , branch points [ p = 0 . 1331 ] , and Sholl analysis [ p = 0 . 1501 ] showed no difference between β - catex3 iDCX and control cells at 13dpT ( control : n = 18 cells from 4 animals , β - catex3 iDCX : n = 20 cells from 4 animals )

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

G Quantification of dendritic length [p=0.1857], branch points [p=0.1331], and Sholl analysis [p=0.1501] showed no difference between β-catex3 iDCX and control cells at 13dpT (control: n= 18 cells from 4 animals, β-catex3 iDCX: n= 20 cells from 4 animals)


Luciferase - based imaging of drug response in Vegfr3Luc ; Tyr : CreERT2 ; BrafV600E ; Ptenflox / flox mice . Panels labeled as " basal " and " induced " correspond to the bioluminescence of animals prior and 5 weeks after administration of 4OH - tamoxifen ( 5 mM , topical administration , 3 consecutive days ) for the induction of melanomas . ( Right panels ) Treatment with anti - PD - L1 antibody ( αPD - L1 ; clone 10F . 9G2 , 3 weeks ) or the corresponding control IgG ( 200 ug / dose , twice per week , 3 weeks ) ; vemurafenib ( Vem , 50 mg / Kg , oral once per day , 3 weeks ) or BO - 110 ( BO , 0 . 8 mg / kg , twice per week , 3 weeks ) . Scale : p / s / cm2 / sr ( x106 ) .

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

Luciferase-based imaging of drug response in Vegfr3Luc;Tyr:CreERT2;BrafV600E; Ptenflox/flox mice. Panels labeled as "basal" and "induced" correspond to the bioluminescence of animals prior and 5 weeks after administration of 4OH-tamoxifen (5 mM, topical administration, 3 consecutive days) for the induction of melanomas. (Right panels) Treatment with anti-PD-L1 antibody (αPD-L1; clone 10F.9G2, 3 weeks) or the corresponding control IgG (200 ug/dose, twice per week, 3 weeks); vemurafenib (Vem, 50 mg/Kg, oral once per day, 3 weeks) or BO-110 (BO, 0.8 mg/kg, twice per week, 3 weeks). Scale: p/s/cm2/sr (x106).


Treatment with BO - 110 of human patient - derived xenografts ( PDX ) implanted in Vegfr3Luc nu / nu . 42 days after implantation ( when systemic luciferase was detected ) , animals were randomized for treatment with vehicle ( V ) or with 0 . 8 mg / kg BO - 110 ( BO , twice per week ) , and luciferase emission was acquired at the indicated times . Scale , p / s / cm2 / sr ( x106 ) .

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

Treatment with BO-110 of human patient-derived xenografts (PDX) implanted in Vegfr3Luc nu/nu. 42 days after implantation (when systemic luciferase was detected), animals were randomized for treatment with vehicle (V) or with 0.8 mg/kg BO-110 (BO, twice per week), and luciferase emission was acquired at the indicated times. Scale, p/s/cm2/sr (x106).


qRT - PCR analysis of relative mRNA levels of MDA5 16 h after treatment of HLEC with 0 . 5 ug / ml BO - 110 ( BO ) , 10 µM vemurafenib ( Vem ) , or vehicle control ( V ) . Data correspond to the mean ± SD of 3 biological replicates . Statistical significance was determined by t - test . qRT - PCR analysis of relative mRNA levels of VEGFR3 16 h after treatment of HLEC with 0 . 5 or 1 ug / ml BO - 110 ( VO ) , 10 µM vemurafenib ( Vem ) , or the corresponding vehicle control ( V ) . Data correspond to the mean ± SD of 3 biological replicates . Statistical significance was determined by ANOVA .

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

qRT-PCR analysis of relative mRNA levels of MDA5 16 h after treatment of HLEC with 0.5 ug/ml BO-110 (BO), 10 µM vemurafenib (Vem), or vehicle control (V). Data correspond to the mean ± SD of 3 biological replicates. Statistical significance was determined by t-test.   qRT-PCR analysis of relative mRNA levels of VEGFR3 16 h after treatment of HLEC with 0.5 or 1 ug/ml BO-110 (VO), 10 µM vemurafenib (Vem), or the corresponding vehicle control (V). Data correspond to the mean ± SD of 3 biological replicates. Statistical significance was determined by ANOVA.


Luciferase signal driven by FLT4 ( VEGFR3 ) - promoter transduced into HLEC treated with vehicle ( v ) or BO - 110 ( BO ) at the indicated doses ( ug / ml ) as indicated in methods . Results were normalized to vehicle control . N = 4 biological replicates . Error bars correspond to mean ± SD . Statistical significance was determined by ANOVA .

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

Luciferase signal driven by FLT4 (VEGFR3)-promoter transduced into HLEC treated with vehicle (v) or BO-110 (BO) at the indicated doses (ug/ml) as indicated in methods. Results were normalized to vehicle control. N=4 biological replicates. Error bars correspond to mean ± SD. Statistical significance was determined by ANOVA.


Analysis of apoptotic cells at the indicated time points . HLEC cells were treated with vehicle ( V ) or 0 . 5 µg / ml BO - 110 ( BO ) for the indicated time points . Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods . Data correspond to the mean ± SD of 3 experiments . Statistical significance was determined by t - test .

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Analysis of apoptotic cells at the indicated time points. HLEC cells were treated with vehicle (V) or 0.5 µg/ml BO-110 (BO) for the indicated time points. Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.


Relative mRNA levels of VEGFC and VEGFD in the indicated melanoma cell lines 8 h after treatment with vehicle ( V ) or 0 . 5 µg / ml BO - 110 ( BO ) , as determined by qRT - PCR . Data correspond to the mean ± SD of 3 experiments . Statistical significance was determined by t - test .

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Relative mRNA levels of VEGFC and VEGFD in the indicated melanoma cell lines 8 h after treatment with vehicle (V) or 0.5 µg/ml BO-110 (BO), as determined by qRT-PCR. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.


Inhibitory effect of the indicated doses of BO - 110 ( in µg / ml ) or vehicle ( V ) on MDK mRNA expression determined by qRT - PCR in SK - Mel - 147 ( 16 h after treatment ) . Data correspond to average mRNA levels in three experiments with technical replicates normalized to vehicle control ± SD . Statistical significance was determined by ANOVA . qRT - PCR analysis of relative mRNA levels of MDA5 16 h after treatment of SK - Mel - 147 with the indicated doses of BO - 110 ( in µg / ml ) ( BO ) . Data correspond to the mean ± SD of three experiments with three technical replicates . Statistical significance was determined by ANOVA .

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Inhibitory effect of the indicated doses of BO-110 (in µg/ml) or vehicle (V) on MDK mRNA expression determined by qRT-PCR in SK-Mel-147 (16 h after treatment). Data correspond to average mRNA levels in three experiments with technical replicates normalized to vehicle control ± SD. Statistical significance was determined by ANOVA.   qRT-PCR analysis of relative mRNA levels of MDA5 16 h after treatment of SK-Mel-147 with the indicated doses of BO-110 (in µg/ml) (BO). Data correspond to the mean ± SD of three experiments with three technical replicates. Statistical significance was determined by ANOVA.


Analysis of apoptotic cells at the indicated time points . SK - Mel - 147 cells were treated with vehicle ( V ) or 0 . 5 µg / ml BO - 110 ( BO ) for the indicated time points . Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods . Data correspond to the mean ± SD of 3 experiments . Statistical significance was determined by t - test .

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Analysis of apoptotic cells at the indicated time points. SK-Mel-147 cells were treated with vehicle (V) or 0.5 µg/ml BO-110 (BO) for the indicated time points. Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.


Immunohistochemical analysis of MDK repression ( pink staining ) in SK - Mel - 147 xenografts and PDX lesions after treatment with BO - 110 ( BO , 0 . 8 mg / kg , 2 doses / week ) . Histological staining in tumors extracted from animals treated with vehicle control ( V ) are included as a reference . Nuclei were counterstained in with hematoxylin .

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Immunohistochemical analysis of MDK repression (pink staining) in SK-Mel-147 xenografts and PDX lesions after treatment with BO-110 (BO, 0.8 mg/kg, 2 doses/week). Histological staining in tumors extracted from animals treated with vehicle control (V) are included as a reference. Nuclei were counterstained in with hematoxylin.


Type I IFN mRNA induction ( IFNA2 and IFNB1 ) in SK - Mel - 147 melanoma cells treated for 16 h with the indicated amounts of BO - 110 ( in mg / ml ) . Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control . Statistical significance was determined by ANOVA .

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Type I IFN mRNA induction (IFNA2 and IFNB1) in SK-Mel-147 melanoma cells treated for 16 h with the indicated amounts of BO-110 (in mg/ml). Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control. Statistical significance was determined by ANOVA.


Heatmap depicting expression changes in interferon - related genes ( GO : 0034340 ) in SK - Mel - 147 melanoma cells ( left panel ) and HLEC ( right panel ) treated with vehicle or 0 . 5 g / ml of BO - 110 for 10 hours . CD274 , LAG3 and PDCD1 genes were also included as a reference .

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Heatmap depicting expression changes in interferon-related genes (GO:0034340) in SK-Mel-147 melanoma cells (left panel) and HLEC (right panel) treated with vehicle or 0.5 g/ml of BO-110 for 10 hours. CD274, LAG3 and PDCD1 genes were also included as a reference.


IFNB1 mRNA induction analyzed by qPCR at the indicated times after BO - 110 treatment ( 0 . 5 µg / ml ) of SK - Mel - 147 melanoma cells or HLEC ( left and right graphs , respectively ) . Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control .

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IFNB1 mRNA induction analyzed by qPCR at the indicated times after BO-110 treatment (0.5 µg/ml) of SK-Mel-147 melanoma cells or HLEC (left and right graphs, respectively). Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control.


Quantification of the impact of BO - 110 as single agent or in the presence of the indicated blocking antibodies for type I interferon ( IFNB1 or IFNAR1 ) . Upper graphs show the effect of these agents on MDK mRNA levels in SK - Mel - 147 melanoma cells . Similar treatments were performed on HLEC for the analysis of VEGFR3 mRNA ( middle graphs ) and tube formation capacity ( lower graphs ) . Data correspond to the mean ± SD of 3 biological replicates in triplicate .

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Quantification of the impact of BO-110 as single agent or in the presence of the indicated blocking antibodies for type I interferon (IFNB1 or IFNAR1). Upper graphs show the effect of these agents on MDK mRNA levels in SK-Mel-147 melanoma cells. Similar treatments were performed on HLEC for the analysis of VEGFR3 mRNA (middle graphs) and tube formation capacity (lower graphs). Data correspond to the mean ± SD of 3 biological replicates in triplicate.


Growth of B16 melanoma xenografts in siblings of Ifnar1 + / + , Ifnar1 + / - or Ifnar1 - / - mice . Treatment started 10 days after tumor cell implantation . BO - 110 was administered at 0 . 8 mg / kg , every third day for 2 weeks . N = 6 mice per condition . Graphs show the mean tumor size ± SD at each time point . Statistical significance was determined by Two - Way ANOVA .

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Growth of B16 melanoma xenografts in siblings of Ifnar1+/+, Ifnar1+/- or Ifnar1-/- mice. Treatment started 10 days after tumor cell implantation. BO-110 was administered at 0.8 mg/kg, every third day for 2 weeks. N= 6 mice per condition. Graphs show the mean tumor size ± SD at each time point. Statistical significance was determined by Two-Way ANOVA.


Impact of BO - 110 on tumor - bearing mice implanted with syngeneic B16R2L ( 5 x 105 cells ) . When tumors have an average size on 100 mm3 , mice were randomized into two groups and treated with BO - 110 ( BO ) or vehicle ( V ) , every second day for 2 weeks . The arrow indicates the start of the treatment . Tumor size was measured with a caliper at the indicated time points . , N = 4 mice per condition . Statistical significance was determined by Two - Way - ANOVA .

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Impact of BO-110 on tumor-bearing mice implanted with syngeneic B16R2L (5 x 105 cells). When tumors have an average size on 100 mm3, mice were randomized into two groups and treated with BO-110 (BO) or vehicle (V), every second day for 2 weeks. The arrow indicates the start of the treatment. Tumor size was measured with a caliper at the indicated time points. , N= 4 mice per condition. Statistical significance was determined by Two-Way-ANOVA.


Vegfr3Luc emission in the inguinal and brachial lymph nodes of mice in experiment ( A ) . N = 4 mice per condition . Statistical significance was determined by Two - Way - ANOVA .

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Vegfr3Luc emission in the inguinal and brachial lymph nodes of mice in experiment (A). N= 4 mice per condition. Statistical significance was determined by Two-Way-ANOVA.


G . ELISA analysis of Mdk blood levels in mice in experiment ( A ) . H . ELISA analysis of mouse Ifn - β blood levels in mice in experiment ( A ) . , N = 4 mice per condition . Statistical significance was determined by Two - Way - ANOVA .

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G. ELISA analysis of Mdk blood levels in mice in experiment (A). H. ELISA analysis of mouse Ifn-β blood levels in mice in experiment (A). , N= 4 mice per condition. Statistical significance was determined by Two-Way-ANOVA.


Efficacy of BO - 110 as an adjuvant ( preventing relapse after surgical removal of the primary lesion ) . Shown are representative examples of Vegfr3Luc mice implanted with mCherry - SK - Mel - 147 and imaged for luciferase emission ( prior to and after tumor removal . Animals were left to recover from surgery ( 4 days ) and then treated for 2 weeks ( 4 dosis ) with 0 . 8 mg / Kg BO - 110 or vehicle control ( n = 8 for control and n = 10 for treatment arm ) . Scale , p / s / cm2 / sr x106 . mCherry emission from tumor cells of the animals in ( A ) . Scale , p / s / cm2 / sr x109 .

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Efficacy of BO-110 as an adjuvant (preventing relapse after surgical removal of the primary lesion). Shown are representative examples of Vegfr3Luc mice implanted with mCherry-SK-Mel-147 and imaged for luciferase emission (prior to and after tumor removal. Animals were left to recover from surgery (4 days) and then treated for 2 weeks (4 dosis) with 0.8 mg/Kg BO-110 or vehicle control (n= 8 for control and n=10 for treatment arm). Scale, p/s/cm2/sr x106.   mCherry emission from tumor cells of the animals in (A). Scale, p/s/cm2/sr x109.


Kaplan - Meier survival curves of animals treated as in ( A ) . 8 / 8 animals treated with vehicle ( V ) control had to be sacrificed for humane reasons 110 days after surgery . 9 / 10 animals in the BO - 110 arm ( BO ) remained tumor - free 8 months after stopping treatment . The grey box marks the period of treatment with BO - 110 . Statistical significance was determined by logrank test .

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Kaplan-Meier survival curves of animals treated as in (A). 8/8 animals treated with vehicle (V) control had to be sacrificed for humane reasons 110 days after surgery. 9/10 animals in the BO-110 arm (BO) remained tumor-free 8 months after stopping treatment. The grey box marks the period of treatment with BO-110. Statistical significance was determined by logrank test.


( a ) Different VPS34 complexes were immunoprecipitated ( IP ) from MEFs in the presence ( N ) or absence ( − A ) of amino acids using the indicated antibodies and assayed for kinase activity ( left , top panel ) . Inputs were immunoblotted using the antibodies indicated ( left , lower panels ) . Quantification of VPS34 activity is from 3 biological repeats ( right panel ; data shown are mean + s . d . ) . AR , autoradiography .

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(a) Different VPS34 complexes were immunoprecipitated (IP) from MEFs in the presence (N) or absence (−A) of amino acids using the indicated antibodies and assayed for kinase activity (left, top panel). Inputs were immunoblotted using the antibodies indicated (left, lower panels). Quantification of VPS34 activity is from 3 biological repeats (right panel; data shown are mean+s.d.). AR, autoradiography.


( b ) Immunoprecipitation of three VPS34 complexes , normalized for VPS34 , was performed using the indicated antibodies under nutrient - rich ( N ) or starvation ( - A ) conditions . VPS34 - binding partners were analysed by western blotting .

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(b) Immunoprecipitation of three VPS34 complexes, normalized for VPS34, was performed using the indicated antibodies under nutrient-rich (N) or starvation (-A) conditions. VPS34-binding partners were analysed by western blotting.


( c ) ATG14L - containing VPS34 complexes were immunopurified from WT , ULK1 − / − 2KD ( ULK def . ) and FIP200 − / − MEFs , and measured for lipid kinase activity as in a .

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(c) ATG14L-containing VPS34 complexes were immunopurified from WT, ULK1−/− 2KD (ULK def.) and FIP200−/− MEFs, and measured for lipid kinase activity as in a.


( d ) VPS34 was immunopurified from WT , ULK - deficient and FIP200 − / − MEFs . A kinase assay was performed as in a .

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(d) VPS34 was immunopurified from WT, ULK-deficient and FIP200−/− MEFs. A kinase assay was performed as in a.


( e ) Immunoprecipitation of Beclin - 1 - containing VPS34 complexes from WT , ULK - deficient and FIP200 − / − MEFs . A kinase assay was performed as in a .

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(e) Immunoprecipitation of Beclin-1-containing VPS34 complexes from WT, ULK-deficient and FIP200−/− MEFs. A kinase assay was performed as in a.


( f ) LC3B puncta and PtdIns ( 3 ) P levels were analysed with anti - LC3B and the biotin - 2XFYVE domain probe . Representative immunofluorescence images of LC3B and 2XFYVE domain binding are shown ( scale bars , 10 μm ) . ( g ) Quantification of LC3B puncta from f . Details of quantification are provided in the Methods ; Data shown in g - i are mean + s . d . from a minimum of 6 unique fields of view from a representative experiment ( see statistical source data in Supplementary Table S1 ) . ( h ) Total PtdIns ( 3 ) P was quantified from the experiment in f . Error bars were calculated as in g . ( i ) Quantification of PtdIns ( 3 ) P that co - localizes with LC3B on amino - acid withdrawal from the experiment described in f .

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(f) LC3B puncta and PtdIns(3)P levels were analysed with anti-LC3B and the biotin-2XFYVE domain probe. Representative immunofluorescence images of LC3B and 2XFYVE domain binding are shown (scale bars, 10 μm). (g) Quantification of LC3B puncta from f. Details of quantification are provided in the Methods; Data shown in g-i are mean+s.d. from a minimum of 6 unique fields of view from a representative experiment (see statistical source data in Supplementary Table S1). (h) Total PtdIns(3)P was quantified from the experiment in f. Error bars were calculated as in g. (i) Quantification of PtdIns(3)P that co-localizes with LC3B on amino-acid withdrawal from the experiment described in f.


( j ) VPS34 activity with or without ULK1 overexpression was assayed . A representative experiment of four repeats is shown .

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(j) VPS34 activity with or without ULK1 overexpression was assayed. A representative experiment of four repeats is shown.


( a ) HEK293 cells were transfected with ATG14L , VPS34 and Beclin - 1 . ATG14L - containing VPS34 complexes were immunopurified and subjected to an in vitro ULK1 kinase assay in the presence of γ - 32P [ ATP ] . Bound ATG14L complexes and soluble ULK1 were separated and phosphorylation was detected by autoradiography ( AR , left panels ) . Western blotting was performed ( right panels ) . Results are representative of two unique experiments .

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(a) HEK293 cells were transfected with ATG14L, VPS34 and Beclin-1. ATG14L-containing VPS34 complexes were immunopurified and subjected to an in vitro ULK1 kinase assay in the presence of γ-32P[ATP]. Bound ATG14L complexes and soluble ULK1 were separated and phosphorylation was detected by autoradiography (AR, left panels). Western blotting was performed (right panels). Results are representative of two unique experiments.


( b ) Full - length murine GST - Beclin - 1 and various truncations ( as labelled ) were subjected to an in vitro HA - ULK1 kinase assay . GST - Beclin - 1 6 ( S / T ) A has serine / threonine residues 4 , 7 , 10 , 14 , 29 and 42 mutated to alanine . ULK1 inputs were determined by western blotting , Beclin - 1 inputs by Coomassie ( Coom ) and target phosphorylation by autoradiography .

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(b) Full-length murine GST-Beclin-1 and various truncations (as labelled) were subjected to an in vitro HA-ULK1 kinase assay. GST-Beclin-1 6(S/T)A has serine/threonine residues 4, 7, 10, 14, 29 and 42 mutated to alanine. ULK1 inputs were determined by western blotting, Beclin-1 inputs by Coomassie (Coom) and target phosphorylation by autoradiography.


( c ) GST - Beclin - 1 ( 1 - 85 ) was subjected to an in vitro ULK1 kinase reaction and analysed by mass spectrometry . Ser 14 , Ser 15 in humans ( outlined ) , is the main phosphorylation site identified ( for mass spectrometry data , see Supplementary Fig . S2b , c ) . Mass spectrometry was performed on a single experiment .

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(c) GST-Beclin-1 (1-85) was subjected to an in vitro ULK1 kinase reaction and analysed by mass spectrometry. Ser 14, Ser 15 in humans (outlined), is the main phosphorylation site identified (for mass spectrometry data, see Supplementary Fig. S2b,c). Mass spectrometry was performed on a single experiment.


( d ) Beclin - 1 Ser 14 is the main in vitro ULK1 phosphorylation site . Beclin - 1 WT , and S4A and S14A mutants were subjected to an in vitro ULK1 kinase assay . The reaction was developed by autoradiography and stained for Beclin - 1 input levels by Coomassie blue . Results are representative of two unique experiments .

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(d) Beclin-1 Ser 14 is the main in vitro ULK1 phosphorylation site. Beclin-1 WT, and S4A and S14A mutants were subjected to an in vitro ULK1 kinase assay. The reaction was developed by autoradiography and stained for Beclin-1 input levels by Coomassie blue. Results are representative of two unique experiments.


( e ) HEK293 cells were transfected with the indicated plasmids under nutrient - rich conditions . Beclin - 1 was immunoprecipitated and immunoblotted with pBeclin - 1 ( Ser 14 ) , or anti - Beclin - 1 as a loading control . ULK1 inputs are included below immunoprecipitated samples .

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(e) HEK293 cells were transfected with the indicated plasmids under nutrient-rich conditions. Beclin-1 was immunoprecipitated and immunoblotted with pBeclin-1(Ser 14), or anti-Beclin-1 as a loading control. ULK1 inputs are included below immunoprecipitated samples.


( f ) Purified GST - Beclin - 1 ( 1 - 85 ) was subjected to in vitro phosphorylation by GST - ULK1 ( top panel ) and GST - ULK2 ( bottom panel ) . Reactions were immunoblotted with the indicated antibodies .

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(f) Purified GST-Beclin-1 (1-85) was subjected to in vitro phosphorylation by GST-ULK1 (top panel) and GST-ULK2 (bottom panel). Reactions were immunoblotted with the indicated antibodies.


( g ) HEK293 cells were transfected with ATG14L , VPS34 and Beclin - 1 and grown under nutrient - rich conditions . ATG14L - containing VPS34 complexes were immunoprecipitated and lipid kinase activity was assayed as described in Fig . 1j . Inputs were immunoblotted with the indicated antibodies . Representative of four unique experiments .

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(g) HEK293 cells were transfected with ATG14L, VPS34 and Beclin-1 and grown under nutrient-rich conditions. ATG14L-containing VPS34 complexes were immunoprecipitated and lipid kinase activity was assayed as described in Fig. 1j. Inputs were immunoblotted with the indicated antibodies. Representative of four unique experiments.


( h ) Stable lines containing Beclin - 1 ( WT or S14A ) were used for Beclin - 1 immunoprecipitation . Binding partners were determined by SDS - PAGE analysis and western blot using the indicated antibodies . Uncropped images of blots are shown in Supplementary Fig . S4 .

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(h) Stable lines containing Beclin-1(WT or S14A) were used for Beclin-1 immunoprecipitation. Binding partners were determined by SDS-PAGE analysis and western blot using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S4.


( a ) WT MEFs were cultured with or without amino acids . ATG14L - associated Beclin - 1 was immunoprecipitated and treated with lambda phosphatase treatment ( PPase ) as indicated . Western blotting ( immunoblotting , IB ) was performed with the indicated antibodies . Beclin - 1 Ser 14 phosphorylation was quantified ( shown under top panel ) and normalized to total Beclin - 1 .

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

(a) WT MEFs were cultured with or without amino acids. ATG14L-associated Beclin-1 was immunoprecipitated and treated with lambda phosphatase treatment (PPase) as indicated. Western blotting (immunoblotting, IB) was performed with the indicated antibodies. Beclin-1 Ser 14 phosphorylation was quantified (shown under top panel) and normalized to total Beclin-1.


( b ) WT MEFs were starved for the indicated times . Beclin - 1 was immunopurified by ATG14L immunoprecipitation and immunoblotted as indicated ( top panels ) . Whole - cell lysates were immunoblotted with pULK1 S757 ( mTORC1 - target site ) , pS6K and ULK1 antibodies ( bottom panels ) . Two unique experiments were performed .

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

(b) WT MEFs were starved for the indicated times. Beclin-1 was immunopurified by ATG14L immunoprecipitation and immunoblotted as indicated (top panels). Whole-cell lysates were immunoblotted with pULK1 S757 (mTORC1-target site), pS6K and ULK1 antibodies (bottom panels). Two unique experiments were performed.


( c ) WT or FIP200 − / − MEFs were incubated under nutrient - rich , amino - acid - deprived or Torin - 1 ( + T , an mTOR inhibitor ) conditions . Beclin - 1 was purified and immunoblotted as in Fig . 3b .

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

(c) WT or FIP200−/− MEFs were incubated under nutrient-rich, amino-acid-deprived or Torin-1 (+T, an mTOR inhibitor) conditions. Beclin-1 was purified and immunoblotted as in Fig. 3b.


( d ) WT or ULK - deficient MEFs were incubated with or without amino acids . Beclin - 1 was purified and immunoblotted as in a . Two unique experiments were performed . Uncropped images of blots are shown in Supplementary Fig . S4 .

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

(d) WT or ULK-deficient MEFs were incubated with or without amino acids. Beclin-1 was purified and immunoblotted as in a. Two unique experiments were performed. Uncropped images of blots are shown in Supplementary Fig. S4.


( a ) Beclin - 1 alone ( lanes 1 - 4 ) or Beclin - 1 and ATG14L ( lanes 5 - 8 ) were overexpressed in HEK293 cells . Beclin - 1 was purified either by direct immunoprecipitation ( lanes 1 - 4 ) or by ATG14L immunoprecipitation ( lanes 5 - 8 ) . Immunoprecipitated samples were subjected to an in vitro ULK1 kinase assay with increasing amounts of ULK1 . Reactions were immunoblotted with the indicated antibodies . Black line denotes discontinuous lanes from the same gel . Two unique experiments were performed .

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

(a) Beclin-1 alone (lanes 1-4) or Beclin-1 and ATG14L (lanes 5-8) were overexpressed in HEK293 cells. Beclin-1 was purified either by direct immunoprecipitation (lanes 1-4) or by ATG14L immunoprecipitation (lanes 5-8). Immunoprecipitated samples were subjected to an in vitro ULK1 kinase assay with increasing amounts of ULK1. Reactions were immunoblotted with the indicated antibodies. Black line denotes discontinuous lanes from the same gel. Two unique experiments were performed.


( b ) Beclin - 1 alone or bound to ATG14L was purified as described in a . Equal amounts of ULK1 were added to each complex and reactions were quenched at the indicated time points . Western blotting was performed with the indicated antibodies .

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(b) Beclin-1 alone or bound to ATG14L was purified as described in a. Equal amounts of ULK1 were added to each complex and reactions were quenched at the indicated time points. Western blotting was performed with the indicated antibodies.


( c ) An ATG14L - FLAG - 6His - inducible U2OS cell line was induced for 16 h in the presence of amino acids . Endogenous Beclin - 1 was immunoprecipitated and immunoblotted as in Fig . 3a . ATG14L input levels were detected by immunoblotting . Two unique experiments were performed .

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

(c) An ATG14L-FLAG-6His-inducible U2OS cell line was induced for 16 h in the presence of amino acids. Endogenous Beclin-1 was immunoprecipitated and immunoblotted as in Fig. 3a. ATG14L input levels were detected by immunoblotting. Two unique experiments were performed.


( d ) HEK293 cells transfected with either ATG14L or Beclin - 1 , or both , in conjunction with ULK1 , were immunoprecipitated as indicated and blotted with the indicated antibodies .

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(d) HEK293 cells transfected with either ATG14L or Beclin-1, or both, in conjunction with ULK1, were immunoprecipitated as indicated and blotted with the indicated antibodies.


( e ) HEK293 cells were transfected with Beclin - 1 and ULK1 in the presence of ATG14L WT or ATG14LΔCCD , which is defective in Beclin - 1 binding , under nutrient - rich conditions . Lysates were resolved by SDS - PAGE and blotted with the indicated antibodies .

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

(e) HEK293 cells were transfected with Beclin-1 and ULK1 in the presence of ATG14L WT or ATG14LΔCCD, which is defective in Beclin-1 binding, under nutrient-rich conditions. Lysates were resolved by SDS-PAGE and blotted with the indicated antibodies.


( f ) HEK293 cells were transfected with ULK1 and Beclin - 1 in conjunction with either ATG14L WT , or one of two mutants ( ΔBATS , ΔN ) that are defective in phagophore localization . Samples were handled as in e . Uncropped images of blots are shown in Supplementary Fig . S4 . Lysates were resolved by SDS - PAGE and blotted with the indicated antibodies .

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

(f) HEK293 cells were transfected with ULK1 and Beclin-1 in conjunction with either ATG14L WT, or one of two mutants (ΔBATS,ΔN) that are defective in phagophore localization. Samples were handled as in e. Uncropped images of blots are shown in Supplementary Fig. S4. Lysates were resolved by SDS-PAGE and blotted with the indicated antibodies.


( a ) HEK293 cells were transfected with Beclin - 1 , with or without UVRAG , in conjunction with ULK1 as indicated in the presence of amino acids . Lysates were immunoblotted with the indicated antibodies . A representative experiment of three repeats is shown .

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

(a) HEK293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated in the presence of amino acids. Lysates were immunoblotted with the indicated antibodies. A representative experiment of three repeats is shown.


( b ) UVRAG bridges the interaction between Beclin - 1 and ULK1 . HEK293 cells were transfected with Beclin - 1 , with or without UVRAG , in conjunction with ULK1 as indicated . Lysates were immunoprecipitated with anti - HA ( Beclin - 1 ) antibody and blotted with the indicated antibodies . A representative experiment of three repeats is shown . Uncropped images of blots are shown in Supplementary Fig . S4 .

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

(b) UVRAG bridges the interaction between Beclin-1 and ULK1. HEK293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated. Lysates were immunoprecipitated with anti-HA(Beclin-1) antibody and blotted with the indicated antibodies. A representative experiment of three repeats is shown. Uncropped images of blots are shown in Supplementary Fig. S4.


( a ) HEK293 cells were transfected with the indicated plasmids . Cells were grown in the presence of amino acids and treated with NH4Cl to block autophagic turnover where indicated . Lysates were immunoblotted with the indicated antibodies .

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(a) HEK293 cells were transfected with the indicated plasmids. Cells were grown in the presence of amino acids and treated with NH4Cl to block autophagic turnover where indicated. Lysates were immunoblotted with the indicated antibodies.


( b ) HEK293 cells transfected with Beclin - 1 ATG14L were grown in the presence or absence of amino acids . Lysates were immunoblotted with the indicated antibodies ( left panel ) and quantified by densitometry ( right panel ) . Data represent mean + s . d . of three unique experiments .

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

(b) HEK293 cells transfected with Beclin-1 ATG14L were grown in the presence or absence of amino acids. Lysates were immunoblotted with the indicated antibodies (left panel) and quantified by densitometry (right panel). Data represent mean+s.d. of three unique experiments.


( c ) Beclin - 1 - shRNA reconstituted lines ( WT or mutant ) and controls ( scramble shRNA or Beclin - 1 shRNA ) were grown with or without amino acids and assessed for autophagy ( left panel ) and Beclin - 1 levels ( right panel ) .

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

(c) Beclin-1-shRNA reconstituted lines (WT or mutant) and controls (scramble shRNA or Beclin-1 shRNA) were grown with or without amino acids and assessed for autophagy (left panel) and Beclin-1 levels (right panel).


( d ) Autophagosome ( denoted by arrowheads ) generation on amino - acid withdrawal was analysed by electron microscopy . Cell lines and conditions from c were used and representative images from the indicated amino - acid - starved lines are shown ; scale bars , 0 . 4 μm .

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

(d) Autophagosome (denoted by arrowheads) generation on amino-acid withdrawal was analysed by electron microscopy. Cell lines and conditions from c were used and representative images from the indicated amino-acid-starved lines are shown; scale bars, 0.4 μm.


( d ) Autophagosome ( denoted by arrowheads ) generation on amino - acid withdrawal was analysed by electron microscopy . Cell lines and conditions from c were used and representative images from the indicated amino - acid - starved lines are shown ; scale bars , 0 . 4 μm . ( e ) Quantification of d . Fold induction was determined by arbitrarily making the nutrient - rich condition 1 ( solid bars ) for each line . Error bars represent the s . d . of the mean value over an average of 20 fields of view within a representative experiment . NS , not significant .

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

(d) Autophagosome (denoted by arrowheads) generation on amino-acid withdrawal was analysed by electron microscopy. Cell lines and conditions from c were used and representative images from the indicated amino-acid-starved lines are shown; scale bars, 0.4 μm. (e) Quantification of d. Fold induction was determined by arbitrarily making the nutrient-rich condition 1 (solid bars) for each line. Error bars represent the s.d. of the mean value over an average of 20 fields of view within a representative experiment. NS, not significant.


( f ) HA - Beclin - 1 WT or S14D was transiently expressed in FIP200 − / − MEFs grown under nutrient - rich conditions . Indirect immunofluorescence was performed using antibodies against endogenous LC3B and HA - Beclin - 1 . Scale bars , 20 μm . ( g ) Quantification of LC3B puncta from confocal in f . In the HA - Beclin - 1 - or HA - Beclin - 1 - S14D - transfected samples , only the HA - positive cells were counted for LC3B puncta . Error bars were processed as in e . Mean value shown ; P values determined by Student ' s t - test using 10 unique fields of view from f . Uncropped images of blots are shown in Supplementary Fig . S4 .

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

(f) HA-Beclin-1 WT or S14D was transiently expressed in FIP200−/− MEFs grown under nutrient-rich conditions. Indirect immunofluorescence was performed using antibodies against endogenous LC3B and HA-Beclin-1. Scale bars, 20 μm. (g) Quantification of LC3B puncta from confocal in f. In the HA-Beclin-1- or HA-Beclin-1-S14D-transfected samples, only the HA-positive cells were counted for LC3B puncta. Error bars were processed as in e. Mean value shown; P values determined by Student's t-test using 10 unique fields of view from f. Uncropped images of blots are shown in Supplementary Fig. S4.


( a ) Bec - 1C . elegans were reconstituted with either WT or mutant GFP - BEC - 1 . Stable worm lines with Bec - 1 rescue were obtained and embryos were stained with anti - PGL - 1 antibody . Arrowheads indicate normal PGL - 1 staining in germline cells . ( b ) Quantification of PGL - 1 puncta outside germline cells ( left panel ) . Error bars represents s . d . between 3 unique embryos in a representative experiment . Reconstituted Bec - 1 ( WT and mutant ) levels in Bec − / − stable worms were compared by western blotting . Mean value presented .

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

(a) Bec-1C. elegans were reconstituted with either WT or mutant GFP-BEC-1. Stable worm lines with Bec-1 rescue were obtained and embryos were stained with anti- PGL-1 antibody. Arrowheads indicate normal PGL-1 staining in germline cells. (b) Quantification of PGL-1 puncta outside germline cells (left panel). Error bars represents s.d. between 3 unique embryos in a representative experiment. Reconstituted Bec-1 (WT and mutant) levels in Bec−/− stable worms were compared by western blotting. Mean value presented.


( c ) Spectrum of defects in PGL granule degradation in bec - 1 mutant rescue embryos . Mutant embryos exhibited either high levels of diffuse PGL - 1 staining ( middle - left panel ; one - third of the embryos ) , or large punctuate PGL - 1 structures in somatic cells ( bottom - left panel ; two - thirds of the embryos ) . Both diffuse or punctuate PGL - 1 staining in somatic cells have been described in autophagy - deficient embryos .

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

(c) Spectrum of defects in PGL granule degradation in bec-1 mutant rescue embryos. Mutant embryos exhibited either high levels of diffuse PGL-1 staining (middle-left panel; one-third of the embryos), or large punctuate PGL-1 structures in somatic cells (bottom-left panel; two-thirds of the embryos). Both diffuse or punctuate PGL-1 staining in somatic cells have been described in autophagy-deficient embryos.


( d ) Embryos from the lines described in a were labelled with anti - LGG - 1 , along with WT and unc - 51 worms . Representative embryos at ∼ 100 cell stage are shown . ( e ) Quantification of LGG - 1 per embryo from labelling in d . Error bars generated as in b . Mean value presented . Uncropped images of blots are shown in Supplementary Fig . S4 . Scale bar , 10 μm .

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

(d) Embryos from the lines described in a were labelled with anti-LGG-1, along with WT and unc-51 worms. Representative embryos at ∼ 100 cell stage are shown. (e) Quantification of LGG-1 per embryo from labelling in d. Error bars generated as in b. Mean value presented. Uncropped images of blots are shown in Supplementary Fig. S4. Scale bar, 10 μm.


( F ) Single tube qPCR experiments showing the down - regulation of the synaptic genes NLGN3 , SLC6A , TRIM9 , SYT1 , SYNGR1 , and SNAP91 in ALSC9orf72 MN ( Welch ' s t - test ) . n = 3 independent cultures for each hiPSC line . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent SEM . Arrow indicates the structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

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

(F) Single tube qPCR experiments showing the down-regulation of the synaptic genes NLGN3, SLC6A, TRIM9, SYT1, SYNGR1, and SNAP91 in ALSC9orf72 MN (Welch's t-test). n=3 independent cultures for each hiPSC line. Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( G - H ) Time course analysis of nuclear pCREBS133 levels and MN survival in ALSC9orf72 and Healthy cultures ( two - way ANOVA ) . n = 3 independent cultures for each hiPSC line ( for each time point ) . Scale bar : 25 µm . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent SEM . Arrow indicates the structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

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

(G-H) Time course analysis of nuclear pCREBS133 levels and MN survival in ALSC9orf72 and Healthy cultures (two-way ANOVA). n=3 independent cultures for each hiPSC line (for each time point). Scale bar: 25 µm. Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( A ) Both K + channel blockers exert a neuroprotective effect in ALSC9orf72 cultures by increasing the neurite length of mutant MN ( one - way ANOVA followed by Dunnett ' s multiple comparison test ) . n = 3 independent treatments with each hiPSC line . Scale bar : 50 µm Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent SEM . Arrow indicates the structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

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

(A) Both K+ channel blockers exert a neuroprotective effect in ALSC9orf72 cultures by increasing the neurite length of mutant MN (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments with each hiPSC line. Scale bar: 50 µm Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( C ) Apamin and XE991 decrease the size of aberrant aggresomes , confirming enhanced autophagy degradation ( one - way ANOVA followed by Dunnett ' s multiple comparison test ) . n = 3 independent treatments with the ALSC9orf72 I line . Scale bar : 5 Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent SEM . Arrow indicates the structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

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

(C) Apamin and XE991 decrease the size of aberrant aggresomes, confirming enhanced autophagy degradation (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments with the ALSC9orf72 I line. Scale bar: 5 Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( E ) The load of aggregated SQSTM1 / p62 is also reduced by the treatments in ALSC9orf72 cultures ( one - way ANOVA followed by Dunnett ' s multiple comparison test ) . n = 3 independent treatments for each cell line . Scale bar : 20 µm . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent SEM . Arrow indicates the structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

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

(E) The load of aggregated SQSTM1/p62 is also reduced by the treatments in ALSC9orf72 cultures (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments for each cell line. Scale bar: 20 µm. Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( F ) Treatment with the K + channel blockers reduces the number of GGGGCC toxic RNA foci in ALSC9orf72 MN . n = 3 independent treatments with each hiPSC line ( one - way ANOVA followed by Dunnett ' s multiple comparison test ) . Scale bar : 5 µm . Dashed line represents the cell soma . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent SEM . Arrow indicates the structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

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

(F) Treatment with the K+ channel blockers reduces the number of GGGGCC toxic RNA foci in ALSC9orf72 MN. n=3 independent treatments with each hiPSC line (one-way ANOVA followed by Dunnett's multiple comparison test). Scale bar: 5 µm. Dashed line represents the cell soma. Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( E ) Apamin and XE991 increase the levels of pCREBS133 in all the ALSC9orf72 lines ( one - way ANOVA followed by Dunnett ' s multiple comparison test ) . n = 3 independent treatments with each hiPSC line . Scale bar : 10 µm . Data information : * p < 0 . 05 ; * * p < 0 . 01 . Error bars represent SEM . Exact p - values are reported in Appendix Table S1 .

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

(E) Apamin and XE991 increase the levels of pCREBS133 in all the ALSC9orf72 lines (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments with each hiPSC line. Scale bar: 10 µm. Data information: *p<0.05; **p<0.01. Error bars represent SEM. Exact p-values are reported in Appendix Table S1.


( F ) ALSC9orf72 MN showed an increased number of Homer1b / c + synaptic contacts after treatment with the K + channel blockers , which in contrast had no significant effect on Healthy MN . n = 3 independent treatments with each hiPSC line ( two - way ANOVA ) . Scale bar : 5 µm Data information : * p < 0 . 05 ; * * p < 0 . 01 . Error bars represent SEM . Exact p - values are reported in Appendix Table S1 .

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

(F) ALSC9orf72 MN showed an increased number of Homer1b/c+ synaptic contacts after treatment with the K+ channel blockers, which in contrast had no significant effect on Healthy MN. n=3 independent treatments with each hiPSC line (two-way ANOVA). Scale bar: 5 µm Data information: *p<0.05; **p<0.01. Error bars represent SEM. Exact p-values are reported in Appendix Table S1.


( K ) Synapse - and neuronal processes - related proteins significantly up - regulated by both treatments .

(K) Synapse- and neuronal processes-related proteins significantly up-regulated by both treatments.


The response of cytosolic and mitochondrial matrix - localized roGFP2 - Tsa2ΔCR ( C ) probes to boli of exogenous H2O2 at the indicated concentrations . Probes were expressed in wild - type BY4742 yeast cells grown to exponential phase in SGal ( - Leu ) medium .

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

The response of cytosolic and mitochondrial matrix-localized roGFP2-Tsa2ΔCR (C) probes to boli of exogenous H2O2 at the indicated concentrations. Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.


The response of cytosolic and mitochondrial matrix - localized Grx1 - roGFP2 ( D ) probes to boli of exogenous H2O2 at the indicated concentrations . Probes were expressed in wild - type BY4742 yeast cells grown to exponential phase in SGal ( - Leu ) medium .

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

The response of cytosolic and mitochondrial matrix-localized Grx1-roGFP2 (D) probes to boli of exogenous H2O2 at the indicated concentrations. Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.


The response of cytosolic and mitochondrial matrix - localized roGFP2 - Tsa2ΔCR ( E ) probes to 10 µM Complex antimycin A ( a complex III inhibitor ) . Probes were expressed in wild - type BY4742 yeast cells grown to exponential phase in SGal ( - Leu ) medium .

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

The response of cytosolic and mitochondrial matrix-localized roGFP2-Tsa2ΔCR (E) probes to 10 µM Complex antimycin A (a complex III inhibitor). Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.


The response of cytosolic and mitochondrial matrix - localized Grx1 - roGFP2 ( F ) probes to 10 µM Complex antimycin A ( a complex III inhibitor ) . Probes were expressed in wild - type BY4742 yeast cells grown to exponential phase in SGal ( - Leu ) medium .

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

The response of cytosolic and mitochondrial matrix-localized Grx1-roGFP2 (F) probes to 10 µM Complex antimycin A (a complex III inhibitor). Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.


( G ) The response of mitochondrial matrix - localized ( left panel ) and cytosolic ( right panel ) roGFP2 - Tsa2ΔCR probes expressed in BY4742 wild - type and Δtsa1Δtsa2 cells , to the addition of exogenous H2O2 at the indicated concentrations . Cells were grown to exponential phase in SGal ( - Leu ) medium . The lighter colored curves are controls , showing the probe response upon the addition of water .

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

(G) The response of mitochondrial matrix-localized (left panel) and cytosolic (right panel) roGFP2-Tsa2ΔCR probes expressed in BY4742 wild-type and Δtsa1Δtsa2 cells, to the addition of exogenous H2O2 at the indicated concentrations. Cells were grown to exponential phase in SGal (-Leu) medium. The lighter colored curves are controls, showing the probe response upon the addition of water.


( H ) The response of mitochondrial matrix - localized ( left panel ) and cytosolic ( right panel ) roGFP2 - Tsa2ΔCR probes , expressed in wild - type and Δtsa1Δtsa2 cells , to the addition of 10 µM antimycin A . Cells were grown to exponential phase in SGal ( - Leu ) medium . The lighter colored curves are controls showing the probe responses upon addition of 0 . 1 % ( v / v ) ethanol .

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

(H) The response of mitochondrial matrix-localized (left panel) and cytosolic (right panel) roGFP2-Tsa2ΔCR probes, expressed in wild-type and Δtsa1Δtsa2 cells, to the addition of 10 µM antimycin A. Cells were grown to exponential phase in SGal (-Leu) medium. The lighter colored curves are controls showing the probe responses upon addition of 0.1% (v/v) ethanol.


( I ) The response of a mitochondrial matrix - localized roGFP2 - Tsa2ΔCR probe , expressed in wild - type and Δpor1 cells , to boli at exogenous H2O2 at the indicated concentrations . Cells were grown to exponential phase in SGal ( - Leu ) medium . Lighter colored curves are controls showing the probe response upon the addition of water .

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

(I) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe, expressed in wild-type and Δpor1 cells, to boli at exogenous H2O2 at the indicated concentrations. Cells were grown to exponential phase in SGal (-Leu) medium. Lighter colored curves are controls showing the probe response upon the addition of water.


( J ) The response of a mitochondrial matrix - localized roGFP2 - Tsa2ΔCR probe , expressed in wild - type and Δpor1 cells to the addition of 10 µM antimycin A . Cells were grown to exponential phase in SGal ( - Leu ) medium . Lighter colored curves are controls showing the probe response upon the addition of 0 . 1 % ( v / v ) ethanol .

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

(J) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe, expressed in wild-type and Δpor1 cells to the addition of 10 µM antimycin A. Cells were grown to exponential phase in SGal (-Leu) medium. Lighter colored curves are controls showing the probe response upon the addition of 0.1% (v/v) ethanol.


( A ) The response of a mitochondrial matrix - localized roGFP2 - Tsa2ΔCR probe , expressed in wild - type and Δprx1 , to the addition of exogenous H2O2 at the indicated concentrations . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase . Lighter colored curves are controls showing the probe response to the addition of water .

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

(A) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe, expressed in wild-type and Δprx1, to the addition of exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Lighter colored curves are controls showing the probe response to the addition of water.


( B , C ) The response of a cytosolic roGFP2 - Tsa2ΔCR probe , expressed in wild - type , Δtrx3 and Δprx1 cells , to the addition of 0 . 1 mM ( B ) or 1 mM ( C ) exogenous H2O2 . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase .

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

(B,C) The response of a cytosolic roGFP2-Tsa2ΔCR probe, expressed in wild-type, Δtrx3 and Δprx1 cells, to the addition of 0.1 mM (B) or 1 mM (C) exogenous H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase.


( D ) The response of a cytosolic roGFP2 - Tsa2ΔCR probe , in Δtrx3 cells transformed with a Trx3 plasmid and Δprx1 cells transformed with a Prx1 plasmid , to the addition of 0 . 1 mM exogenous H2O2 . Cells were grown in SGal medium lacking the appropriate amino acids for plasmid selection and harvested at early exponential phase . The light grey curve in both panels represents the wild - type control , treated with the same concentration of exogenous H2O2 .

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

(D) The response of a cytosolic roGFP2-Tsa2ΔCR probe, in Δtrx3 cells transformed with a Trx3 plasmid and Δprx1 cells transformed with a Prx1 plasmid, to the addition of 0.1 mM exogenous H2O2. Cells were grown in SGal medium lacking the appropriate amino acids for plasmid selection and harvested at early exponential phase. The light grey curve in both panels represents the wild-type control, treated with the same concentration of exogenous H2O2.


( E ) The profile of mRNA expression of the yeast redox or redox - related enzymes in Δprx1 + empty vector cells , compared to Δprx1 + Prx1 - WT cells , both grown to an early exponential phase in SGal ( - Ura ) medium ( n = 3 biological replicates , with cells obtained from 3 independent cultures ) .

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

(E) The profile of mRNA expression of the yeast redox or redox-related enzymes in Δprx1+empty vector cells, compared to Δprx1+Prx1-WT cells, both grown to an early exponential phase in SGal (-Ura) medium (n = 3 biological replicates, with cells obtained from 3 independent cultures).


( F ) The response of a cytosolic roGFP2 - Tsa2ΔCR probe , in Δprx1 cells or Δctt1Δprx1 cells transformed with either an empty plasmid or a Prx1 - WT plasmid to the addition of 0 . 1 mM exogenous H2O2 . Cells were grown in SGal medium lacking the appropriate amino acids for selection and harvested in early exponential

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

(F) The response of a cytosolic roGFP2-Tsa2ΔCR probe, in Δprx1 cells or Δctt1Δprx1 cells transformed with either an empty plasmid or a Prx1-WT plasmid to the addition of 0.1 mM exogenous H2O2. Cells were grown in SGal medium lacking the appropriate amino acids for selection and harvested in early exponential phase


( A ) The response of a mitochondrial matrix - localized Grx1 - roGFP2 sensor expressed in wild - type , Δtsa1Δtsa2 , Δpor1 , Δtrr2 , Δtrx3 or Δprx1 cells to a bolus of exogenous H2O2 at the indicated concentrations . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase .

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

(A) The response of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in wild-type, Δtsa1Δtsa2, Δpor1, Δtrr2, Δtrx3 or Δprx1 cells to a bolus of exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase.


( B ) Left panel : The response of a mitochondrial matrix - localized Grx1 - roGFP2 sensor expressed in wild - type , Δtrr2 , Δtrx3 or Δprx1 cells to a bolus of 2 . 5 mM H2O2 . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase . Right panel : Scheme depicting Prx1 and its peroxidatic cysteine at position 91 .

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

(B) Left panel: The response of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in wild-type, Δtrr2, Δtrx3 or Δprx1 cells to a bolus of 2.5 mM H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Right panel: Scheme depicting Prx1 and its peroxidatic cysteine at position 91.


( C ) The response of a mitochondrial matrix - localized roGFP2 - Tsa2ΔCR probe to exogenous 2 . 5 mM H2O2 . The probe was expressed in wild - type cells transformed with an empty vector or in ∆ prx1 cells transformed with either an empty vector , a vector encoding wild - type Prx1 or a vector encoding Prx1 - C91A , grown in SGal ( - Leu , - Ura ) . ( D ) The response to of a mitochondrial matrix - localized Grx1 - roGFP2 sensor expressed in either wild - type cells transformed with an empty vector or in ∆ prx1 cells transformed with either an empty vector , a Prx1 - WT or a Prx1 - C91A plasmid to exogenous H2O2 at a concentration of 2 . 5 mM . Cells were grown to SGal ( - Leu , - Ura ) medium and harvested at early exponential phase .

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

(C) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe to exogenous 2.5 mM H2O2. The probe was expressed in wild-type cells transformed with an empty vector or in ∆prx1 cells transformed with either an empty vector, a vector encoding wild-type Prx1 or a vector encoding Prx1-C91A, grown in SGal (-Leu, -Ura). (D) The response to of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in either wild-type cells transformed with an empty vector or in ∆prx1 cells transformed with either an empty vector, a Prx1-WT or a Prx1-C91A plasmid to exogenous H2O2 at a concentration of 2.5 mM. Cells were grown to SGal (-Leu, -Ura) medium and harvested at early exponential phase.


( B ) ' Acute stress - washout ' assay . Δprx1 cells containing a Prx1 - WT plasmid and expressing a mitochondrial matrix - localized Grx1 - roGFP2 probe were grown to exponential phase in SGal ( - Leu , - Ura ) medium harvested and treated with either 0 , 1 , 5 , 10 , 25 or 50 mM H2O2 for 10 mins . After removal of H2O2 and resuspension in fresh buffer the response of a mitochondrial matrix - localized Grx1 - roGFP2 probe to the addition of 1 mM H2O2 was measured . The middle panel is an enlargement of the left panel . The graph in the right panel shows the steady state Grx1 - roGFP2 oxidation following H2O2 pre - treatment as well as the maximum Grx1 - roGFP2 oxidation in response to the subsequent second H2O2 treatment .

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

(B) 'Acute stress-washout' assay. Δprx1 cells containing a Prx1-WT plasmid and expressing a mitochondrial matrix-localized Grx1-roGFP2 probe were grown to exponential phase in SGal (-Leu, -Ura) medium harvested and treated with either 0, 1, 5, 10, 25 or 50 mM H2O2 for 10 mins. After removal of H2O2 and resuspension in fresh buffer the response of a mitochondrial matrix-localized Grx1-roGFP2 probe to the addition of 1 mM H2O2 was measured. The middle panel is an enlargement of the left panel. The graph in the right panel shows the steady state Grx1-roGFP2 oxidation following H2O2 pre-treatment as well as the maximum Grx1-roGFP2 oxidation in response to the subsequent second H2O2 treatment.


' Acute stress - washout ' assay . Δprx1 cells expressing a mitochondrial matrix - localized Grx1 - roGFP2 probe were grown to exponential phase in SGal ( - Leu , - Ura ) medium harvested and treated with either 0 , 1 , 5 , 10 , 25 or 50 mM H2O2 for 10 mins . ( C ) The same experiment as in ( B ) performed with Δprx1 cells transformed with a Prx1 - C91A plasmid .

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

'Acute stress-washout' assay. Δprx1 cells expressing a mitochondrial matrix-localized Grx1-roGFP2 probe were grown to exponential phase in SGal (-Leu, -Ura) medium harvested and treated with either 0, 1, 5, 10, 25 or 50 mM H2O2 for 10 mins. (C) The same experiment as in (B) performed with Δprx1 cells transformed with a Prx1-C91A plasmid.


( D ) ∆ prx1 cells transformed with a vector encoding wild - type Prx1 , a vector encoding the matrix - targeted D - aminooxidase ( su9 - DAO ) and expressing a mitochondrial matrix - localized Grx1 - roGFP2 were grown to exponential phase in SGal ( - Leu , - Ura , - His ) and then pre - treated with 0 . 15 M D - alanine for 0 , 0 . 5 , 1 . 5 hours . Afterwards , the cells were washed and the response of Grx1 - roGFP2 to the addition of 1 mM H2O2 was measured .

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(D) ∆prx1 cells transformed with a vector encoding wild-type Prx1, a vector encoding the matrix-targeted D-aminooxidase (su9-DAO) and expressing a mitochondrial matrix-localized Grx1-roGFP2 were grown to exponential phase in SGal (-Leu, -Ura, -His) and then pre-treated with 0.15 M D-alanine for 0, 0.5, 1.5 hours. Afterwards, the cells were washed and the response of Grx1-roGFP2 to the addition of 1 mM H2O2 was measured.


( E ) Left panel : the response of mitochondrial matrix - localized roGFP2 - Prx1 and roGFP2 probes expressed in wild - type cells to exogenous H2O2 at the indicated concentrations . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase . Right panel : model illustrating that hyperoxidation of the Prx1 moiety in the roGFP2 - Prx1 sensor leads to a roGFP2 - like behavior .

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(E) Left panel: the response of mitochondrial matrix-localized roGFP2-Prx1 and roGFP2 probes expressed in wild-type cells to exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Right panel: model illustrating that hyperoxidation of the Prx1 moiety in the roGFP2-Prx1 sensor leads to a roGFP2-like behavior.


( H ) Redox shift assays of Prx1 . Δprx1 cells transformed with a plasmid encoding wild - type Prx1 were grown to early exponential phase in SGal ( - Ura ) medium . Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins . Exposure to H2O2 oxidizes the single cysteine of Prx1 to a state that cannot be reduced with TCEP .

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(H) Redox shift assays of Prx1. Δprx1 cells transformed with a plasmid encoding wild-type Prx1 were grown to early exponential phase in SGal (-Ura) medium. Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins. Exposure to H2O2 oxidizes the single cysteine of Prx1 to a state that cannot be reduced with TCEP.


( J ) The response of a mitochondrial matrix - localized Grx1 - roGFP2 , expressed in wild - type cells grown to the addition of exogenous 1 mM H2O2 . Cells were grown in SGal ( - Leu ) and harvested at early exponential phase . Left panel , the pre - treatment efficiently inhibits the ability of Prx1 to transfer oxidizing equivalents to EGSH . Right panel , newly synthesized Prx1 is required to recover the transfer of oxidizing equivalents to EGSH in the mitochondrial matrix .

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(J) The response of a mitochondrial matrix-localized Grx1-roGFP2, expressed in wild-type cells grown to the addition of exogenous 1 mM H2O2. Cells were grown in SGal (-Leu) and harvested at early exponential phase. Left panel, the pre-treatment efficiently inhibits the ability of Prx1 to transfer oxidizing equivalents to EGSH. Right panel, newly synthesized Prx1 is required to recover the transfer of oxidizing equivalents to EGSH in the mitochondrial matrix.