E . HeLa were transfected with miR - 586 mimics or inhibitors as indicated . Forty - eight hours after transfection , total was extracted subjected to real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - t - ) .

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

E. HeLa cells were transfected with miR-586 mimics or inhibitors as indicated. Forty-eight hours after transfection, total RNA was extracted and subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


F . copy numbers of miR - 586 lncRNA - MIF transcript per in indicated were quantified using a quantitative real - time RT - PCR . Data shown are mean ± SD ( n = 3 ) .

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

F. The copy numbers of miR-586 and lncRNA-MIF transcript per cell in the indicated cells were quantified using a quantitative real-time RT-PCR assay. Data shown are mean ± SD (n = 3).


G . HeLa were infected with lentiviruses expressing control shRNA or lncRNA - MIF shRNA . Forty - eight hours after infection , total was subjected to real - time RT - PCR . HeLa were infected with lentiviruses expressing lncRNA - MIF or control . Forty - eight hours after infection , total was analyzed by real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

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 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

G. HeLa cells were infected with lentiviruses expressing control shRNA or lncRNA-MIF shRNA. Forty-eight hours after infection, total RNA was subjected to real-time RT-PCR analysis. HeLa cells were infected with lentiviruses expressing lncRNA-MIF or control RNA. Forty-eight hours after infection, total RNA was analyzed by real-time RT-PCR. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).


H . HeLa expressing lncRNA - MIF or control were transfected with miR - 586 mimics or NC mimics . Twenty - four hours after transfection , lysates were analyzed by Western blotting .

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

H. HeLa cells expressing lncRNA-MIF or control RNA were transfected with miR-586 mimics or NC mimics. Twenty-four hours after transfection, cell lysates were analyzed by Western blotting.


I . HeLa expressing lncRNA - MIF or control shRNA were transfected with miR - 586 inhibitor or NC inhibitor . Twenty - four hours after transfection , lysates were analyzed by Western blotting .

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

I. HeLa cells expressing lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor. Twenty-four hours after transfection, cell lysates were analyzed by Western blotting.


K . HeLa were infected with lentiviruses expressing control , lncRNA - MIF , lncRNA - MIF harboring nonsense mutation or miR - 586 binding defective lncRNA - MIF . Forty - eight hours after transfection , lysates were analyzed by Western blotting .

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

K. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, lncRNA-MIF harboring nonsense mutation or miR-586 binding defective lncRNA-MIF. Forty-eight hours after transfection, cell lysates were analyzed by Western blotting.


A . HeLa were infected with lentiviruses expressing control , lncRNA - MIF or lncRNA - MIF - AS . By Forty - eight hours infection , were treated with MG132 or DMSO ( vehicle ) for 6 h . lysates were then analyzed by Western blotting with indicated antibodies .

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

A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS. By Forty-eight hours infection, cells were treated with MG132 or DMSO (vehicle) for 6 h. Cell lysates were then analyzed by Western blotting with the indicated antibodies.


B . HeLa were infected with lentiviruses expressing control , lncRNA - MIF or lncRNA - MIF - AS . Forty - eight hours after infection , total was subjected to analyzing of lncRNA - MIF lncRNA - MIF - AS by real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - t - ) .

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

B. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS. Forty-eight hours after infection, total RNA was subjected to analyzing the levels of lncRNA-MIF and lncRNA-MIF-AS by real-time RT-PCR. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


C . HeLa were infected with lentiviruses expressing control , lncRNA - MIF or lncRNA - MIF - AS . By 48 h infection , total was subjected to real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - t - ) .

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

C. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS. By 48 h infection, total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


D . HeLa were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . By 48 h infection , were treated with MG132 or DMSO for 6 h . lysates were then analyzed by Western blot with indicated antibodies .

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

D. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. By 48 h infection, cells were treated with MG132 or DMSO for 6 h. Cell lysates were then analyzed by Western blot analysis with the indicated antibodies.


E . HeLa were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . Forty - eight hours after infection , total was subjected to real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - t - ) .

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

E. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. Forty-eight hours after infection, total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


A . HeLa were infected with lentiviruses expressing control , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , acidification of culture medium was evaluated by visually inspecting color of medium . Yellow medium indicates presence of more lactate .

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

A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, acidification of the culture medium was evaluated by visually inspecting the color of the medium. Yellow medium indicates the presence of more lactate.


B . HeLa were infected with lentiviruses expressing control , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , lysates were analyzed by Western blotting with indicated antibodies .

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

B. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, cell lysates were analyzed by Western blotting with the indicated antibodies.


C . HeLa expressing either lncRNA - MIF or control were transfected with miR - 586 mimics or negative control mimics ( NC mimics ) as indicated . Twenty - four hours after transfection , of lactate in culture medium were measured normalized to number . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - t - ) .

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

C. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or negative control mimics (NC mimics) as indicated. Twenty-four hours after transfection, levels of lactate in the culture medium were measured and normalized to cell number. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


D . HeLa expressing either lncRNA - MIF or control were transfected with miR - 586 mimics or negative control mimics ( NC mimics ) as indicated . Twenty - four hours after transfection , intracellular glucose were measured normalized on . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - t - ) .

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

D. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or negative control mimics (NC mimics) as indicated. Twenty-four hours after transfection, intracellular glucose levels were measured and normalized based on protein concentration. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


E . HeLa expressing either lncRNA - MIF or control shRNA were transfected with miR - 586 inhibitor or NC inhibitor as indicated . Twenty - four hours after transfection , of lactate in culture medium were measured normalized to number . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - t - ) .

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

E. HeLa cells expressing either lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor as indicated. Twenty-four hours after transfection, levels of lactate in the culture medium were measured and normalized to cell number. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


F . HeLa expressing either lncRNA - MIF or control shRNA were transfected with miR - 586 inhibitor or NC inhibitor as indicated . Twenty - four hours after transfection , intracellular glucose were measured normalized on . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - t - ) .

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

F. HeLa cells expressing either lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor as indicated. Twenty-four hours after transfection, intracellular glucose levels were measured and normalized based on protein concentration. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


A . HeLa were infected with lentiviruses expressing control , lncRNA - MIF or lncRNA - MIF - AS as indicated . Forty - eight hours after infection , growth curves were measured for indicated periods of time . Data shown are mean ± SD ( n = 3 ) .

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

A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, growth curves were measured for the indicated periods of time. Data shown are mean ± SD (n = 3).


B . HeLa were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . Forty - eight hours after infection , growth curves were measured for indicated periods of time . Data shown are mean ± SD ( n = 3 ) .

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

B. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. Forty-eight hours after infection, growth curves were measured for the indicated periods of time. Data shown are mean ± SD (n = 3).


C . HeLa were infected with lentiviruses expressing control , lncRNA - MIF or lncRNA - MIF - AS as indicated . Forty - eight hours after infection , were stained with PI analyzed by flow cytometry to identify phases of cycle .

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

C. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle.


D . HeLa were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . Forty - eight hours after infection , were stained with PI analyzed by flow cytometry to identify phases of cycle .

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

D. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. Forty-eight hours after infection, cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle.


E . numbers of ( Fig 7C ) in G1 , S or G2 / M phase were analyzed by FlowJo 7 . 6 software . Data shown are mean ± SD ( n = 3 ) .

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

E. The percentage numbers of cells (Fig 7C) in G1, S or G2/M phase were analyzed by FlowJo 7.6 software. Data shown are mean ± SD (n = 3).


F . numbers of ( Fig 7D ) in G1 , S or G2 / M phase were analyzed by FlowJo 7 . 6 software . Data shown are mean ± SD ( n = 3 ) .

F. The percentage numbers of cells (Fig 7D) in G1, S or G2/M phase were analyzed by FlowJo 7.6 software. Data shown are mean ± SD (n = 3).


A . HeLa expressing either lncRNA - MIF or control were transfected with miR - 586 mimics or NC mimics as indicated . Colonies were stained with crystal violet counted after 14 days incubation .

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

A. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or NC mimics as indicated. Colonies were stained with crystal violet and counted after 14 days incubation.


B . HeLa expressing either lncRNA - MIF shRNA or control shRNA were transfected with miR - 586 inhibitors or NC inhibitors as indicated . Colonies were stained with crystal violet counted after 14 days incubation .

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

B. HeLa cells expressing either lncRNA-MIF shRNA or control shRNA were transfected with miR-586 inhibitors or NC inhibitors as indicated. Colonies were stained with crystal violet and counted after 14 days incubation.


C . 2 × 106 HeLa expressing either lncRNA - MIF , lncRNA - MIF control , lncRNA - MIF - shRNA or lncRNA - MIF control shRNA were individually injected subcutaneously into flanks of nude mice ( n = 7 for each group ) . Representative of xenografttumors in situ were taken 3 after injection . D . Three post graft , mice were euthanized , tumors were excised weighed , mean was plotted on graph . Data shown are mean ± SD ( n = 7 ; * * P < 0 . 01 , two - t - ) .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 2×106 HeLa cells expressing either lncRNA-MIF, lncRNA-MIF control RNA, lncRNA-MIF-shRNA or lncRNA-MIF control shRNA were individually injected subcutaneously into flanks of nude mice (n = 7 for each group). Representative photographs of xenografttumors in situ were taken 3 weeks after injection.D. Three weeks post graft, mice were euthanized, tumors were excised and weighed, and the mean tumor weight was plotted on the graph. Data shown are mean ± SD (n = 7; **P < 0.01, two-tailed t-test).


A Peroxisomes were induced by growing WT expressing Pot1 ‐ GFP in oleate medium to mid ‐ log ‐ phase , then transferred to SD ‐ N starvation medium with or without GABA to trigger pexophagy for 6 h . GFP cleavage was analyzed at indicated time points by immunoblotting .

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

A Peroxisomes were induced by growing the WT strain expressing Pot1‐GFP in oleate medium to mid‐log‐phase, then transferred to SD‐N starvation medium with or without GABA to trigger pexophagy for 6 h. GFP cleavage was analyzed at the indicated time points by immunoblotting.


B Mitochondria were induced by growing WT expressing OM45 ‐ GFP in YPL medium to mid ‐ log ‐ phase subsequently transferring to either SD ‐ N with or without GABA to trigger mitophagy for 12 h . GFP cleavage was analyzed at indicated time points by immunoblotting .

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

B Mitochondria were induced by growing the WT strain expressing OM45‐GFP in YPL medium to mid‐log‐phase and subsequently transferring cells to either SD‐N with or without GABA to trigger mitophagy for 12 h. GFP cleavage was analyzed at the indicated time points by immunoblotting.


C Mitophagy was monitored by microscopy using a WT expressing OM45 ‐ GFP grown in YPL medium for 12 h to mid ‐ log ‐ phase in presence of FM4 ‐ 64 , transferred to either SD ‐ N medium with or without GABA for 24 h . Bar , 5 μm .

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

C Mitophagy was monitored by fluorescence microscopy using a WT strain expressing OM45‐GFP grown in YPL medium for 12 h to mid‐log‐phase in the presence of FM4‐64, and transferred to either SD‐N medium with or without GABA for 24 h. Bar, 5 μm.


D Cvt pathway was monitored using WT in SD medium with or without GABA , grown to mid ‐ log ‐ phase , after which samples were analyzed for Ape1 maturation .

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

D The Cvt pathway was monitored using the WT strain in SD medium with or without GABA, grown to mid‐log‐phase, after which samples were analyzed for Ape1 maturation.


E Ribophagy was monitored by growing WT expressing Rpl25 ‐ GFP in SD medium to mid ‐ log ‐ phase transferring to SD ‐ N either with or without GABA for 24 h .

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 B-CONTROLLED_VAR O O O O

E Ribophagy was monitored by growing the WT strain expressing Rpl25‐GFP in SD medium to mid‐log‐phase and transferring cells to SD‐N either with or without GABA for 24 h.


F Autophagy was monitored by growing WT expressing GFP ‐ Atg8 in SD medium to mid ‐ log ‐ phase transferring to SD ‐ N either with or without GABA for 6 h .

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

F Autophagy was monitored by growing the WT strain expressing GFP‐Atg8 in SD medium to mid‐log‐phase and transferring cells to SD‐N either with or without GABA for 6 h.


APeroxisomes were induced in oleate medium pexophagy was monitored as described for Fig 1A .

APeroxisomes were induced in oleate medium and pexophagy was monitored as described for Fig 1A.


B Pexophagy was monitored by microscopy using a WT expressing Pot1 ‐ GFP grown in oleate medium to mid ‐ log ‐ phase in presence of FM4 ‐ 64 , transferred to either SD ‐ N medium with or without GABA or to SD ‐ N with GABA rapamycin for 6 h . Bar , 5 μm .

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

B Pexophagy was monitored by fluorescence microscopy using a WT strain expressing Pot1‐GFP grown in oleate medium to mid‐log‐phase in the presence of FM4‐64, and transferred to either SD‐N medium with or without GABA or to SD‐N with GABA and rapamycin for 6 h. Bar, 5 μm.


C Mitochondria were induced in YPL medium mitophagy was assessed as described for Fig .

C Mitochondria were induced in YPL medium and mitophagy was assessed as described for Fig .


A WT expressing OM45 ‐ GFP , along with uga2 ∆ over ‐ expressing GAD1 gene expressing OM45 ‐ GFP were grown in YPL medium to mid ‐ log ‐ phase . To monitor mitophagy , strains were transferred to SD ‐ N starvation medium ( with or without rapamycin ) .

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

A WT cells expressing OM45‐GFP, along with the uga2∆ strain over‐expressing the GAD1 gene and expressing OM45‐GFP were grown in YPL medium to mid‐log‐phase. To monitor mitophagy, strains were transferred to SD‐N starvation medium (with or without rapamycin).


B WT along with uga2 ∆ over ‐ expressing GAD1 gene was grown in oleate medium pexophagy was monitored as described in Fig , with or without rapamycin . Samples were taken at indicated time points , Pot1 degradation was analyzed by immunoblotting ( 45 kD ) .

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

B WT strain along with the uga2∆ strain over‐expressing the GAD1 gene was grown in oleate medium and pexophagy was monitored as described in Fig , with or without rapamycin. Samples were taken at the indicated time points, and Pot1 degradation was analyzed by immunoblotting (45 kD).


C To monitor autophagy , WT expressing GFP ‐ Atg8 along with uga2 ∆ over ‐ expressing GAD1 gene expressing GFP ‐ Atg8 were grown in SD medium transferred to SD ‐ N .

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

C To monitor autophagy, WT cells expressing GFP‐Atg8 along with the uga2∆ strain over‐expressing the GAD1 gene and expressing GFP‐Atg8 were grown in SD medium and transferred to SD‐N.


A , B WT were cultured under pexophagy ( A ) or mitophagy ( B ) conditions with or without GABA rapamycin . S6 phosphorylation at indicated time points was analyzed by immunoblotting with a loading control .

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

A,B WT cells were cultured under pexophagy (A) or mitophagy (B) conditions with or without GABA and rapamycin. S6 phosphorylation at the indicated time points was analyzed by immunoblotting with a loading control.


C Samples were analyzed for Pot1 degradation by immunoblotting ( 45 kD ) .

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

C Samples were analyzed for Pot1 degradation by immunoblotting (45 kD).


D GFP production during mitophagy was analyzed by immunoblotting .

D GFP production during mitophagy was analyzed by immunoblotting.


A S6 phosphorylation after 6 h in SD ‐ N was analyzed by immunoblotting with a loading control .

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

A S6 phosphorylation after 6 h in SD‐N was analyzed by immunoblotting with a loading control.


B GFP production monitoring autophagy at indicated time points was analyzed by immunoblotting .

B GFP production monitoring autophagy at the indicated time points was analyzed by immunoblotting.


A , B WT , WT with GABA , WT with GABA 10 mM GSH WT with GABA 200 nM rapamycin were tested for intracellular ROS under ( A ) pexophagy ( B ) mitophagy conditions . After 24 h incubation , were stained with DHR − 123 propidium iodide for 1 h . Living were analyzed for DHR − 123 by flow cytometry . Data represent mean + s . d . ( n = 3 ) . * P < 0 . 005 , * * P < 0 .

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

A,B WT, WT with GABA, WT with GABA and 10 mM GSH and WT with GABA and 200 nM rapamycin were tested for intracellular ROS levels under (A) pexophagy and (B) mitophagy conditions. After 24 h incubation, cells were stained with DHR−123 and propidium iodide for 1 h. Living cells were analyzed for DHR−123 fluorescence by flow cytometry. Data represent mean + s.d. (n = 3). *P < 0.005, **P < 0.01


C , D Yeast stained with 5 μM propidium iodide were used to differentiate between living dead under ( C ) pexophagy or ( D ) mitophagy conditions . Significant differences between treatments strains were determined using an unpaired two ‐ t ‐ . * * P < 0 . 01 .

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

C,D Yeast cells stained with 5 μM propidium iodide were used to differentiate between living and dead cells under (C) pexophagy or (D) mitophagy conditions. Significant differences between the treatments and strains were determined using an unpaired two‐tailed t‐test. **P < 0.01.


E Pexophagy was monitored by degradation of Pot1 ‐ GFP analyzed for GFP cleavage by immunoblotting .

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

E Pexophagy assay was monitored by the degradation of Pot1‐GFP and analyzed for GFP cleavage by immunoblotting.


F Mitophagy was monitored by degradation of Om45 ‐ GFP analyzed for GFP cleavage by immunoblotting .

F Mitophagy assay was monitored by the degradation of Om45‐GFP and analyzed for GFP cleavage by immunoblotting.


A , B Example of Parkin ‐ expressing HeLa analyzed using a tandem fluorochrome ( mito ‐ RFP ‐ GFP ) mitophagy under ( A ) control conditions or ( B ) displaying mitophagy depicted by red mitochondrial structures localized to lysosomes . Bar , 10 μm .

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

A,B Example images of Parkin‐expressing HeLa cells analyzed using a tandem fluorochrome protein (mito‐RFP‐GFP) mitophagy assay under (A) control conditions or (B) displaying mitophagy depicted by the red mitochondrial structures localized to lysosomes. Bar, 10 μm.


C of displaying mitophagy + s . d . , * * P < 0 . 01 using an unpaired two ‐ t ‐ , n > 80 .

O O O O O 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 Percentage of cells displaying mitophagy + s.d., **P < 0.01 using an unpaired two‐tailed t‐test, n > 80.


A Electron microscopy of mitochondria from WT ( n = 44 ) SSADH ‐ deficient mice ( Aldh5a1 − / − ) ( n = 80 ) were calculated for .

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

A Electron microscopy images of mitochondria from WT (n = 44) and SSADH‐deficient mice (Aldh5a1−/−) (n = 80) were calculated for area size.


B Electron microscopy showing typical sizes of WT Aldh5a1 − / − mice liver mitochondria . Bar , 0 . 5 μm .

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 Electron microscopy images showing typical sizes of WT and Aldh5a1−/− mice liver mitochondria. Bar, 0.5 μm.


C of mitochondrial numbers from electron microscopy of liver from WT ( n = 31 ) Aldh5a1 − / − mice treated with vehicle ( n = 39 ) or rapamycin ( n = 34 ) ( 5 mg / kg per day ) via intraperitoneal injections for 3 successive days starting at day 7 of life .

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

C Quantification of mitochondrial numbers from electron microscopy images of liver from WT (n = 31) and Aldh5a1−/− mice treated with vehicle (n = 39) or rapamycin (n = 34) (5 mg/kg body weight per day) via intraperitoneal injections for 3 successive days starting at day 7 of life.


D of mitochondrial numbers from electron microscopy of brain from WT ( n = 23 ) Aldh5a1 − / − mice treated with vehicle ( n = 30 ) or rapamycin ( n = 41 ) ( 5 mg / kg per day ) via intraperitoneal injections for 3 successive days starting at day 7 of life .

D Quantification of mitochondrial numbers from electron microscopy images of brain from WT (n = 23) and Aldh5a1−/− mice treated with vehicle (n = 30) or rapamycin (n = 41) (5 mg/kg body weight per day) via intraperitoneal injections for 3 successive days starting at day 7 of life.


E Aldh5a1 − / − mice were treated with vehicle or rapamycin ( 10 mg / kg per day ) via intraperitoneal injections for 10 successive days starting at day 10 of life . WT mice served as non ‐ disease controls ( set to 1 ) . After sacrifice , liver homogenates were used to measure SOD enzyme activity using a colorimetric SOD activity .

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

E Aldh5a1−/− mice were treated with vehicle or rapamycin (10 mg/kg body weight per day) via intraperitoneal injections for 10 successive days starting at day 10 of life. WT mice served as non‐disease controls (set to 1). After sacrifice, liver homogenates were used to measure SOD enzyme activity using a colorimetric SOD activity assay.


F Mitochondrial SOD2 were quantified from liver microsections using immunofluorescence microscopy automated image ( WT set to 1 ) .

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

F Mitochondrial SOD2 protein levels were quantified from liver microsections using immunofluorescence microscopy and automated image analysis (WT set to 1).


G Immunofluorescence showing typical ( DAPI , blue ) SOD2 ( red ) from WT , Aldh5a1 − / − mice treated with vehicle Aldh5a1 − / − mice treated with rapamycin . Bar , 10 μm .

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

G Immunofluorescence images showing typical nuclear staining (DAPI, blue) and SOD2 staining (red) from WT, Aldh5a1−/− mice treated with vehicle and Aldh5a1−/− mice treated with rapamycin. Bar, 10 μm.


A of S6 phosphorylation of liver lysates from WT ( n = 5 ) Aldh5a1 − / − mice treated with vehicle ( n = 4 ) or rapamycin ( n = 5 ) after normalization ( WT set to 1 ) .

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

A Quantification of S6 phosphorylation of liver lysates from WT (n = 5) and Aldh5a1−/− mice treated with vehicle (n = 4) or rapamycin (n = 5) after normalization (WT set to 1).


B S6 phosphorylation of liver lysates analyzed by immunoblotting .

B S6 phosphorylation of liver lysates analyzed by immunoblotting.


C of S6 phosphorylation of brain lysates from WT ( n = 2 ) Aldh5a1 − / − mice treated with vehicle ( n = 3 ) or rapamycin ( n = 3 ) after normalization ( WT set to 1 ) .

C Quantification of S6 phosphorylation of brain lysates from WT (n = 2) and Aldh5a1−/− mice treated with vehicle (n = 3) or rapamycin (n = 3) after normalization (WT set to 1).


D S6 phosphorylation of brain lysates analyzed by immunoblotting .

D S6 phosphorylation of brain lysates analyzed by immunoblotting.


( A ) Raji stably expressing HIV - 1 YU - 2 ENV ( Raji - Env ) were stained with bNAbs ( 10 - 1074 or 3BNC117 ) or isotype control ( mGO53 ) analyzed by flow cytometry . numbers indicate % of positive . One representative experiment is shown .

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

(A) Raji cells stably expressing HIV-1 YU-2 ENV (Raji-Env) were stained with bNAbs (10-1074 or 3BNC117) or isotype control (mGO53) and analyzed by flow cytometry. The numbers indicate the % of positive cells. One representative experiment is shown.


( B ) Binding of 24 indicated antibodies to surface of Raji - Env . Results are expressed as median ( MFI ) of . n = 6 independent experiments . Data information : Error bars indicate . Significance was determined by comparing each antibody to mGO53 . Only significant comparisons are depicted ; * P < 0 . 05 , Mann - Whitney .

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

(B) Binding of the 24 indicated antibodies to the surface of Raji-Env cells. Results are expressed as the median fluorescence intensity (MFI) of staining. n=6 independent experiments. Data information: Error bars indicate SEM. Significance was determined by comparing each antibody to mGO53. Only significant comparisons are depicted; *P<0.05, Mann-Whitney test.


( C ) Raji - Env were incubated with indicated antibodies either normal ( NHS ) or heat - inactivated ( HIHS ) human serum . After 1h , surface of C3 were analyzed by flow cytometry . One representative experiment is shown .

O O O O O O O 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

(C) Raji-Env cells were incubated with indicated antibodies and either normal (NHS) or heat-inactivated (HIHS) human serum. After 1h, surface levels of C3 were analyzed by flow cytometry. One representative experiment is shown.


( D ) C3 deposition by 24 indicated antibodies . For each antibody , a normalized MFI is calculated by subtracting MFI of " no antibody " condition . ( nd . not done ) . n = 3 donors of serum . Data information : Error bars indicate . Significance was determined by comparing each antibody to mGO53 . Only significant comparisons are depicted ; * P < 0 . 05 , Mann - Whitney .

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

(D) C3 deposition by the 24 indicated antibodies. For each antibody, a normalized MFI is calculated by subtracting the MFI of the "no antibody" condition. (nd. not done). n=3 donors of serum. Data information: Error bars indicate SEM. Significance was determined by comparing each antibody to mGO53. Only significant comparisons are depicted; *P<0.05, Mann-Whitney test.


( E ) Raji - Env were incubated with indicated antibodies either normal ( NHS ) or heat - inactivated ( HIHS ) human serum . After 24h , was determined by flow cytometry . numbers indicate of dead . One representative experiment is shown .

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

(E) Raji-Env cells were incubated with indicated antibodies and either normal (NHS) or heat-inactivated (HIHS) human serum. After 24h, cell death was determined by flow cytometry. The numbers indicate the percentage of dead cells. One representative experiment is shown.


( F ) CDC induction by 24 indicated antibodies . % of CDC was calculated as relative of dead compared to " no antibody " condition . n = 3 donors of serum . Data information : Error bars indicate . Significance was determined by comparing each antibody to mGO53 . Only significant comparisons are depicted ; * P < 0 . 05 , Mann - Whitney .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CDC induction by the 24 indicated antibodies. The % of CDC was calculated as the relative percentage of dead cells compared to the "no antibody" condition. n=3 donors of serum. Data information: Error bars indicate SEM. Significance was determined by comparing each antibody to mGO53. Only significant comparisons are depicted; *P<0.05, Mann-Whitney test.


( G ) Raji - Env were incubated for 24h with NHS indicated concentrations of WT or E430G mutant bNAbs ( 10 - 1074 , 3BNC117 or PGDM1400 ) or isotype control ( mGO53 ) . was measured by flow cytometry % of CDC was calculated as relative % of dead compared to " no antibody " condition . n = 3 donors of serum .

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 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

(G) Raji-Env cells were incubated for 24h with NHS and indicated concentrations of WT or E430G mutant bNAbs (10-1074, 3BNC117 or PGDM1400) or isotype control (mGO53). Cell death was measured by flow cytometry and the % of CDC was calculated as the relative % of dead cells compared to the "no antibody" condition. n=3 donors of serum.


( A ) Activated CD4 T were infected by HIV - 1 ( NLAD8 ) subjected to antibody binding . numbers indicate % of Gag + that are decorated by indicated antibodies . One representative experiment is shown .

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

(A) Activated primary CD4 T cells were infected by HIV-1 (strain NLAD8) and subjected to antibody binding assay. The numbers indicate the % of Gag+ cells that are decorated by the indicated antibodies. One representative experiment is shown.


( B ) Antibody binding at surface of HIV - 1 - infected CD4 T . Results are expressed as median ( MFI ) of on infected ( Gag + ) . Each dot represents a single donor of CD4 T . n = 9 donors of CD4 T . Data information : Error bars indicate . * P < 0 . 05 , * * P < 0 . 01 , Wilcoxon . Only significant comparisons are depicted .

O O O O 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

(B) Antibody binding at the surface of HIV-1-infected primary CD4 T cells. Results are expressed as median fluorescence (MFI) of staining on infected (Gag+) cells. Each dot represents a single donor of CD4 T cells. n=9 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted.


( C ) HIV - 1 - infected ( NLAD8 ) CD4 T were cultured with indicated antibody NHS . After 24h , surface of C3 frequency of infected ( Gag + ) were determined by flow cytometry . One representative experiment is shown .

O 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-MEASURED_VAR O O O O O O O O O O O O O O

(C) HIV-1-infected (strain NLAD8) CD4 T cells were cultured with the indicated antibody and NHS. After 24h, the surface levels of C3 and the frequency of infected cells (Gag+) were determined by flow cytometry. One representative experiment is shown.


( D ) C3 surface at surface of HIV - 1 - infected CD4 T . Results are expressed as MFI of on infected ( Gag + ) . Each dot represents a single donor of CD4 T . n = 7 donors of CD4 T . Data information : Error bars indicate . * P < 0 . 05 , * * P < 0 . 01 , Wilcoxon . Only significant comparisons are depicted .

(D) C3 surface levels at the surface of HIV-1-infected primary CD4 T cells. Results are expressed as MFI of staining on infected (Gag+) cells. Each dot represents a single donor of CD4 T cells. n=7 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted.


( E ) HIV - 1 - infected CD4 T ( NLAD8 ) were cultured with indicated antibody normal ( NHS ) or heat - inactivated ( HIHS ) human serum . After 24h , surface of C5b - 9 ( MAC ) on infected ( Gag + ) were determined by flow cytometry . One representative experiment is shown .

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

(E) HIV-1-infected CD4 T cells (strain NLAD8) were cultured with the indicated antibody and normal (NHS) or heat-inactivated (HIHS) human serum. After 24h, the surface levels of C5b-9 (MAC) on infected (Gag+) cells were determined by flow cytometry. One representative experiment is shown.


( F ) C5b - 9 ( MAC ) surface on HIV - 1 - infected ( NLAD8 ) . Results are expressed as MFI of on infected ( Gag + ) . Each dot represents a single donor of CD4 T . n = 6 donors of CD4 T . Data information : Error bars indicate . * P < 0 . 05 , * * P < 0 . 01 , Wilcoxon . Only significant comparisons are depicted .

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

(F) C5b-9 (MAC) surface levels on HIV-1-infected cells (strain NLAD8). Results are expressed as MFI of staining on infected (Gag+) cells. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted.


( G ) HIV - 1 - infected CD4 T ( NLAD8 ) were cultured with indicated antibody NHS or HIHS to determine complement - mediated cytotoxicity . After 24h , of infected ( Gag + ) in each condition was determined by flow cytometry . One representative experiment is shown .

O O O O O O O O O O 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

(G) HIV-1-infected CD4 T cells (strain NLAD8) were cultured with the indicated antibody and NHS or HIHS to determine complement-mediated cytotoxicity. After 24h, the percentage of infected (Gag+) cells in each condition was determined by flow cytometry. One representative experiment is shown.


( H ) Complement - mediated cytotoxicity of HIV - 1 - infected CD4 T ( NLAD8 ) . For each antibody , % of CDC was calculated at 24h as % of disappearance of Gag + in NHS compared to HIHS . Each dot represents a single donor of CD4 T . n = 7 donors of CD4 T . Data information : Error bars indicate . * P < 0 . 05 , * * P < 0 . 01 , Wilcoxon . Only significant comparisons are depicted .

O O O O O O O O O O O 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

(H) Complement-mediated cytotoxicity of HIV-1-infected CD4 T cells (strain NLAD8). For each antibody, the % of CDC was calculated at 24h as the % of disappearance of Gag+ cells in NHS compared to HIHS. Each dot represents a single donor of CD4 T cells. n=7 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted.


( A ) Raji - Env were subcloned to obtain lines expressing increasing amounts of Env . Env surface were determined by binding of 10 - 1074 measured by flow cytometry . One representative experiment is shown .

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

(A) Raji-Env cells were subcloned to obtain cell lines expressing increasing amounts of Env. Env surface levels were determined by the binding of 10-1074 and measured by flow cytometry. One representative experiment is shown.


( B ) Parental Raji different Raji - Env clones were cultured with 10 - 1074 or an isotype control ( mGO53 ) NHS for 24h . % of CDC was calculated as relative of dead compared to " no antibody " condition . n = 3 donors of serum .

O O O O O O O O B-MEASURED_VAR 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

(B) Parental Raji cells and the different Raji-Env clones were cultured with 10-1074 or an isotype control (mGO53) and NHS for 24h. The % of CDC was calculated as the relative percentage of dead cells compared to the "no antibody" condition. n=3 donors of serum.


( C ) Non - infected ( NI ) HIV - 1 - infected CD4 T ( NLAD8 ) as well as three Raji - Env clones ( D1 , D2 D4 ) were incubated with 10 - 1074 antibody surface were analyzed by flow cytometry . Results are expressed as median ( MFI ) of in total ( Raji , n = 3 independent experiments ) or in infected ( Gag + ) ( CD4 T , n = 5 donors ) .

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

(C) Non-infected (NI) and HIV-1-infected primary CD4 T cells (strain NLAD8) as well as three Raji-Env clones (D1, D2 and D4) were incubated with 10-1074 and antibody surface levels were analyzed by flow cytometry. Results are expressed as median fluorescence (MFI) of staining in total cells (Raji, n=3 independent experiments) or in infected (Gag+) cells (CD4 T cells, n=5 donors).


( D ) HIV - or HIV ∆ Vpu - infected CD4 T ( NLAD8 ) were subjected to antibody binding with indicated antibodies surface were determined by flow cytometry ( left panel ) . obtained with mGO53 was similar to background signal observed on unstained . Infected were cultured with indicated antibodies NHS for 24h C3 surface on infected ( Gag + ) were determined by flow cytometry ( right panel ) . One representative experiment is shown .

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 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

(D) HIV- or HIV∆Vpu-infected CD4 T cells (strain NLAD8) were subjected to antibody binding with indicated antibodies and surface levels were determined by flow cytometry (left panel). The staining obtained with mGO53 was similar to the background signal observed on unstained cells. Infected cells were cultured with the indicated antibodies and NHS for 24h and the C3 surface levels on infected (Gag+) cells were determined by flow cytometry (right panel). One representative experiment is shown.


( E ) HIV - or HIV ∆ Vpu - infected CD4 T ( NLAD8 ) were incubated with NHS either 10 - 1074 ( left panel ) or 3BNC117 ( right panel ) . Both WT or E430G - mutated antibodies were used . After 24h , C3 surface on infected were analyzed by flow cytometry . Results are expressed as MFI of on infected ( Gag + ) . MFI of C3 on Gag - was included as a control . Each dot represents a single donor of CD4 T . n = 6 donors of CD4 T .

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 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-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

(E) HIV- or HIV∆Vpu-infected CD4 T cells (strain NLAD8) were incubated with NHS and either 10-1074 (left panel) or 3BNC117 (right panel). Both WT or E430G-mutated antibodies were used. After 24h, C3 surface levels on infected cells were analyzed by flow cytometry. Results are expressed as MFI of staining on infected (Gag+) cells. The MFI of C3 staining on Gag- cells was included as a control. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.


( A ) CD4 T were nucleofected with a control ( CRISPR Control ) or anti - CD59 ( CRISPR CD59 ) guide / Cas9 . CD59 surface were analyzed by flow cytometry 7 days after nucleofection . Isotype control ( grey ) CD59 stainings ( blue ) . One representative experiment is shown on left . Numbers indicate % of CD59 + ( middle ) or MFI of in CD59 - ( Right ) . Each dot represents a single donor of CD4 T . n = 6 donors of CD4 T .

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

(A) Primary CD4 T cells were nucleofected with a control (CRISPR Control) or anti-CD59 (CRISPR CD59) guide RNA/Cas9 complex. CD59 surface levels were analyzed by flow cytometry 7 days after nucleofection. Isotype control (grey) and CD59 stainings (blue). One representative experiment is shown on the left. Numbers indicate the % of CD59+ cells (middle) or the MFI of staining in CD59- cells (Right). Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.


( B ) NLAD8 ∆ Vpu - infected CD59 KO CD4 T were cultured with indicated antibodies NHS or HIHS for 24h . Numbers indicate of infected ( Gag + ) . One representative experiment is shown .

O O O O 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) NLAD8∆Vpu-infected CD59 KO primary CD4 T cells were cultured with indicated antibodies and NHS or HIHS for 24h. Numbers indicate the percentage of infected (Gag+) cells. One representative experiment is shown.


( C ) CD59 - KO ( CRISPR CD59 ) or control ( CRISPR Control ) CD4 T infected with HIV WT ( left ) or HIVΔVpu ( right ) ( NLAD8 ) were cultured with indicated antibody , NHS or HIHS for 24h . % of CDC was calculated as % of disappearance of Gag + after culture with NHS compared to HIHS . Each dot represents a single donor of CD4 T . n = at least 6 donors of CD4 T .

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 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

(C) CD59-KO (CRISPR CD59) or control (CRISPR Control) CD4 T cells infected with HIV WT (left) or HIVΔVpu (right) (strain NLAD8) were cultured with the indicated antibody, and NHS or HIHS for 24h. The % of CDC was calculated as the % of disappearance of Gag+ cells after culture with NHS compared to HIHS. Each dot represents a single donor of CD4 T cells. n= at least 6 donors of CD4 T cells.


( D ) CD59 surface on non - infected ( NI ) or HIV - 1 - infected ( NLAD8 ) CD4 T were analyzed by flow cytometry . Results are expressed as MFI of with an isotype control or anti - CD59 antibody . n = 4 donors of CD4 T .

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

(D) CD59 surface levels on non-infected (NI) or HIV-1-infected (strain NLAD8) primary CD4 T cells were analyzed by flow cytometry. Results are expressed as the MFI of staining with an isotype control or the anti-CD59 antibody. n=4 donors of CD4 T cells.


( B ) HIV - 1 - infected CD4 T were cultured with indicated antibodies ( E430G ) with NHS or HIHS . frequency of infected was assessed by flow cytometry in live at days 1 , 2 , 3 6 . Numbers indicate of infected ( Gag + ) . One representative experiment is shown .

O O O O O O O O O O O O O O O O O O O O O O O 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) HIV-1-infected primary CD4 T cells were cultured with the indicated antibodies (E430G) and with NHS or HIHS. The frequency of infected cells was assessed by flow cytometry in live cells at days 1, 2, 3 and 6. Numbers indicate the percentage of infected (Gag+) cells. One representative experiment is shown.


( C ) Frequency of live HIV - 1 - infected ( NLAD8 WT or ΔVpu ) 6 days after incubation with indicated antibodies , with NHS or HIHS . Each donor of CD4 T is represented by a different symbol . n = 8 donors of CD4 T .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Frequency of live HIV-1-infected cells (NLAD8 WT or ΔVpu strain) 6 days after incubation with the indicated antibodies, and with NHS or HIHS. Each donor of CD4 T cells is represented by a different symbol. n=8 donors of CD4 T cells.


( D ) Elimination of HIV - 1 - infected ( NLAD8 WT or ΔVpu ) over culture with indicated antibody ( E430G ) , NHS or HIHS . frequency of % infected ( Gag + ) is determinined by flow cytometry . mean relative of infected compared to HIHS condition is depicted . n = 6 - 8 donors of CD4 T .

O O O O O O O O O O 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

(D) Elimination of HIV-1-infected cells (strain NLAD8 WT or ΔVpu) over culture with the indicated antibody (E430G), and NHS or HIHS. The frequency of % infected (Gag+) cells is determinined by flow cytometry. The mean relative percentage of infected cells compared to the HIHS condition is depicted. n=6-8 donors of CD4 T cells.


( A ) CD4 T infected with HIV - 1 ( Transmitted / Founder strains CH058 CH077 , vKB18 ) were incubated with indicated bNAbs surface were analyzed by flow cytometry . numbers indicate % of bNAb + among infected ( Gag + ) . percentages of infected were 27 % , 30 % 6 % for CH058 , CH077 vKB18 , respectively . One representative experiment is shown .

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

(A) Primary CD4 T cells infected with HIV-1 (Transmitted/Founder strains CH058 and CH077, and the primary strain vKB18) were incubated with the indicated bNAbs and surface levels were analyzed by flow cytometry. The numbers indicate the % of bNAb+ cells among infected (Gag+) cells. The percentages of infected cells were 27%, 30% and 6% for CH058, CH077 and vKB18, respectively. One representative experiment is shown.


( B ) CH058 - infected CD4 T were cultured with indicated WT or E430G mutant antibody NHS . After 24h , C3 surface on infected ( Gag + ) were determined by flow cytometry . One representative experiment is shown .

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-MEASURED_VAR O O O O O O O O O O O O O O

(B) CH058-infected CD4 T cells were cultured with the indicated WT or E430G mutant antibody and NHS. After 24h, the C3 surface levels on infected (Gag+) cells were determined by flow cytometry. One representative experiment is shown.


( C ) CH058 - , CH077 - vKB18 - infected CD4 T were incubated with indicated antibodies NHS . After 24h , C3 surface on infected were analyzed by flow cytometry . Results are expressed as MFI of on infected ( Gag + ) . Each dot represents a donor of CD4 T . n = 6 donors of CD4 T .

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

(C) CH058-, CH077- and vKB18-infected primary CD4 T cells were incubated with the indicated antibodies and NHS. After 24h, C3 surface levels on infected cells were analyzed by flow cytometry. Results are expressed as MFI of staining on infected (Gag+) cells. Each dot represents a donor of CD4 T cells. n=6 donors of CD4 T cells.


( D ) CD4 T infected with indicated HIV - 1 strains ( CH058 , CH077 or vKB18 ) were cultured with indicated WT or E430G mutant antibodies NHS or HIHS to determine complement - mediated cytotoxicity . After 24h , of infected ( Gag + ) in each condition was determined by flow cytometry . For each antibody , % of CDC was calculated at 24h as % of disappearance of Gag + in NHS compared to HIHS . Each dot represents a single donor of CD4 T . n = 6 donors of CD4 T .

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

(D) Primary CD4 T cells infected with the indicated HIV-1 strains (CH058, CH077 or vKB18) were cultured with the indicated WT or E430G mutant antibodies and NHS or HIHS to determine complement-mediated cytotoxicity. After 24h, the percentage of infected (Gag+) cells in each condition was determined by flow cytometry. For each antibody, the % of CDC was calculated at 24h as the % of disappearance of Gag+ cells in NHS compared to HIHS. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.


( A ) HIV - 1 - infected CD4 T ( CH058 ) were incubated with sera of HIV negative ( HIV - ; n = 9 ) or ART - treated HIV - 1 - infected ( HIV + ; n = 9 ) individuals ( serum dilution 1 : 1000 ) . of IgG binding on infected ( Gag + ) were analyzed by flow cytometry . Results are expressed as MFI on infected ( Gag + ) . Error bars indicate . * * * * P < 0 , 0001 ; Mann - Whitney .

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

(A) HIV-1-infected primary CD4 T cells (strain CH058) were incubated with sera of HIV negative (HIV-; n=9) or ART-treated HIV-1-infected (HIV+; n=9) individuals (serum dilution 1:1000). The levels of IgG binding on infected (Gag+) cells were analyzed by flow cytometry. Results are expressed as the MFI on infected (Gag+) cells. Error bars indicate SEM. ****P<0,0001; Mann-Whitney test.


( B ) CH058 - infected CD4 T were cultured for 24h with sera of healthy ( HIV - ; n = 9 ) or ART - treated HIV - 1 - infected ( HIV + ; n = 9 ) individuals ( serum dilution 1 : 100 ) 50 % NHS from a healthy donor . C3 surface on infected ( Gag + ) were analyzed by flow cytometry . Results are expressed as MFI on infected ( Gag + ) . Error bars indicate . * * * * P < 0 , 0001 ; Mann - Whitney .

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

(B) CH058-infected primary CD4 T cells were cultured for 24h with sera of healthy (HIV-; n=9) or ART-treated HIV-1-infected (HIV+; n=9) individuals (serum dilution 1:100) and 50% NHS from a healthy donor. C3 surface levels on infected (Gag+) cells were analyzed by flow cytometry. Results are expressed as the MFI on infected (Gag+) cells. Error bars indicate SEM. ****P<0,0001; Mann-Whitney test.


( C ) CD4 T not infected ( NI ) or infected with WT , ∆ Nef , ∆ Vpu or ∆ Nef ∆ Vpu HIV - 1 ( CH058 ) were incubated with serum of HIV - 1 - infected ART - treated individuals ( sKB104 or sKB105 , dilution 1 : 1000 ) . of IgG binding on infected ( Gag + ) were analyzed by flow cytometry . One representative experiment is shown on left . On right , results are expressed as MFI of . Each dot represents a single donor of CD4 T . n = 6 donors of CD4 T . Error bars indicate . * P < 0 . 05 , Wilcoxon . Only significant comparisons are depicted .

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

(C) CD4 T cells not infected (NI) or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058) were incubated with the serum of HIV-1-infected ART-treated individuals (sKB104 or sKB105, dilution 1:1000). The levels of IgG binding on infected (Gag+) cells were analyzed by flow cytometry. One representative experiment is shown on the left. On the right, results are expressed as MFI of staining. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells. Error bars indicate SEM. *P<0.05, Wilcoxon test. Only significant comparisons are depicted.


( D ) CD4 T either not infected ( NI ) or infected with WT , ∆ Nef , ∆ Vpu or ∆ Nef ∆ Vpu HIV - 1 ( CH058 ) were incubated with 50 % NHS from a healthy donor serum of HIV - 1 - infected ART - treated individuals ( sKB104 or sKB105 , dilution 1 : 100 ) . of C3 deposition on infected ( Gag + ) were analyzed by flow cytometry . One representative experiment is shown ( left panel ) . Results are expressed as MFI of ( right panel ) . Each dot represents a single donor of CD4 T . n = 6 donors of CD4 T .

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

(D) Primary CD4 T cells either not infected (NI) or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058) were incubated with 50% NHS from a healthy donor and serum of HIV-1-infected ART-treated individuals (sKB104 or sKB105, dilution 1:100). The levels of C3 deposition on infected (Gag+) cells were analyzed by flow cytometry. One representative experiment is shown (left panel). Results are expressed as MFI of staining (right panel). Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.


( E ) Correlation between ' sera ( sK104 sKB105 ) antibody binding C3 deposition on CD4 T either mock - infected or infected with WT , ∆ Nef , ∆ Vpu or ∆ Nef ∆ Vpu HIV - 1 ( CH058 ) . Each dot is mean of 6 donors of CD4 T . Correlation was analyzed by Spearman correlation . Correlation coefficient ( r ) p - value are indicated .

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

(E) Correlation between patients' sera (sK104 and sKB105) antibody binding and C3 deposition on CD4 T cells either mock-infected or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058). Each dot is the mean of 6 donors of CD4 T cells. Correlation was analyzed by Spearman correlation. Correlation coefficient (r) and p-value are indicated.


A T98G were challenged or not with dsDNAs or with 2 ' 3 ' cGAMP for 6 h prior to whole extraction Western Blot ( WB ) using indicated antibodies .

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

A T98G cells were challenged or not with dsDNAs or with 2'3'cGAMP for 6 h prior to whole cell extraction and Western Blot (WB) analysis using indicated antibodies.


C Gli4 were treated as in A prior to WB using indicated antibodies .

C Gli4 cells were treated as in A prior to WB analysis using indicated antibodies.


F T98G were challenged or not with dsDNA for 6 h , in presence or not of NU7441 - PKcs inhibitor , prior WB using indicated antibodies .

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

F T98G cells were challenged or not with dsDNA for 6 h, in the presence or not of the NU7441 DNA-PKcs inhibitor, prior WB analysis using indicated antibodies.


I T98G were treated or not with 0 . 16 µM camptothecin ( CPT ) for 48 h prior to immunofluorescence using dsDNA - 53BP1 - specific antibodies , DAPI ( n = 3 independent experiments ) .

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

I T98G cells were treated or not with 0.16 µM camptothecin (CPT) for 48 h prior to immunofluorescence analysis using dsDNA- and 53BP1-specific antibodies, and DAPI nuclear staining (n=3 independent experiments).


J Whole extracts from T98G treated or not for 72 h with 0 . 16 µM CPT were analyzed by WB using indicated antibodies .

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

J Whole cell extracts from T98G cells treated or not for 72 h with 0.16 µM CPT were analyzed by WB using indicated antibodies.


A T98G were treated or not with dsDNA for 6 h prior to IF using - PKcs - pDNA - PKcs - specific antibodies , DAPI ( n = 3 independent experiments ) .

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

A T98G cells were treated or not with dsDNA for 6 h prior to IF analysis using DNA-PKcs- and pDNA-PKcs-specific antibodies, and DAPI nuclear staining (n=3 independent experiments).


B Whole extracts from T98G were incubated with 80nt - long biotinylated ssDNA or dsDNA prior to pull - down using streptavidin - affinity beads . Input eluates were analyzed by WB using indicated antibodies .

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 O O O O O O

B Whole cell extracts from T98G cells were incubated with 80nt-long biotinylated ssDNA or dsDNA prior to pull-down using streptavidin-affinity beads. Input and eluates were analyzed by WB using indicated antibodies.


C T98G were treated with non - targeting ( CTRL ) or KU70 - targeting siRNAs prior to whole extract preparation , pull - down as in B . Inputs eluates were analyzed by WB using indicated antibodies .

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

C T98G cells were treated with non-targeting (CTRL) or KU70-targeting siRNAs prior to whole cell extract preparation, and pull-down as in B. Inputs and eluates were analyzed by WB using indicated antibodies.


A T98G stably expressing cGAS ( T98GcGAS ) or not ( T98GEmpty ) were transfected or not with dsDNA for 6 h prior to whole extraction WB using indicated antibodies .

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

A T98G cells stably expressing cGAS (T98GcGAS) or not (T98GEmpty) were transfected or not with dsDNA for 6 h prior to whole cell extraction and WB analysis using indicated antibodies.


C T98GEmpty T98GcGAS were transfected or not with biotinylated dsDNA prior to whole extraction pull - down using streptavidin - affinity beads . Inputs eluates were analyzed by WB using indicated antibodies .

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

C T98GEmpty and T98GcGAS were transfected or not with biotinylated dsDNA prior to whole cell extraction and pull-down using streptavidin-affinity beads. Inputs and eluates were analyzed by WB using indicated antibodies.


J T98GcGAS were transfected or not with dsDNA in presence or not of NU7441 , prior to WB using indicated antibodies .

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

J T98GcGAS were transfected or not with dsDNA in presence or not of NU7441, prior to WB analysis using indicated antibodies.


K THP - 1 were transfected or not with dsDNA in presence or not of NU7441 , prior to WB using indicated antibodies .

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

K THP-1 were transfected or not with dsDNA in presence or not of NU7441, prior to WB analysis using indicated antibodies.


F T98GEmpty T98GcGAS stably expressing a GFP reporter ( T98G - GFPEmpty T98G - GFPcGAS , respectively ) were xenotransplanted into head of tg ( mfap4 : RFP ) zebrafish at 3 days post fertilization ( dpf ) . Zebrafish were imaged daily over 3 days . graph represents mean ( ± ) of growth normalized by on day of transplantation ( n = 21 T98G - GFPEmpty n = 29 T98G - GFPcGAS ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 T98GEmpty and T98GcGAS stably expressing a GFP reporter (T98G-GFPEmpty and T98G-GFPcGAS, respectively) were xenotransplanted into the head of tg(mfap4:RFP) zebrafish line at 3 days post fertilization (dpf). Zebrafish embryos were imaged daily over 3 days. The graph represents the mean (±SEM) percentage of tumor growth normalized by the area on the day of transplantation (n=21 T98G-GFPEmpty and n=29 T98G-GFPcGAS embryos).


A Graph represents mean ( ± ) fold migration of THP - 1 through a 3 µm transwell insert when conditioned media from T98GEmpty or T98GcGAS was applied to lower chamber for 6 h . CCL2 was used as positive control ( n = 8 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 Graph represents the mean (± SEM) fold migration of THP-1 cells through a 3 µm transwell insert when conditioned media from T98GEmpty or T98GcGAS was applied to lower chamber for 6 h. CCL2 was used as positive control (n=8 biological replicates).


E Graph presents of macrophages recruited at site 24 h post xenotransplantation in D ( n = 21 T98G - GFPEmpty n = 29 T98G - GFPcGAS ) .

O O O O O 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 Graph presents the quantification of macrophages recruited at tumor site 24 h post xenotransplantation in D (n=21 T98G-GFPEmpty and n=29 T98G-GFPcGAS embryos).


G Graph presents survival rate of glioblastoma from A that present PRKDClow / MB21D1low , PRKDChigh / MB21D1low PRKDChigh / MB21D1high expression .

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

G Graph presents the survival rate of glioblastoma patients from A that present PRKDClow/MB21D1low, PRKDChigh/MB21D1low and PRKDChigh/MB21D1high expression.


H Violin plots present expression of PRKDC MB21D1 in datasets analyzed in A , on grade ( II to IV ) .

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

H Violin plots present the expression of PRKDC and MB21D1 in datasets analyzed in A, based on tumor grade (II to IV).


3D - brain model , horizontal brain section illustrating transgenic human P301Ltau expression in entorhinal cortex ( green , EC ) of ECrTgTaumouse lines , propagation of transgenic tau to dentate gyrus ( DG ) . Tau composition in ECrTgTau control mouse lines investigated .

O O O O 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

3D-brain model, horizontal brain section illustrating transgenic human P301Ltau expression in the entorhinal cortex (green, EC) of the ECrTgTaumouse lines, and the propagation of transgenic tau to the dentate gyrus (DG). Tau composition in ECrTgTau and control mouse lines investigated.


Immunostained horizontal sections show expression of human P301Ltau in ECneurons in absence of endogenous mousetau ( ECrTgTau - Mapt0 / 0 ) . in situ hybridization of humantau mRNA combined with immunofluorescent labeling ( Immuno - FISH ) of humantau ( huTau ) verifies P301Ltau transgene expression in EC . Scale bars 50 μm .

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

Immunostained horizontal sections show the expression of human P301Ltau in ECneurons in absence of endogenous mousetau (ECrTgTau-Mapt0/0). Fluorescence in situ hybridization of humantau mRNA combined with immunofluorescent labeling (Immuno-FISH) of humantau protein (huTau) verifies P301Ltau transgene expression in the EC. Scale bars 50 μm.


Propagation of humantau to neurons in DG ( arrows ) in ECrTgTau - Mapt0 / 0 mice . Close - ups show DG neurons from three ECrTgTau - Mapt0 / 0 mice ( DG I - III ) . Immuno - FISH proofs absence of humantau expression in DG neurons , which have huTau but no humantau mRNA . Scale bars 50 μm .

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

Propagation of humantau protein to neurons in the DG (white arrows) in ECrTgTau-Mapt0/0 mice. Close-ups show DG neurons from three ECrTgTau-Mapt0/0 mice (DG I-III). Immuno-FISH proofs the absence of humantau expression in DG neurons, which have huTau protein but no humantau mRNA. Scale bars 50 μm.


Immunostained horizontal sections of ECrTgTaumice show expression of human P301Ltau in ECneurons in presence of endogenous mousetau . Close - ups show DGneurons from three ECrTgTaumice ( DG I - III ) Immuno - FISH proofs absence of humantau expression in these DGneurons . Scale bars 50 μm .

Immunostained horizontal sections of ECrTgTaumice show the expression of human P301Ltau in ECneurons in presence of endogenous mousetau. Close-ups show DGneurons from three ECrTgTaumice (DG I-III) and Immuno-FISH proofs the absence of humantau expression in these DGneurons. Scale bars 50 μm.


Human P301Ltau propagation to DG neurons ( arrows ) in presence of endogenous mouse tau in ECrTgTau mice . Scale bars 50 µm .

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

Human P301Ltau propagation to DG neurons (white arrows) in presence of endogenous mouse tau in ECrTgTau mice. Scale bars 50 µm.


Human ( huTau , antibody Tau13 ) total tau ( hu + moTau , DAKO ) in entorhinal cortex ( EC ) extracts from 18 - month - oldmice show equal human P301Ltau expression in ECrTgTau ECrTgTau - Mapt0 / 0 mice ( p = 0 . 201 , n = 3 mice / group , one - way ANOVA with Bonferroni correction )

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 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

Human (huTau, antibody Tau13) and total tau (hu+moTau, DAKO) levels in entorhinal cortex (EC) extracts from 18-month-oldmice show equal human P301Ltau expression in ECrTgTau and ECrTgTau-Mapt0/0 mice (p=0.201, n=3 mice/group, one-way ANOVA with Bonferroni correction)


number of human tau - positive bodies in DG ( p = 0 . 58 , n = 4 sections 3 mice / group ) human tau in hippocampal ( HPC ) extracts ( p = 0 . 14 , n = 3 mice / group ) were similar in ECrTgTau - Mapt0 / 0 ECrTgTau mice ( two - Student ' s T - ) .

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

The number of human tau-positive cell bodies in the DG (p=0.58, n=4 sections and 3 mice/group) and human tau in hippocampal (HPC) extracts (p=0.14, n=3 mice/group) were similar in ECrTgTau-Mapt0/0 and ECrTgTau mice (two-tailed Student's T-test).


cortical neuron cultures that were transduced with AAVeGFP - 2a - P301Ltau at 7 DIV , fixed immunostained for GFP humantau ( Tau13 antibody ) at 14 DIV , show tau donor ( GFP + , huTau + ; ~ 10 % neurons ) a small number of tau recipient neurons ( GFP - , huTau + ; ~ 1 % neurons ) . Western blot of whole lysates verified efficient cleavage ( ~ 95 % ) of eGFP P301Ltau by 2a peptide ( n = 3 ) .

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

Primary cortical neuron cultures that were transduced with AAVeGFP-2a-P301Ltau at 7 DIV, and fixed and immunostained for GFP and humantau (Tau13 antibody) at 14 DIV, show tau donor (GFP+, huTau+; ~10% neurons) and a small number of tau recipient neurons (GFP-, huTau+; ~1% neurons). Western blot of whole cell lysates verified efficient cleavage (~95%) of eGFP and P301Ltau by the 2a peptide (n=3).


Eight after AAV injection into right EC of aged Mapt0 / 0 mice ( = 3 ) , immunostained brain sections showed that huTauP301L ( red ) propagated to a few DG " recipient neurons " ( arrows ) . Scale bar 50 µm .

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

Eight weeks after AAV injection into right EC of aged Mapt0/0 mice (=3), immunostained brain sections showed that huTauP301L (red) propagated to a few DG "recipient neurons" (white arrows). Scale bar 50 µm.


Unilateral AAV - mediated human P301L tau expression in EC DG of - matched WT mice ( n = 3 ) . Representative of brain sections show donor neurons in injected EC DG , a few tau recipient neurons ( arrows ) adjacent to AAV injection site . Scale bar 50 µm . In hemisphere of same brain section , some tau recipient neurons ( arrows ) were also present in ( non - injected ) axonal projection areas in EC ( GFP - filled terminal ends ) . Scale bar 100 µm .

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 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-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

Unilateral AAV-mediated human P301L tau expression in the EC and DG of age-matched WT mice (n=3). Representative images of brain sections show donor neurons in the injected EC and DG, and a few tau recipient neurons (white arrows) adjacent to the AAV injection site. Scale bar 50 µm.In the contralateral hemisphere of the same brain section, some tau recipient neurons (white arrows) were also present in the (non-injected) axonal projection areas in the contralateral EC (GFP-filled terminal ends). Scale bar 100 µm.


A . Brain sections from 18 - month - old ECrTgTau - Mapt0 / 0 ECrTgTau mice were co - immuolabeled for human tau misfolded tau ( Alz50 ) . Misfolded tau was only found in EC DG neurons ( arrows ) of ECrTgTau but not ECrTgTau - Mapt0 / 0 ( n = 4 sections / mouse , 3 mice / group ) . Scale bars 50 μm .

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

A. Brain sections from 18-month-old ECrTgTau-Mapt0/0 and ECrTgTau mice were co-immuolabeled for human tau and misfolded tau (Alz50). Misfolded tau was only found in EC and DG neurons (white arrows) of ECrTgTau but not ECrTgTau-Mapt0/0 animals (n=4 sections/mouse, 3 mice/group). Scale bars 50 μm.


B + C . Immunofluorescence labeling stereological counting of microglia in entorhinal cortex ( B ) astrocytes in hippocampus ( C ) indicated early signs of neurodegeneration in ECrTgTaumice . significantly increased number of Iba1 - positive microglia in EC layer II / III of ECrTgtau mice ( compared to WT ) was partially rescued in ECrTgTau - Mapt0 / 0 mice ( non - significant ) . number of GFAP - positive astrocytes was similar across genotypes ( non - significant ) . Mean ± , n = 4 sections per mouse , 3 mice / group , one - way ANOVA with Bonferroni correction . Scale bars 100 μm .

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

B+C. Immunofluorescence labeling and stereological counting of microglia in entorhinal cortex (B) and astrocytes in hippocampus (C) indicated early signs of neurodegeneration in ECrTgTaumice. The significantly increased number of Iba1-positive microglia in the EC layer II/III of ECrTgtau mice (compared to WT) was partially rescued in ECrTgTau-Mapt0/0 mice (non-significant). The number of GFAP-positive astrocytes was similar across all genotypes (non-significant). Mean±SEM, n=4 sections per mouse, 3 mice/group, one-way ANOVA with Bonferroni correction. Scale bars 100 μm.


Human , mouse total tau in cortical TBS - extracts of rTg4510 , rTg4510 - Mapt0 / 0 , control mice : amount of human tau ( Tau13 antibody ) was comparable in rTg4510 rTg4510 - Mapt0 / 0 , moTau ( Tau / 5 ) was comparable in WT rTg4510 , total tau ( hu + moTau , DAKO antibody ) were ( expected ) highest in rTg4510 mice . Mean ± , n = 3 mice / group , non - significant .

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

Human, mouse and total tau protein levels in cortical TBS-extracts of rTg4510, rTg4510-Mapt0/0, and control mice: The amount of human tau (Tau13 antibody) was comparable in rTg4510 and rTg4510-Mapt0/0, moTau (Tau/5) was comparable in WT and rTg4510, and total tau levels (hu+moTau, DAKO antibody) were (expected) highest in rTg4510 mice. Mean±SEM, n=3 mice/group, non-significant.


Whole brain weights of 9 - month - old revealed pronounced brain matter loss in rTg4510 compared to WT mice ( loss > 16 % ) , which was rescued in rTg4510 - Mapt0 / 0 mice to > 96 % . Mean ± , n = 5 mice / group .

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

Whole brain weights of 9-month-old animals revealed pronounced brain matter loss in rTg4510 compared to WT mice (weight loss >16%), which was rescued in rTg4510-Mapt0/0 mice to >96%. Mean±SEM, n=5 mice/group.


Cortical thickness measured adjacent to HPC , from CTX surface to corpus callosum , was decreased in rTg4510 mice by ≈ 25 % compared to WT mice . rTg4510 - Mapt0 / 0 showed no CTX thinning compared to Mapt0 / 0 or WT mcie . Mean ± , n = 3 mice / group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_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

Cortical thickness measured adjacent to HPC, from CTX surface to corpus callosum, was decreased in rTg4510 mice by ≈25% compared to WT mice. rTg4510-Mapt0/0 showed no CTX thinning compared to Mapt0/0 or WT mcie. Mean±SEM, n=3 mice/group.


number of neurons ( NeuN + ) in cortex of rTg4510 mice was significantly reduced to ≈ 67 % compared to both WT rTg4510 - Mapt0 / 0 . Mean ± , n = 3 mice / group .

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

The number of neurons (NeuN+ cells) in the cortex of rTg4510 mice was significantly reduced to ≈67% compared to both WT and rTg4510-Mapt0/0. Mean±SEM, n=3 mice/group.


volume of hippocampal region CA1 , with CT most effected regions in rTg4510 mice , was significantly reduced in rTg4510 by ~ 70 % volume ; rTg4510 - Mapt0 / 0 had significantly larger CA1 volume left ( reduced by only ~ 40 % ) . Mean ± , n = 3 mice / group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 volume of hippocampal region CA1, with CT the most effected regions in rTg4510 mice, was significantly reduced in rTg4510 by ~70% volume; rTg4510-Mapt0/0 had significantly larger CA1 volume left (reduced by only ~40%). Mean±SEM, n=3 mice/group.


rTg4510 showed strong signs of neuroinflammation with extremely high numbers of activated astroglia ( GFAP + , red ) microglia ( Iba1 + , ) in CTX compared to WT mice . Both astro - microgliosis was reduced by ~ 50 % in rTg4510 - Mapt0 / 0 mice . Mean ± , n = 3 sections / mouse 5 mice per / group . For panels : two - Student ' s T - one - way ANOVA with Bonferroni for multiple comparison , ns not significant , scale bars 100 µm .

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 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

rTg4510 showed strong signs of neuroinflammation with extremely high numbers of activated astroglia (GFAP+, red) and microglia (Iba1+, white) in the CTX compared to WT mice. Both astro- and microgliosis was reduced by ~50% in rTg4510-Mapt0/0 mice. Mean±SEM, n=3 sections/mouse and 5 mice per/group. For all panels: two-tailed Student's T-test and one-way ANOVA with Bonferroni for multiple comparison, ns not significant, scale bars 100 µm.


Cortical extracts from rTg4510 - Mapt0 / 0 brains had significantly less phospho - tau ( CP13 , PHF1 , 12E8 ) than rTg4510 extracts . Compared to WT mice , both transgenic tau lines had high of phospho - tau . Mean ± , n = 3 mice / group .

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

Cortical extracts from rTg4510-Mapt0/0 brains had significantly less phospho-tau (CP13, PHF1, 12E8) than rTg4510 extracts. Compared to WT mice, both transgenic tau lines had high levels of phospho-tau. Mean±SEM, n=3 mice/group.


Representative of gallyas silver stained aggregated tau in cortices from 12 - month - old mice unravel stunning differences in degree of tau pathology in rTg4510 compared to rTg4510 - Mapt0 / 0 mice . Mean ± , n = 3 mice / group .

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

Representative images of gallyas silver stained aggregated tau in cortices from 12-month-old mice unravel stunning differences in the degree of tau pathology in rTg4510 compared to rTg4510-Mapt0/0 mice. Mean±SEM, n=3 mice/group.


Higher magnification of silver ( 12 month - old ) Thioflavine - S ( 9month - old ) stained cortices show mature tangles ( arrows in Thio - S stain ) in rTg4510 rTg4510 - Mapt0 / 0 mice ; enhanced pathological changes such as neuritic tau accumulation neuropil vacuolation around NFTs are found only in rTg4510 mice . Stereological counting revealed similar numbers of cortical NFTs between in rTg4510 rTg4510 - Mapt0 / 0 mice at 9 12 month of . Because of pronounced neuronal only in rTg4510 mice , of tangle - bearing neurons was ≈ 1 . 6 to 1 . 8 - fold higher higher in 9 12 - month old rTg4510 mice . Mean ± , n = 3 sections / mouse , 3 mice / group .

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

Higher magnification images of silver (12 month-old) and Thioflavine-S (9month-old) stained cortices show mature tangles (white arrows in Thio-S stain) in rTg4510 and rTg4510-Mapt0/0 mice; enhanced pathological changes such as neuritic tau accumulation and neuropil vacuolation around NFTs are found only in rTg4510 mice. Stereological counting revealed similar numbers of cortical NFTs between in rTg4510 and rTg4510-Mapt0/0 mice at 9 and 12 month of age. Because of the pronounced neuronal death only in rTg4510 mice, the percentage of tangle-bearing neurons was ≈1.6 to 1.8-fold higher higher in 9 and 12-month old rTg4510 mice. Mean±SEM, n=3 sections/mouse, 3 mice/group.


Immuno - FISH for huTau mRNA ( green ) phospho - tau ( PHF1 , red ) shows obvious differences in distribution of neurons in cortex layer II / III : 9 - month - old rTg4510 mice had more neurons filled with NFT - like phosphorylated tau ( PHF1 + , red ) , rTg4510 - Mapt0 / 0 mice had significantly more huTau mRNA positive neurons ( FISH + ) . rTg4510 - Mapt0 / 0 mice had also more neurons still expressing both PHF1 huTau mRNA ( PHF1 + FISH + ) , suggesting a reduced neurotoxicity of P301Ltau expression in rTg4510 - Mapt0 / 0 mice . Mean ± , n = 3 sections per mouse , 3 mice / group . For panels : two - Student ' s T - one - way ANOVA with Bonferroni for multiple comparison , ns not significant , scale bars 100 μm .

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

Immuno-FISH for huTau mRNA (green) and phospho-tau (PHF1, red) shows obvious differences in the distribution of neurons in cortex layer II/III: 9-month-old rTg4510 mice had more neurons filled with NFT-like phosphorylated tau (PHF1+, red), and rTg4510-Mapt0/0 mice had significantly more huTau mRNA positive neurons (FISH+). rTg4510-Mapt0/0 mice had also more neurons still expressing both PHF1 and huTau mRNA (PHF1+ FISH+), suggesting a reduced neurotoxicity of P301Ltau expression in rTg4510-Mapt0/0 mice. Mean±SEM, n=3 sections per mouse, 3 mice/group. For all panels: two-tailed Student's T-test and one-way ANOVA with Bonferroni for multiple comparison, ns not significant, scale bars 100 μm.


Immuno - FISH showing EC neurons having both misfolded somatic tau ( Alz50 , red ) human tau mRNA ( green ; arrows ) in rTg4510 - Mapt0 / 0 but rarely in rTg4510 mice . n = 3 sections , 2 mice / group . Scale bars 50 μm .

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 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

Immuno-FISH showing EC neurons having both misfolded somatic tau (Alz50, red) and human tau mRNA (green; white arrows) in rTg4510-Mapt0/0 but rarely in rTg4510 mice. n=3 sections, 2 mice/group. Scale bars 50 μm.


Extraction of cortices revealed similar human tau ( Tau13 ) in TBS - extracts ( not significant ) but significantly more human tau in TritonX - 100 ( TX - 100 ) extracts of rTg4510 - Mapt0 / 0 compared to rTg4510 mice . Mean ± , n = 3 mice / group , two - Student ' s T , ns non significant .

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

Extraction of cortices revealed similar human tau (Tau13) in TBS-extracts (not significant) but significantly more human tau in TritonX-100 (TX-100) extracts of rTg4510-Mapt0/0 compared to rTg4510 mice. Mean±SEM, n =3 mice/group, two-tailed Student's T test, ns non significant.


Native gel electrophoresis of cortical TBS - extracts showed small differences in HMW ( oligomeric ) human tau between rTg4510 rTg4510 - Mapt0 / 0 brains . Western blot lanes were averaged across ~ 2 / 3 of width ( black rectangular arrow in Tau13 blot ) . mean ± ( n = 3 mice / group ) of these averages was plotted as longitudinal lane profiles . Differences in HMW tau are indicated by red black arrows .

O O O 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 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

Native gel electrophoresis of cortical TBS-extracts showed small differences in HMW (oligomeric) human tau between rTg4510 and rTg4510-Mapt0/0 brains. Western blot lanes were averaged across ~2/3 of the width (black rectangular and arrow in Tau13 blot). The mean±SEM (n=3 mice/group) of these averages was plotted as longitudinal lane profiles. Differences in HMW tau are indicated by red and black arrows.


TBS - brain extracts were applied to a HEK293 tau aggregation seeding ( Holmes et al , 2014 , Sanders et al , 2014 ) , in which TauRDP301S - CFP TauRDP301S - YFP are co - expressed intracellularly . formation of intracellular TauRDP301S leads to Foerster resonance energy transfer ( FRET ) activity between co - aggregated CFP YFP - tags correlates with tau aggregation seeding activity of applied brain extracts . After 24 hours , treated with extract ( 0 . 5 µg 1 . 0 µg total per 96 well ) from 9 - month - old rTg4510 had significantly more intracellular YFP - positive ( arrows ) compared to rTg4510 - Mapt0 / 0 extracts ; FRET activity of TauRDP301S appeared similar for both rTg4510 rTg4510 - Mapt0 / 0 . WT Mapt0 / 0 extracts never showed seeding activity . Addition of lipofectamine corrected for differences in uptake of tau led to similar differences in seeding activity between rTg4510 rTg4510 - Mapt0 / 0 mice . Two - Student ' s T - , mean ± ns , not significant , Insets 100 µm , Scale bars 50 µm .

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

TBS-brain extracts were applied to a HEK293 cell tau aggregation seeding assay(Holmes et al, 2014, Sanders et al, 2014), in which TauRDP301S-CFP and TauRDP301S-YFP are co-expressed intracellularly. The formation of intracellular fluorescent TauRDP301S aggregates leads to Foerster resonance energy transfer (FRET) activity between co-aggregated CFP and YFP-tags and correlates with the tau aggregation seeding activity of the applied brain extracts. After 24 hours, cells treated with extract (0.5 µg and 1.0 µg total protein per 96 well) from 9-month-old rTg4510 had significantly more intracellular YFP-positive (white arrows) aggregates compared to rTg4510-Mapt0/0 extracts; FRET activity of TauRDP301S aggregates appeared similar for both rTg4510 and rTg4510-Mapt0/0. WT and Mapt0/0 extracts never showed seeding activity. Addition of lipofectamine corrected for differences in cellular uptake of tau and led to similar differences in seeding activity between rTg4510 and rTg4510-Mapt0/0 mice. Two-tailed Student's T-test, mean±SEM ns, not significant, Insets 100 µm, Scale bars 50 µm.


A - D LSM of proximity ligation ( PLA ) dots composed of VEGFR3 phosphorylated tyrosine ( p - Tyr ) on cross - sections through jugular lymph sac / primordial thoracic ( jls / pTD ) of E13 . 5 control ILK K . O . . Scale bars : 10 µm . E - H LSM of PLA dots composed of VEGFR2 phosphorylated tyrosine ( p - Tyr ) on cross - sections through jls / pTD of E13 . 5 control ILK K . O . . Scale bars : 10 µm . I of PLA dots indicating VEGFR3 with phosphorylated tyrosine ( p - Tyr ) per LEC of E13 . 5 control or ILK K . O . ( n = 9 per genotype ) , * P = 0 . 022 . J of PLA dots indicating VEGFR2 with phosphorylated tyrosine ( p - Tyr ) per LEC of E13 . 5 control or ILK K . O . ( n = 3 per genotype ) . K of total number of PLA dots indicating both VEGFR2 ( left ) VEGFR3 ( right ) with phosphorylated tyrosine ( p - Tyr ) per jls / pTD section of E13 . 5 control or ILK K . O . ( n = 3 per genotype ) , * P = 0 . 005 ( VEGFR2 / p - Tyr in ILK K . O . versus VEGFR3 / p - Tyr in ILK K . O . ) , * P = 0 . 013 ( VEGFR3 / p - Tyr in control versus VEGFR3 / p - Tyr in ILK K . 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 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 B-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 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 O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O

A-D LSM images of proximity ligation assay (PLA) dots composed of VEGFR3 and phosphorylated tyrosine (p-Tyr) on cross-sections through the jugular lymph sac / primordial thoracic duct (jls/pTD) of E13.5 control and ILK K.O. embryos. Scale bars: 10 µm. E-H LSM images of PLA dots composed of VEGFR2 and phosphorylated tyrosine (p-Tyr) on cross-sections through the jls/pTD of E13.5 control and ILK K.O. embryos. Scale bars: 10 µm. I Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK K.O. embryos (n = 9 embryos per genotype), *P = 0.022. J Quantification of the PLA dots indicating VEGFR2 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK K.O. embryos (n = 3 embryos per genotype). K Quantification of the total number of PLA dots indicating both VEGFR2 (left) and VEGFR3 (right) with phosphorylated tyrosine (p-Tyr) per jls/pTD section of E13.5 control or ILK K.O. embryos (n = 3 embryos per genotype), *P = 0.005 (VEGFR2/p-Tyr in ILK K.O. versus VEGFR3/p-Tyr in ILK K.O.), *P = 0.013 (VEGFR3/p-Tyr in control versus VEGFR3/p-Tyr in ILK K.O.)


LSM of cross - sections through jugular lymph sac / primordial thoracic ( jls / pTD ) of an E13 . 5 control stained for surface VEGFR3 β1 integrin . Scale bar : 10

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

LSM images of cross-sections through the jugular lymph sac / primordial thoracic duct (jls/pTD) of an E13.5 control embryo stained for surface VEGFR3 and β1 integrin. Scale bar: 10 µm


LSM of proximity ligation ( PLA ) dots ( arrows ) composed of VEGFR3 β1 integrin on cross - sections through jls / pTD of E13 . 5 control ILK K . O . . Scale bars : 10 µm of VEGFR3 / β1 integrin PLA dots normalised to Lyve1 - positive of control or ILK K . O . ( n = 6 per genotype ) ,

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

LSM images of proximity ligation assay (PLA) dots (arrows) composed of VEGFR3 and β1 integrin on cross-sections through the jls/pTD of E13.5 control and ILK K.O. embryos. Scale bars: 10 µm Quantification of VEGFR3/β1 integrin PLA dots normalised to Lyve1-positive area of control or ILK K.O. embryos (n = 6 embryos per genotype),