( E ) Kymographs and quantification of anterograde , retrograde , mean velocity , and linear flow rate of BDNF - mCherry axonal trafficking into WT ( n = 894 vesicles / 94 axons ) , HTTSA ( n = 812 vesicles / 82 axons ) , or HTTSD ( n = 787 vesicles / 94 axons ) cortical neurons transfected with siCtl or siMecp2 , which significantly silenced Mecp2 ( 3 independent experiments ; one - way ANOVA with Tukey ' s multiple comparisons ) . ( F ) Cell viability in WT , HTTSA , or HTTSD neurons transfected with siCtl or siMecp2 measured by MTT assay ( WT siCtl : n = 3 ; WT siMecp2 : n = 3 ; HTTSD siMecp2 : n = 3 ; HTTSA siMecp2 : n = 2 ) . No differences were observed between the different groups ( 3 independent experiments ; one - way ANOVA Kruskal - Wallis test , p = 0 . 5701 ) Data information : Scale bars = 20 µm . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant . Data are presented as the means ± SEM .

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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kymographs and quantification of anterograde, retrograde, mean velocity, and linear flow rate of BDNF-mCherry axonal trafficking into WT (n= 894 vesicles/ 94 axons), HTTSA (n= 812 vesicles/ 82 axons), or HTTSD (n=787 vesicles/ 94 axons) cortical neurons transfected with siCtl or siMecp2, which significantly silenced Mecp2 (3 independent experiments; one-way ANOVA with Tukey's multiple comparisons). (F) Cell viability in WT, HTTSA, or HTTSD neurons transfected with siCtl or siMecp2 measured by MTT assay (WT siCtl: n=3; WT siMecp2: n=3; HTTSD siMecp2: n=3; HTTSA siMecp2: n=2). No differences were observed between the different groups (3 independent experiments; one-way ANOVA Kruskal-Wallis test, p=0.5701) Data information: Scale bars = 20 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Data are presented as the means ± SEM.


( A ) Left : The ratio between the striatal and the cortical BDNF determined by quantifying the level of BDNF proteins at the cortex and the striatum of 55 day - old WT ( n = 8 ) , Mecp2 KO ( n = 6 ) , Mecp2 KO / HTTSD ( mimicking the absence of phosphorylation ) ( n = 4 ) and Mecp2 KO / HTTSA mice ( mimicking constitutive phosphorylation ) ( n = 5 ) . Since striatal BDNF depends only on BDNF transport from the cortex , this ratio reflects BDNF transport through corticostriatal pathway ( Mann Whitney test ) . Right : Quantitative analysis of phospho TrkB protein level in striatum of KO WT mice and KO HTTSD mice by immunoblotting . The relative expression levels of phospho TrkB were normalized against GAPDH and are presented as the ratio . ( n = 6 mice per group ) ( Mann Whitney test ) Middle : Quantitative analysis of PSD - 95 protein level in striatum of Mecp2 KO / HTT WT mice and KO / HTTSD mice by immunoblotting . The relative expression levels of PSD - 95 were normalized against GAPDH and are presented as the ratio . ( n = 6 mice per group ) ( Mann Whitney test ) Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant .

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

(A) Left: The ratio between the striatal and the cortical BDNF determined by quantifying the level of BDNF proteins at the cortex and the striatum of 55 day-old WT (n=8), Mecp2 KO (n=6), Mecp2 KO/HTTSD (mimicking the absence of phosphorylation) (n=4) and Mecp2 KO/HTTSA mice (mimicking constitutive phosphorylation) (n=5). Since striatal BDNF depends only on BDNF transport from the cortex, this ratio reflects BDNF transport through corticostriatal pathway (Mann Whitney test). Right: Quantitative analysis of phospho TrkB protein level in striatum of KO WT mice and KO HTTSD mice by immunoblotting. The relative expression levels of phospho TrkB were normalized against GAPDH and are presented as the ratio. (n = 6 mice per group) (Mann Whitney test) Middle: Quantitative analysis of PSD-95 protein level in striatum of Mecp2 KO/HTT WT mice and KO/HTTSD mice by immunoblotting. The relative expression levels of PSD-95 were normalized against GAPDH and are presented as the ratio. (n = 6 mice per group) (Mann Whitney test) Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.


( B ) Left : We investigated the behavior of Mecp2 KO mice at 35 , 45 , and 55 days of age and assessed their survival . Right : Mecp2 KO / HTTSD mice ( n = 30 ) and WT mice ( n = 17 ) had a significantly longer lifespan than KO ( n = 22 ) or KO / HTTSA mice ( n = 21 ) ( Kaplan - Meier survival test ) . Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant .

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

(B) Left: We investigated the behavior of Mecp2 KO mice at 35, 45, and 55 days of age and assessed their survival. Right: Mecp2 KO/HTTSD mice (n = 30) and WT mice (n = 17) had a significantly longer lifespan than KO (n=22) or KO/HTTSA mice (n = 21) (Kaplan-Meier survival test). Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.


( C ) Body weight of 10 WT , 24 KO , 32 KO / HTTSD , and 24 KO / HTTSA mice at P30 and P50 ( one - way ANOVA with Tukey ' s comparison ) . Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant .

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

(C) Body weight of 10 WT, 24 KO, 32 KO/HTTSD, and 24 KO/HTTSA mice at P30 and P50 (one-way ANOVA with Tukey's comparison). Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.


( D ) Frequency of apnea of 9 WT , 10 KO , 14 KO / HTTSD , and 9 KO / HTTSA mice at P35 and P55 . ( Kruskal - Wallis test with Dunn ' s comparison ) . Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant .

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

(D) Frequency of apnea of 9 WT, 10 KO, 14 KO/HTTSD, and 9 KO/HTTSA mice at P35 and P55. (Kruskal-Wallis test with Dunn's comparison). Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.


( E ) Mecp2 KO / HTTSD mice ( n = 19 ) performed as well as WT ( n = 17 ) at P35 on the accelerating rotarod test and continued to outperform Mecp2 KO ( n = 16 ) and Mecp2 KO / HTTSA ( n = 13 ) at P55 ( one - way ANOVA with Fisher ' s LSD test ) . Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant .

O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mecp2 KO/HTTSD mice (n=19) performed as well as WT (n=17) at P35 on the accelerating rotarod test and continued to outperform Mecp2 KO (n=16) and Mecp2 KO/ HTTSA (n=13) at P55 (one-way ANOVA with Fisher's LSD test). Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.


( F ) Time before the onset of spontaneous locomotor activity of 17 WT , 20 KO , 19 KO / HTTSD , and 17 KO / HTTSA mice at P55 . ( Kruskal - Wallis test with Dunn ' s comparison ) . Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ns = not significant .

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

(F) Time before the onset of spontaneous locomotor activity of 17 WT, 20 KO, 19 KO/HTTSD, and 17 KO/HTTSA mice at P55. (Kruskal-Wallis test with Dunn's comparison). Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 ns = not significant.


( A ) Western blot analysis of DIV 5 cortical neurons transfected with siMecp2 or siControl ( siCtl ) and treated with 1 µM FK506 or vehicle for 1 h , and quantification of pS421 HTT ( n = 9 per group ) . Data are presented as means ± SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Mann - Whitney test .

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

(A) Western blot analysis of DIV 5 cortical neurons transfected with siMecp2 or siControl (siCtl) and treated with 1 µM FK506 or vehicle for 1 h, and quantification of pS421 HTT (n=9 per group). Data are presented as means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Mann-Whitney test.


( B ) Representative kymographs showing axonal trafficking of BDNF - mCherry - containing vesicles in cortical neurons transfected with siMecp2 or siControl ( siCtl ) and treated with 1 µM FK506 or vehicle for 1 h . Scale bar = 20 µm .

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

(B) Representative kymographs showing axonal trafficking of BDNF-mCherry-containing vesicles in cortical neurons transfected with siMecp2 or siControl (siCtl) and treated with 1 µM FK506 or vehicle for 1 h. Scale bar = 20 µm.


( C ) Quantification of anterograde , retrograde , mean velocity , linear flow rate and number of BDNF - mCherry - containing vesicles from the data in ( A ) ( siMecp2 + Vehicle : n = 801 vesicles / 95 axons ; siMecp2 + FK506 : n = 1020 vesicles / 116 axons ; siCtl + Vehicle : n = 780 vesicles / 83 axons ; siCtl + FK506 : n = 1029 vesicles / 102 axons ) . Data are presented as the mean ± SEM of at least three independent experiments ( one - way ANOVA with Tukey ' s comparison ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant .

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

(C) Quantification of anterograde, retrograde, mean velocity, linear flow rate and number of BDNF-mCherry-containing vesicles from the data in (A) (siMecp2+Vehicle: n=801 vesicles/95 axons; siMecp2+FK506: n=1020 vesicles/116 axons; siCtl+Vehicle: n=780 vesicles/83 axons; siCtl+FK506: n=1029 vesicles/102 axons). Data are presented as the mean ± SEM of at least three independent experiments (one-way ANOVA with Tukey's comparison). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.


( A ) Western blot of HTT S421 phosphorylation . We treated 30 - day - old Mecp2 KO mice intraperitoneally with FK506 ( 5 mg / kg ) or vehicle and analyzed brain extracts for endogenous HTT phosphorylation by western blotting , 2 h after administration , using an anti - phospho - HTT - S421 specific antibody . The D7F7 antibody recognizes total HTT . The relative protein level of phospho HTT was normalized on total HTT protein level and are presented as the ratio ( KO FK506 n = 4 , KO Vehicle n = 4 ) . Data are presented as means ± SEM , * p < 0 . 05 , Mann Whitney test . )

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

(A) Western blot of HTT S421 phosphorylation. We treated 30-day-old Mecp2 KO mice intraperitoneally with FK506 (5 mg/kg) or vehicle and analyzed brain extracts for endogenous HTT phosphorylation by western blotting, 2 h after administration, using an anti-phospho-HTT-S421 specific antibody. The D7F7 antibody recognizes total HTT. The relative protein level of phospho HTT was normalized on total HTT protein level and are presented as the ratio (KO FK506 n = 4, KO Vehicle n = 4). Data are presented as means ± SEM, *p < 0.05, Mann Whitney test.)


We treated 30 - day - old Mecp2 KO mice ( n = 10 ) and Mecp2 KO / HTTSA mice ( n = 10 ) with 5mg / kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests . ( B ) FK506 - treated KO mice lived longer than vehicle - treated KO mice and FK506 - treated KO HTTSA mice ( Kaplan - Meier survival test ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant . Data are means ± SEM .

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-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 B-CONTROLLED_VAR 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

We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (B) FK506-treated KO mice lived longer than vehicle-treated KO mice and FK506-treated KO HTTSA mice (Kaplan-Meier survival test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.


We treated 30 - day - old Mecp2 KO mice ( n = 10 ) and Mecp2 KO / HTTSA mice ( n = 10 ) with 5mg / kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests . ( C ) Body weight of FK506 - treated KO , FK506 - treated KO HTTSA and vehicle - treated KO mice at P35 and P55 ( Mann - Whitney test ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant . Data are means ± SEM .

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

We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (C) Body weight of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice at P35 and P55 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.


We treated 30 - day - old Mecp2 KO mice ( n = 10 ) and Mecp2 KO / HTTSA mice ( n = 10 ) with 5mg / kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests . ( D ) Frequency of apnea of FK506 - treated KO , FK506 - treated KO HTTSA and vehicle - treated KO mice at P35 and P55 ( Mann - Whitney test ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant . Data are means ± SEM .

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

We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (D) Frequency of apnea of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice at P35 and P55 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.


We treated 30 - day - old Mecp2 KO mice ( n = 10 ) and Mecp2 KO / HTTSA mice ( n = 10 ) with 5mg / kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests . ( E ) Motor coordination of FK506 - treated KO , FK506 - treated KO HTTSA and vehicle - treated KO mice on the accelerating rotarod test at P30 and P50 ( Mann - Whitney test ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant . Data are means ± SEM .

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

We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (E) Motor coordination of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice on the accelerating rotarod test at P30 and P50 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.


We treated 30 - day - old Mecp2 KO mice ( n = 10 ) and Mecp2 KO / HTTSA mice ( n = 10 ) with 5mg / kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests . ( F ) Forelimb strength of FK506 - treated KO , FK506 - treated KO HTTSA and vehicle - treated KO mice assessed by the grip strength test at P40 and P60 ( Mann - Whitney test ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant . Data are means ± SEM .

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

We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (F) Forelimb strength of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice assessed by the grip strength test at P40 and P60 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.


Figure 1 . Helix - 7 dynamically interacts with the miRNA seed region . A ) Cartoon representation of the Ago2 - miRNA crystal structure . Ago2 is colored gray except for helix - 7 , which is yellow . miRNA guide colored red . C ) Close - up view of the unpaired complex shows helix - 7 breaks nucleobase stacking in the miRNA seed region by intercalating between g6 and g7 .

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

Figure 1. Helix-7 dynamically interacts with the miRNA seed region. A) Cartoon representation of the Ago2-miRNA crystal structure. Ago2 is colored gray except for helix-7, which is yellow. miRNA guide colored red.C) Close-up view of the unpaired complex shows helix-7 breaks nucleobase stacking in the miRNA seed region by intercalating between g6 and g7.


B ) Crystal structure of the Ago2 - miRNA - target RNA ternary complex . Target RNA is colored blue . D ) Close - up view of the Ago2 - miRNA - target complex shows helix - 7 docks into the minor groove of the guide : target duplex , directly contacting base pairs at positions g6 and g7 .

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

B) Crystal structure of the Ago2-miRNA-target RNA ternary complex. Target RNA is colored blue. D) Close-up view of the Ago2-miRNA-target complex shows helix-7 docks into the minor groove of the guide:target duplex, directly contacting base pairs at positions g6 and g7.


Figure 2 . Helix - 7 is not required for target recognition . A ) Plot of g2 - g8 target RNA bound ( 0 . 1 nM ) versus Ago2 - miR122 concentration for wild type ( WT ) , helix - 7 double point mutant ( MI - AA ) , and Ηhelix - 7 Ago2 . Average values from at least three independent experiments ± standard deviation ( SD ) are plotted . Top panel shows target RNA paired to seed region of the guide RNA ( miRNA - 122 ) .

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

Figure 2. Helix-7 is not required for target recognition. A) Plot of g2-g8 target RNA bound (0.1 nM) versus Ago2-miR122 concentration for wild type (WT), helix-7 double point mutant (MI-AA), and Ηhelix-7 Ago2. Average values from at least three independent experiments ± standard deviation (SD) are plotted. Top panel shows target RNA paired to seed region of the guide RNA (miRNA-122).


B ) Dissociation of a 32P - labeled target RNA ( 0 . 1 nM ) from the Ago2 - miR122 complex ( 1 nM ) was monitored in the presence of unlabeled target RNA ( 100 nM ) . Fraction of the target RNA bound to Ago2 - miR122 is plotted as a function of time for WT and Ηhelix - 7 Ago2 . Average values from at least three independent experiments ± SD were fit to single exponential decays .

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B) Dissociation of a 32P-labeled target RNA (0.1 nM) from the Ago2-miR122 complex (1 nM) was monitored in the presence of unlabeled target RNA (100 nM). Fraction of the target RNA bound to Ago2-miR122 is plotted as a function of time for WT and Ηhelix-7 Ago2. Average values from at least three independent experiments ± SD were fit to single exponential decays.


C ) Representative fluorescence time traces ( with a resolution of 100 ms for wt , 300 ms for MI - AA and Ηhelix - 7 ) show docking and dissociation of wild type ( top ) , MI - AA ( middle ) , and Ηhelix - 7 ( bottom ) Ago2 - miRNA at single spots in the microfluidic chamber .

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C) Representative fluorescence time traces (with a resolution of 100 ms for wt, 300 ms for MI-AA and Ηhelix-7) show docking and dissociation of wild type (top), MI-AA (middle), and Ηhelix-7 (bottom) Ago2-miRNA at single spots in the microfluidic chamber.


D ) Dwell time histograms obtained for N = 4 , 5 and 6 , with wild type Ago2 ; histograms were fit with single exponential decay . The first column of the data was not included in the analysis to avoid potential artifacts arising from the limit of the time resolution . The bin size is 0 . 5 sec for N = 4 and 1 sec for N = 5 and 6 .

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D) Dwell time histograms obtained for N = 4, 5 and 6, with wild type Ago2; histograms were fit with single exponential decay. The first column of the data was not included in the analysis to avoid potential artifacts arising from the limit of the time resolution. The bin size is 0.5 sec for N =4 and 1 sec for N =5 and 6.


E ) Bar plot showing the dependence of dwell time on N for wild type Ago2 . The dashed red line indicates the observation time limit ( 300 s ) , which is constrained by photobleaching . For all bar plots ( E , G and I ) error bars are the SD of three independent experiments carried out on different days .

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E) Bar plot showing the dependence of dwell time on N for wild type Ago2. The dashed red line indicates the observation time limit (300 s), which is constrained by photobleaching.For all bar plots (E, G and I) error bars are the SD of three independent experiments carried out on different days.


F ) Dwell time histogram for different N ' s with the MI - AA helix - 7 mutant . The dwell time histogram for N = 6 fit with double exponential decay ( green ; R2 = 0 . 98 ) ; The dwell time for N = 6 with single exponential fit was 15 . 2 ± 3 . 41 sec , R2 = 0 . 96 ) .

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F) Dwell time histogram for different N's with the MI-AA helix-7 mutant. The dwell time histogram for N = 6 fit with double exponential decay (green; R2 = 0.98); The dwell time for N = 6 with single exponential fit was 15.2 ± 3.41 sec, R2 = 0.96).


G ) Bar plot for dwell times of MI - AA Ago2 as a function of N . For all bar plots ( E , G and I ) error bars are the SD of three independent experiments carried out on different days .

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G) Bar plot for dwell times of MI-AA Ago2 as a function of N. For all bar plots (E, G and I) error bars are the SD of three independent experiments carried out on different days.


H ) Dwell time histogram for different N ' s with the ∆ helix - 7 mutant . The dwell time histograms for N = 5 and N = 6 were fit with double exponential decays ( green ; R2 = 0 . 97 for N = 5 and R2 = 0 . 96 for N = 6 ) ; The dwell time for N = 5 and N = 6 with single exponential fit was 9 . 1 ± 0 . 91 sec ( R2 = 0 . 86 ) and 22 . 3 ± 5 . 08 sec ( R2 = 0 . 90 ) respectively ) .

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H) Dwell time histogram for different N's with the ∆helix-7 mutant. The dwell time histograms for N = 5 and N = 6 were fit with double exponential decays (green; R2 = 0.97 for N = 5 and R2 = 0.96 for N = 6); The dwell time for N = 5 and N = 6 with single exponential fit was 9.1 ± 0.91 sec (R2 = 0.86) and 22.3 ± 5.08 sec (R2 = 0.90) respectively).


I ) Bar plot for dwell times of ∆ helix - 7 Ago2 as a function of N . For all bar plots ( E , G and I ) error bars are the SD of three independent experiments carried out on different days .

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I) Bar plot for dwell times of ∆helix-7 Ago2 as a function of N. For all bar plots (E, G and I) error bars are the SD of three independent experiments carried out on different days.


Figure 4 . Helix - 7 discourages non - Watson Crick pairing at g6 and g7 . A - C ) Plots of target RNA ( 0 . 1 nM ) bound to Ago2 versus Ago2 - miR122 ( wild type and Ηhelix - 7 ) concentration for targets with an increasing number of G : U wobble pairs in the seed 3 ' end . D ) Plot of target RNA ( 0 . 1 nM ) bound to Ago2 versus Ago2 - miR122 concentration for a target with mismatches at positions g6 - g8 . For A - E , average values from at least three independent experiments ± SD are plotted .

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Figure 4. Helix-7 discourages non-Watson Crick pairing at g6 and g7. A-C) Plots of target RNA (0.1 nM) bound to Ago2 versus Ago2-miR122 (wild type and Ηhelix-7) concentration for targets with an increasing number of G:U wobble pairs in the seed 3' end. D) Plot of target RNA (0.1 nM) bound to Ago2 versus Ago2-miR122 concentration for a target with mismatches at positions g6-g8. For A-E, average values from at least three independent experiments ± SD are plotted.


E ) Bar plots of the Kd of wild type ( left ) Ηhelix - 7 ( right ) Ago2 - miR122 for indicated target RNAs . For A - E , average values from at least three independent experiments ± SD are plotted .

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E) Bar plots of the Kd of wild type (left) Ηhelix-7 (right) Ago2-miR122 for indicated target RNAs. For A-E, average values from at least three independent experiments ± SD are plotted.


Figure 5 . ΗHelix - 7 Ago2 is sensitive to inhibition by off - target RNAs . A ) Ago2 - miRNA122 ( 1 nM ) was incubated with a 32P - labeled , seed - matched target RNA ( 0 . 1 nM ) in the presence of increasing concentrations of unlabeled a competitor RNA ( top panel ) . Fraction target RNA bound to Ago2 is plotted as a function of off - target competitor RNA concentration . B ) Fraction of target RNA bound to Ago2 in the presence of increasing concentrations of unlabeled total cellular RNA . For A - B , average values from at least three independent experiments ± SD are plotted .

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Figure 5. ΗHelix-7 Ago2 is sensitive to inhibition by off-target RNAs. A) Ago2-miRNA122 (1 nM) was incubated with a 32P-labeled, seed-matched target RNA (0.1 nM) in the presence of increasing concentrations of unlabeled a competitor RNA (top panel). Fraction target RNA bound to Ago2 is plotted as a function of off-target competitor RNA concentration. B) Fraction of target RNA bound to Ago2 in the presence of increasing concentrations of unlabeled total cellular RNA. For A-B, average values from at least three independent experiments ± SD are plotted.


Figure 6 . Helix - 7 mutants display reduced target - binding rates . A ) Ago2 - miR122 complexes were mixed with a 32P - labeled seed matched target RNA ; bound and free RNAs were then separated using a filter - binding apparatus at various times . Representative time course for target RNA ( 0 . 1 nM ) binding to wild type , MI - AA , and Δhelix - 7 Ago2 ( 1 nM ) . Average values from at least three independent experiments ± SD are plotted .

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Figure 6. Helix-7 mutants display reduced target-binding rates. A) Ago2-miR122 complexes were mixed with a 32P-labeled seed matched target RNA; bound and free RNAs were then separated using a filter-binding apparatus at various times. Representative time course for target RNA (0.1 nM) binding to wild type, MI-AA, and Δhelix-7 Ago2 (1 nM). Average values from at least three independent experiments ± SD are plotted.


B ) Observed binding rates ( kon , obs ) plotted as a function of Ago2 - miR122 concentration . Average values ± SD are plotted .

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B) Observed binding rates (kon, obs) plotted as a function of Ago2-miR122 concentration. Average values ± SD are plotted.


C ) Arrival time distribution of single Ago2 - miRNA complexes binding immobilized targets in a microfluidic chamber . The value is the average of three independent measurements .

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C) Arrival time distribution of single Ago2-miRNA complexes binding immobilized targets in a microfluidic chamber. The value is the average of three independent measurements.


Figure 7 . Structure of a helix - 7 Ago2 mutant . A ) Superposition of wild type ( white ) and MI - AA ( gray ) Ago2 . Helix - 7 colored yellow ; ordered region of the guide RNAs colored in red .

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Figure 7. Structure of a helix-7 Ago2 mutant. A) Superposition of wild type (white) and MI-AA (gray) Ago2. Helix-7 colored yellow; ordered region of the guide RNAs colored in red.


B ) Close - up view shows helix - 7 tilts away from the seed region in the MI - AA mutant .

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B) Close-up view shows helix-7 tilts away from the seed region in the MI-AA mutant.


C ) Side - by - side comparison of the seed region in wild type ( left ) and MI - AA ( right ) Ago2 crystal structures .

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C) Side-by-side comparison of the seed region in wild type (left) and MI-AA (right) Ago2 crystal structures.


SIRT1 activity is required for resveratrol - induced autophagy but not for spermidine - mediated autophagy induction in mammalian cultured cells . ( A - C ) Human colon carcinoma HCT 116 cells were left untransfected ( Co , control ) or transfected with an irrelevant siRNA ( UNR , unrelated ) or siRNAs specific for ATG5 , ATG7 , or SIRT1 and then retransfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and left untreated or treated for 4 h with 100 - µM resveratrol ( Resv ) or spermidine ( Spd ) . The same experiment was performed in the presence of bafilomycin A1 ( BafA1 ) , which inhibits the fusion between lysosomes and autophagosomes , to evaluate the autophagic flux . ( A ) Representative images . ( B ) Quantitative data .

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SIRT1 activity is required for resveratrol-induced autophagy but not for spermidine-mediated autophagy induction in mammalian cultured cells. (A-C) Human colon carcinoma HCT 116 cells were left untransfected (Co, control) or transfected with an irrelevant siRNA (UNR, unrelated) or siRNAs specific for ATG5, ATG7, or SIRT1 and then retransfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and left untreated or treated for 4 h with 100-µM resveratrol (Resv) or spermidine (Spd). The same experiment was performed in the presence of bafilomycin A1 (BafA1), which inhibits the fusion between lysosomes and autophagosomes, to evaluate the autophagic flux. (A) Representative images. (B) Quantitative data.


SIRT1 activity is required for resveratrol - induced autophagy but not for spermidine - mediated autophagy induction in mammalian cultured cells . ( A - C ) Human colon carcinoma HCT 116 cells were left untransfected ( Co , control ) or transfected with an irrelevant siRNA ( UNR , unrelated ) or siRNAs specific for ATG5 , ATG7 , or SIRT1 and then retransfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and left untreated or treated for 4 h with 100 - µM resveratrol ( Resv ) or spermidine ( Spd ) . The same experiment was performed in the presence of bafilomycin A1 ( BafA1 ) , which inhibits the fusion between lysosomes and autophagosomes , to evaluate the autophagic flux . ( A ) Representative images . ( B ) Quantitative data . ( C ) Representative immunoblots of HCT 116 cells transfected either with an unrelated siRNA or with a SIRT1 - specific siRNA showing LC3 lipidation after treatment with 100 - µM spermidine in the presence or absence of bafilomycin A1 .

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SIRT1 activity is required for resveratrol-induced autophagy but not for spermidine-mediated autophagy induction in mammalian cultured cells. (A-C) Human colon carcinoma HCT 116 cells were left untransfected (Co, control) or transfected with an irrelevant siRNA (UNR, unrelated) or siRNAs specific for ATG5, ATG7, or SIRT1 and then retransfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and left untreated or treated for 4 h with 100-µM resveratrol (Resv) or spermidine (Spd). The same experiment was performed in the presence of bafilomycin A1 (BafA1), which inhibits the fusion between lysosomes and autophagosomes, to evaluate the autophagic flux. (A) Representative images. (B) Quantitative data. (C) Representative immunoblots of HCT 116 cells transfected either with an unrelated siRNA or with a SIRT1-specific siRNA showing LC3 lipidation after treatment with 100-µM spermidine in the presence or absence of bafilomycin A1.


( D - F ) HCT 116 cells were left transfected with a GFP - LC3 plasmid , cultured in complete medium for 24 h , and treated with either vehicle ( Co ) , 100 - µM resveratrol , or 100 - µM spermidine in the presence or absence of the SIRT1 inhibitor EX527 for 4 h . ( D ) Representative images . ( E ) Quantitative data . ( B and E ) Bars depict the percentages of cells showing accumulation of GFP - LC3 in puncta ( GFP - LC3vac ; means ± SEM ; n = 3 ; * , P < 0 . 05 ) . ( F ) Representative immunoblots showing LC3 lipidation in HCT 116 cells treated with 100 - µM spermidine in the presence or absence of EX527 . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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(D-F) HCT 116 cells were left transfected with a GFP-LC3 plasmid, cultured in complete medium for 24 h, and treated with either vehicle (Co), 100-µM resveratrol, or 100-µM spermidine in the presence or absence of the SIRT1 inhibitor EX527 for 4 h. (D) Representative images. (E) Quantitative data. (B and E) Bars depict the percentages of cells showing accumulation of GFP-LC3 in puncta (GFP-LC3vac; means ± SEM; n = 3; *, P < 0.05). (F) Representative immunoblots showing LC3 lipidation in HCT 116 cells treated with 100-µM spermidine in the presence or absence of EX527. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


The lifespan - extending and autophagy - inducing effects of spermidine in yeast are not mediated by Sir2 . ( A - E ) EGFP - Atg8p was ectopically expressed in wild - type ( WT ) or Δsir2 S . cerevisiae undergoing chronological aging on small synthetic 2 % glucose media with or without ( Co , control ) supplementation of 4 - mM spermidine ( Spd ) . ( A ) Representative images . EGFP - Atg8p localization ( bottom ) was visualized by fluorescence microscopy . Yeast cells undergoing autophagy ( in which EGFP - Atg8p exhibits a prominent vacuolar localization ) are indicated by arrows . Yeast morphology was monitored by differential interference contrast ( DIC ; top ) .

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The lifespan-extending and autophagy-inducing effects of spermidine in yeast are not mediated by Sir2. (A-E) EGFP-Atg8p was ectopically expressed in wild-type (WT) or Δsir2 S. cerevisiae undergoing chronological aging on small synthetic 2% glucose media with or without (Co, control) supplementation of 4-mM spermidine (Spd). (A) Representative images. EGFP-Atg8p localization (bottom) was visualized by fluorescence microscopy. Yeast cells undergoing autophagy (in which EGFP-Atg8p exhibits a prominent vacuolar localization) are indicated by arrows. Yeast morphology was monitored by differential interference contrast (DIC; top).


( B ) Representative immunoblots against EGFP . Free EGFP indicates the vacuolar degradation of EGFP - Atg8p fusion , thereby representing the autophagic flux . Notice that both WT and Δsir2 yeast cells show similar free EGFP levels after spermidine - mediated autophagy induction .

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(B) Representative immunoblots against EGFP. Free EGFP indicates the vacuolar degradation of EGFP-Atg8p fusion, thereby representing the autophagic flux. Notice that both WT and Δsir2 yeast cells show similar free EGFP levels after spermidine-mediated autophagy induction.


( C ) Relative alkaline phosphatase ( ALP ) activity indicative of autophagy . n = 3 .

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(C) Relative alkaline phosphatase (ALP) activity indicative of autophagy. n = 3.


( E ) Quantification of reactive oxygen species . Bars indicate the percentages of cells exhibiting the reactive oxygen species - mediated conversion of dihydroethidine ( DHE ) into ethidium ( Eth ; n = 4 ) . Data represent means ± SEM ; * , P < 0 . 001 as compared with untreated cells of the same genotype . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase . RFU , relative fluorescence unit .

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(E) Quantification of reactive oxygen species. Bars indicate the percentages of cells exhibiting the reactive oxygen species-mediated conversion of dihydroethidine (DHE) into ethidium (Eth; n = 4). Data represent means ± SEM; *, P < 0.001 as compared with untreated cells of the same genotype. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. RFU, relative fluorescence unit.


The life - extending and autophagy - inducing effects of spermidine in C . elegans are not mediated by Sir2 . ( A ) Fluorescence microscopy of C . elegans transgenic embryos expressing a full - length plgg - 1DsRed : : LGG - 1 fusion protein indicative of autophagic activity . Two representative pictures of wild - type ( WT ) and sir - 2 . 1 embryos untreated ( Co , control ) or treated with 0 . 2 - mM spermidine ( Spd ) supplementation of food are shown . ( B ) Quantification of autophagic activity through the measurement of DsRed : : LGG - 1 pixel intensity from images of WT animals shown in A . Data represent means ± SEM ( n = 3 ) with ≥ 25 images processed for each trial .

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The life-extending and autophagy-inducing effects of spermidine in C. elegans are not mediated by Sir2. (A) Fluorescence microscopy of C. elegans transgenic embryos expressing a full-length plgg-1DsRed::LGG-1 fusion protein indicative of autophagic activity. Two representative pictures of wild-type (WT) and sir-2.1 embryos untreated (Co, control) or treated with 0.2-mM spermidine (Spd) supplementation of food are shown. (B) Quantification of autophagic activity through the measurement of DsRed::LGG-1 pixel intensity from images of WT animals shown in A. Data represent means ± SEM (n = 3) with ≥25 images processed for each trial.


C ) Survival of WT C . elegans during aging with and without ( control ) supplementation of food ( UV - killed E . coli ) with 0 . 2 - mM spermidine ( n = 110 ; P < 0 . 005 ) . ( D ) Survival of sir - 2 . 1 C . elegans ( ok434 phenotype ) during aging with and without ( control ) supplementation of food ( UV - killed E . coli ) with 0 . 2 - mM spermidine ( n = 110 ; P < 0 . 01 ) . P - values were calculated using the log - rank test as described in Materials and methods .

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C) Survival of WT C. elegans during aging with and without (control) supplementation of food (UV-killed E. coli) with 0.2-mM spermidine (n = 110; P < 0.005). (D) Survival of sir-2.1 C. elegans (ok434 phenotype) during aging with and without (control) supplementation of food (UV-killed E. coli) with 0.2-mM spermidine (n = 110; P < 0.01). P-values were calculated using the log-rank test as described in Materials and methods.


Convergent alterations in the phosphoproteome status after resveratrol and / or spermidine treatment . ( A - C ) Human colon carcinoma HCT 116 cells were treated for 2 h with vehicle ( Co , control ) , 100 - µM resveratrol ( Resv ) , and 100 - µM spermidine ( Spd ) , alone or in combination ( Resv + Spd ) . ( A ) Representative phosphoprotein arrays are shown .

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Convergent alterations in the phosphoproteome status after resveratrol and/or spermidine treatment. (A-C) Human colon carcinoma HCT 116 cells were treated for 2 h with vehicle (Co, control), 100-µM resveratrol (Resv), and 100-µM spermidine (Spd), alone or in combination (Resv + Spd). (A) Representative phosphoprotein arrays are shown.


( B ) Clustering analysis for the effects on protein kinase phosphorylation .

(B) Clustering analysis for the effects on protein kinase phosphorylation.


( C ) Representative immunoblots of selected kinases whose phosphorylation status was unaffected ( PRKAA1 , RPS6KB1 , and acetyl - CoA carboxylase a [ ACACA ] ) or affected by resveratrol or / and spermidine treatment ( PTKB , AKT1 , MAPK8 , and CDKN1B ) , validating phosphoprotein array data .

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(C) Representative immunoblots of selected kinases whose phosphorylation status was unaffected (PRKAA1, RPS6KB1, and acetyl-CoA carboxylase a [ACACA]) or affected by resveratrol or/and spermidine treatment (PTKB, AKT1, MAPK8, and CDKN1B), validating phosphoprotein array data.


( D ) Human colorectal carcinoma HCT 116 cells were transfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and then treated with either vehicle or the indicated dose of resveratrol or spermidine , alone or in combination , for 2 h . Quantitative data . Bars depict the percentages ( means ± SD ; n = 3 ; * , P < 0 . 05 ) of cells showing the accumulation of GFP - LC3 in puncta ( GFP - LC3vac ) . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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(D) Human colorectal carcinoma HCT 116 cells were transfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and then treated with either vehicle or the indicated dose of resveratrol or spermidine, alone or in combination, for 2 h. Quantitative data. Bars depict the percentages (means ± SD; n = 3; *, P < 0.05) of cells showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


Convergent acetylproteome modification after resveratrol or spermidine treatment . ( A - C ) Colon carcinoma HCT 116 cells were cultured for 2 wk in three different SILAC media containing different arginine and lysine isotopes . Cells were treated with 100 - µM resveratrol ( Resv ) or spermidine ( Spd ) for 2 h , fractioned into cytoplasmic , nuclear , and mitochondrial extracts , processed for acetyl lysine peptide enrichment , and analyzed by MS . ( A ) Hierarchical clustering of drug - specific organellar distributions of all acetylated sites quantified in at least one fraction . Fold changes are calculated relative to untreated cells . Only sites regulated > 1 . 5 - fold were included in statistical analyses . ( B ) Graphical representation of peptides whose acetylation status was affected in a convergent or divergent way . n = 560 .

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Convergent acetylproteome modification after resveratrol or spermidine treatment. (A-C) Colon carcinoma HCT 116 cells were cultured for 2 wk in three different SILAC media containing different arginine and lysine isotopes. Cells were treated with 100-µM resveratrol (Resv) or spermidine (Spd) for 2 h, fractioned into cytoplasmic, nuclear, and mitochondrial extracts, processed for acetyl lysine peptide enrichment, and analyzed by MS. (A) Hierarchical clustering of drug-specific organellar distributions of all acetylated sites quantified in at least one fraction. Fold changes are calculated relative to untreated cells. Only sites regulated >1.5-fold were included in statistical analyses. (B) Graphical representation of peptides whose acetylation status was affected in a convergent or divergent way. n = 560.


Significant motifs among sites undergoing acetylation . ( A ) Hierarchical clustering of the organellar distributions of sites whose acetylation was increased by > 1 . 5 - fold in response to resveratrol or spermidine , at least in one organellar fraction . ( B ) Consensus acetylation motifs identified upon resveratrol ( left ) or spermidine ( right ) treatment are depicted using the MotifX algorithm ( Schwartz and Gygi , 2005 ) . Among sites that were hyperacetylated in response to both agents , the K ( F / Y ) motif is significantly enriched when tested against the whole proteome ( P < 0 . 00001 ) . ( C ) When testing against the largest acetylation site dataset from Choudhary et al . ( 2009 ) ( acetylation background dataset [ ABD ] ) , the SxK motif is significant ( P < 0 . 0001 ) for sites whose acetylation increased upon spermidine treatment . No general consensus motifs were found for sites whose acetylation increased in response to both agents .

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Significant motifs among sites undergoing acetylation. (A) Hierarchical clustering of the organellar distributions of sites whose acetylation was increased by >1.5-fold in response to resveratrol or spermidine, at least in one organellar fraction. (B) Consensus acetylation motifs identified upon resveratrol (left) or spermidine (right) treatment are depicted using the MotifX algorithm (Schwartz and Gygi, 2005). Among sites that were hyperacetylated in response to both agents, the K(F/Y) motif is significantly enriched when tested against the whole proteome (P < 0.00001). (C) When testing against the largest acetylation site dataset from Choudhary et al. (2009) (acetylation background dataset [ABD]), the SxK motif is significant (P < 0.0001) for sites whose acetylation increased upon spermidine treatment. No general consensus motifs were found for sites whose acetylation increased in response to both agents.


Significant motifs among sites undergoing deacetylation . ( A ) Hierarchical clustering of the organellar distributions of sites whose deacetylation was increased by > 1 . 5 - fold in response to resveratrol or spermidine , at least in one organellar fraction . ( B ) Among sites that were hypoacetylated in response to both agents , the KP motif is significantly enriched when tested against ABD ( P < 0 . 001 ) . ( C and D ) When tested against the ABD ( C ) or the whole proteome ( D ) , the KP motif is significant for sites undergoing hypoacetylation upon spermidine treatment ( P < 0 . 0001 ) . No general consensus motifs were found for sites whose acetylation decreased in response to both agents .

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Significant motifs among sites undergoing deacetylation. (A) Hierarchical clustering of the organellar distributions of sites whose deacetylation was increased by >1.5-fold in response to resveratrol or spermidine, at least in one organellar fraction. (B) Among sites that were hypoacetylated in response to both agents, the KP motif is significantly enriched when tested against ABD (P < 0.001). (C and D) When tested against the ABD (C) or the whole proteome (D), the KP motif is significant for sites undergoing hypoacetylation upon spermidine treatment (P < 0.0001). No general consensus motifs were found for sites whose acetylation decreased in response to both agents.


Autophagy can be efficiently regulated by cytoplasmic ( de ) acetylation reactions . ( A and B ) HCT 116 cells were transfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and enucleated to obtain cytoplasts . The cytoplasts were treated with either vehicle ( Co , control ) , 100 - µM resveratrol ( Resv ) , or 100 - µM spermidine ( Spd ) for 4 h . 1 - µM rapamycin ( Rapa ) was used as a positive control . ( A ) Representative images of cytoplasts indicative of autophagic activity . Arrows indicate GFP - LC3 - transfected cytoplasts , whereas insets show nonenucleated transfected cells . ( B ) Quantitative data . Bars show the percentages of cells or cytoplasts showing the accumulation of GFP - LC3 in puncta ( GFP - LC3vac ) .

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Autophagy can be efficiently regulated by cytoplasmic (de)acetylation reactions. (A and B) HCT 116 cells were transfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and enucleated to obtain cytoplasts. The cytoplasts were treated with either vehicle (Co, control), 100-µM resveratrol (Resv), or 100-µM spermidine (Spd) for 4 h. 1-µM rapamycin (Rapa) was used as a positive control. (A) Representative images of cytoplasts indicative of autophagic activity. Arrows indicate GFP-LC3-transfected cytoplasts, whereas insets show nonenucleated transfected cells. (B) Quantitative data. Bars show the percentages of cells or cytoplasts showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac).


( C - E ) HCT 116 cells were cotransfected with a plasmid for the expression of RFP - LC3 together with an empty vector ( pcDNA3 ) or plasmids encoding wild - type ( WT ) SIRT1 or a SIRT1 variant with a mutation in the nuclear localization signal fused to GFP ( WT GFP - SIRT1 and mtNLS GFP - SIRT1 , respectively ) for 12 or 24 h , fixed , and analyzed by fluorescence microscopy . ( C ) Representative images indicative of 24 - h autophagic activity .

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(C-E) HCT 116 cells were cotransfected with a plasmid for the expression of RFP-LC3 together with an empty vector (pcDNA3) or plasmids encoding wild-type (WT) SIRT1 or a SIRT1 variant with a mutation in the nuclear localization signal fused to GFP (WT GFP-SIRT1 and mtNLS GFP-SIRT1, respectively) for 12 or 24 h, fixed, and analyzed by fluorescence microscopy. (C) Representative images indicative of 24-h autophagic activity.


( D ) Quantitative data . Bars depict the percentages of cells exhibiting the accumulation of RFP - LC3 in puncta ( RFP - LC3vac ) . ( B and D ) Means ± SEM ; n = 3 ; * , P < 0 . 05 .

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(D) Quantitative data. Bars depict the percentages of cells exhibiting the accumulation of RFP-LC3 in puncta (RFP-LC3vac). (B and D) Means ± SEM; n = 3; *, P < 0.05.


( E ) Representative immunoblots for 24 - h endogenous LC3 lipidation . The asterisk stands for a nonspecific band . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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(E) Representative immunoblots for 24-h endogenous LC3 lipidation. The asterisk stands for a nonspecific band. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


Low doses of resveratrol and spermidine synergistically induce autophagy in vitro and in vivo . ( A ) Human colorectal carcinoma HCT 116 cells were transfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and then treated with either vehicle ( Co , control ) or the indicated dose of resveratrol ( Resv ) or spermidine ( Spd ) , alone or in combination , for 2 h . ( top ) Quantitative data . Bars depict the percentages ( means ± SD ; n = 3 ; * * , P < 0 . 05 ) of cells showing the accumulation of GFP - LC3 in puncta ( GFP - LC3vac ) . ( bottom ) Representative immunoblots showing endogenous LC3 lipidation .

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Low doses of resveratrol and spermidine synergistically induce autophagy in vitro and in vivo. (A) Human colorectal carcinoma HCT 116 cells were transfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and then treated with either vehicle (Co, control) or the indicated dose of resveratrol (Resv) or spermidine (Spd), alone or in combination, for 2 h. (top) Quantitative data. Bars depict the percentages (means ± SD; n = 3; **, P < 0.05) of cells showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac). (bottom) Representative immunoblots showing endogenous LC3 lipidation.


B ) Representative immunoblots showing endogenous LC3 lipidation in the presence of bafilomycin A1 ( BafA1 ) . ( C - E ) Transgenic C57BL / 6 mice expressing a GFP - LC3 fusion protein were injected with resveratrol and spermidine at the indicated concentrations . 3 h later , mice were killed , and tissues were processed for immunofluorescence microscopy determinations of GFP - LC3vac .

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B) Representative immunoblots showing endogenous LC3 lipidation in the presence of bafilomycin A1 (BafA1). (C-E) Transgenic C57BL/6 mice expressing a GFP-LC3 fusion protein were injected with resveratrol and spermidine at the indicated concentrations. 3 h later, mice were killed, and tissues were processed for immunofluorescence microscopy determinations of GFP-LC3vac.


( D ) Quantitative results . Bars represent the percentages ( means ± SEM ; n = 3 ; * , P < 0 . 05 ; and * * , P < 0 . 01 as compared with the same tissue from untreated animals ) of cells exhibiting GFP - LC3vac .

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(D) Quantitative results. Bars represent the percentages (means ± SEM; n = 3; *, P < 0.05; and **, P < 0.01 as compared with the same tissue from untreated animals) of cells exhibiting GFP-LC3vac.


( E ) Representative immunoblots showing endogenous LC3 lipidation and p62 protein content in WT C57BL / 6 mouse tissues . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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(E) Representative immunoblots showing endogenous LC3 lipidation and p62 protein content in WT C57BL/6 mouse tissues. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


A ) Correlations of mean fold - changes of the proteins changing PD - dependently in the Columbia and LCC cohorts . Only proteins quantified in both cohorts are shown ( n = 330 ) . Proteins overlapping between the two cohorts are labeled with their name .

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A) Correlations of mean fold-changes of the proteins changing PD-dependently in the Columbia and LCC cohorts. Only proteins quantified in both cohorts are shown (n=330). Proteins overlapping between the two cohorts are labeled with their name.


B ) Mean fold - changes for each of the 33 proteins that were LRRK2 - dependently regulated in both cohorts using a pairwise t - test comparing the four subgroups ( HC , NMC , iPD and LRRK2 PD ) .

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B) Mean fold-changes for each of the 33 proteins that were LRRK2-dependently regulated in both cohorts using a pairwise t-test comparing the four subgroups (HC, NMC, iPD and LRRK2 PD).


A ) Pearson correlation scores and associated p - values [ - log10 ] of all protein intensities with the MoCA total score . Either all PD patients ( left ) , iPD patients ( middle ) or LRRK2 PD patients ( right ) were included in the analysis . Significantly correlated proteins with an FDR of 5 % after Benjamini - Hochberg correction are highlighted in red .

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A) Pearson correlation scores and associated p-values [-log10] of all protein intensities with the MoCA total score. Either all PD patients (left), iPD patients (middle) or LRRK2 PD patients (right) were included in the analysis. Significantly correlated proteins with an FDR of 5% after Benjamini-Hochberg correction are highlighted in red.


B ) Pearson correlation scores and associated p - values [ - log10 ] of all protein intensities with the UPDRS - III score . Either all PD patients ( left ) , iPD patients ( middle ) or LRRK2 PD patients ( right ) were included . Significantly correlated proteins with an FDR of 5 % after Benjamini - Hochberg correction are highlighted in red .

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B) Pearson correlation scores and associated p-values [-log10] of all protein intensities with the UPDRS-III score. Either all PD patients (left), iPD patients (middle) or LRRK2 PD patients (right) were included. Significantly correlated proteins with an FDR of 5% after Benjamini-Hochberg correction are highlighted in red.


( a , b ) BJAB lymphoma cells stably expressing mCherry - GFP - LC3 were serially cultured at log phase followed by fluorescence - activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry / GFP ( a ) . The high and low 20 % were sorted ( a ) ,

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(a,b) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP (a). The high and low 20% were sorted (a),


( a , b ) BJAB lymphoma cells stably expressing mCherry - GFP - LC3 were serially cultured at log phase followed by fluorescence - activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry / GFP ( a ) . The high and low 20 % were sorted ( a ) , re - plated and treated with lysosomal protease inhibitors pepstatin and E - 64d for 1 h ; lysates were then immunoblotted for the indicated proteins ( b ) . ( c ) Densitometry of LC3 - II and p62 western blots ( normalized to actin and hour 0 , mean ± s . e . m . , n = 3 blots from 2 independent experiments , * P = 0 . 051 , * * P = 0 . 0091 ) .

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(a,b) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP (a). The high and low 20% were sorted (a), re-plated and treated with lysosomal protease inhibitors pepstatin and E-64d for 1 h; lysates were then immunoblotted for the indicated proteins (b). (c) Densitometry of LC3-II and p62 western blots (normalized to actin and hour 0, mean ± s.e.m., n = 3 blots from 2 independent experiments, *P = 0.051, **P = 0.0091).


( d ) ; autophagic LC3 puncta were assessed by quantitative

(d); autophagic LC3 puncta were assessed by quantitative microscopy


( d ) ; autophagic LC3 puncta were assessed by quantitative microscopy ( e ; punctate area per cell , mean ± s . e . m . , n = 50 fields , * P = 0 . 010 , * * P = 0 . 053 ) .

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(d); autophagic LC3 puncta were assessed by quantitative microscopy (e; punctate area per cell, mean ± s.e.m., n = 50 fields, *P = 0.010, **P = 0.053).


f ) Electron micrographs of HeLa mCherry - GFP - LC3 cells sorted for autophagic flux as in a . Yellow arrows denote autophagosomes ; red arrows indicate autolysosomes . ( g ) Quantification of autophagosomes and autolysomes from electron micrographs ( mean ± s . e . m . , n = 50 fields ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

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f) Electron micrographs of HeLa mCherry-GFP-LC3 cells sorted for autophagic flux as in a. Yellow arrows denote autophagosomes; red arrows indicate autolysosomes. (g) Quantification of autophagosomes and autolysomes from electron micrographs (mean ± s.e.m., n = 50 fields). Uncropped images of blots are shown in Supplementary Fig. 6.


a - c ) BJAB lymphoma cells stably expressing mCherry - GFP - LC3 were serially cultured at log phase followed by fluorescence - activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry / GFP . Cells were then re - plated in growth medium and autophagic flux was again measured by flow cytometry at the indicated time points ( median ratio of mCherry / GFP fluorescence ± s . e . m . , n = 3 wells ) . Lysates from cells collected at the indicated time points were immunoblotted with the indicated antibodies ( b ) . Representative flow cytometry histograms for cells at 0 h and 24 h after sorting ( c ) .

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a-c) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP. Cells were then re-plated in growth medium and autophagic flux was again measured by flow cytometry at the indicated time points (median ratio of mCherry/GFP fluorescence ± s.e.m., n = 3 wells). Lysates from cells collected at the indicated time points were immunoblotted with the indicated antibodies (b). Representative flow cytometry histograms for cells at 0 h and 24 h after sorting (c).


( d ) BJAB mCherry - GFP - LC3 cells were sorted for autophagic flux as in a ( top and bottom 20 % ) . Following treatment with Fas ligand ( 1 . 5 ng ml − 1 ) or TRAIL ( 4 ng ml − 1 ) , cell viability was determined by MTS assay ( a tetrazolium reduction - based metabolic measurement ) at 24 h ( percentage of untreated control , mean ± s . e . m . , n = 3 wells , * P = 2 . 3 × 10 − 4 , * * P = 0 . 0036 ) .

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(d) BJAB mCherry-GFP-LC3 cells were sorted for autophagic flux as in a (top and bottom 20%). Following treatment with Fas ligand (1.5 ng ml−1) or TRAIL (4 ng ml−1), cell viability was determined by MTS assay (a tetrazolium reduction-based metabolic measurement) at 24 h (percentage of untreated control, mean ± s.e.m., n = 3 wells, *P = 2.3×10−4, **P = 0.0036).


( e ) Long - term growth of BJAB mCherry - GFP - LC3 cells sorted for autophagic flux followed by treatment with Fas ligand ( 4 ng ml − 1 ) or TRAIL ( 15 ng ml − 1 ) for 24 h . Cells were then re - plated and allowed to recover for 5 ( Fas ligand ) or 6 ( TRAIL ) days then assayed for viability ( percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 012 ) .

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(e) Long-term growth of BJAB mCherry-GFP-LC3 cells sorted for autophagic flux followed by treatment with Fas ligand (4 ng ml−1) or TRAIL (15 ng ml−1) for 24 h. Cells were then re-plated and allowed to recover for 5 (Fas ligand) or 6 (TRAIL) days then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.012).


( f ) BJAB mCherry - GFP - LC3 cells were sorted for autophagic flux as above and apoptosis was measured at 1 h by flow cytometry using AnnexinV and DAPI ( mean ± s . e . m . , n = 3 wells , * P = 0 . 0046 ) .

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

(f) BJAB mCherry-GFP-LC3 cells were sorted for autophagic flux as above and apoptosis was measured at 1 h by flow cytometry using AnnexinV and DAPI (mean ± s.e.m., n = 3 wells, *P = 0.0046).


( g ) Cells sorted as in a , were re - plated and , starting at the indicated times following sorting , treated with Fas ligand ( 4 ng ml − 1 ) for 24 h ; cell viability was then determined by MTS ( percentage of untreated control , mean ± s . e . m . , n = 3 wells ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

O 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

(g) Cells sorted as in a, were re-plated and, starting at the indicated times following sorting, treated with Fas ligand (4 ng ml−1) for 24 h; cell viability was then determined by MTS (percentage of untreated control, mean ± s.e.m., n = 3 wells). Uncropped images of blots are shown in Supplementary Fig. 6.


( a ) BJAB and Jurkat cells were treated with vehicle or chloroquine ( BJAB , 20 μM ; Jurkat , 10 μM ) for 16 h followed by Fas ligand ( 1 . 5 ng ml − 1 ) . Cell viability was determined by MTS 24 h later ( percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 0 . 0058 ) .

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

(a) BJAB and Jurkat cells were treated with vehicle or chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by Fas ligand (1.5 ng ml−1). Cell viability was determined by MTS 24 h later (percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 0.0058).


( b ) Immunoblots of cells in a , c probed for the indicated antibodies . CQ ,

(b) Immunoblots of cells in a,c probed for the indicated antibodies. CQ, chloroquine


( c ) BJAB and Jurkat cells were treated with chloroquine ( BJAB , 20 μM ; Jurkat , 10 μM ) for 16 h followed by treatment with Fas ligand ( 4 ng ml − 1 ) for 24 h ( same technical replicate as a ) . Cells were then re - plated at low density in growth media and allowed to recover for 5 days , and then assayed for viability ( percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 0024 ) .

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

(c) BJAB and Jurkat cells were treated with chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by treatment with Fas ligand (4 ng ml−1) for 24 h (same technical replicate as a). Cells were then re-plated at low density in growth media and allowed to recover for 5 days, and then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0024).


( d ) BJAB and Jurkat cells were transduced with the indicated lentiviral shRNA constructs , followed by 3 days of puromycin selection . Selected cells were plated and treated with Fas ligand ( 1 . 5 ng ml − 1 ) or TRAIL ( 4 ng ml − 1 ) for 24 h and viability was assessed by MTS ( percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 , * P = 1 . 7 × 10 − 4 , * * P = 0 . 024 ) .

O O O O O O O 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 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

(d) BJAB and Jurkat cells were transduced with the indicated lentiviral shRNA constructs, followed by 3 days of puromycin selection. Selected cells were plated and treated with Fas ligand (1.5 ng ml−1) or TRAIL (4 ng ml−1) for 24 h and viability was assessed by MTS (percentage of control (no ligand), mean ± s.e.m., n = 3, *P = 1.7×10−4, **P = 0.024).


( e ) Immunoblots for Atg5 , Atg7 and p62 confirm protein depletion and autophagy inhibition in d , f .

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

(e) Immunoblots for Atg5, Atg7 and p62 confirm protein depletion and autophagy inhibition in d,f.


( f ) BJAB and Jurkat cells expressing control or Atg5 shRNA were treated with Fas ligand ( 15 ng ml − 1 ) or TRAIL ( 12 . 5 ng ml − 1 ) for 24 h ( same technical replicate as d ) . Cells were then re - plated at low density and allowed to recover for 5 - 6 days , and then assayed for viability ( percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 0089 ) . Atg7 knockdown data were not included for long - term viability owing to the growth suppressive effect of Atg7 knockdown in the absence of drug treatment . Uncropped images of blots are shown in Supplementary Fig . 6 .

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

(f) BJAB and Jurkat cells expressing control or Atg5 shRNA were treated with Fas ligand (15 ng ml−1) or TRAIL (12.5 ng ml−1) for 24 h (same technical replicate as d). Cells were then re-plated at low density and allowed to recover for 5-6 days, and then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0089). Atg7 knockdown data were not included for long-term viability owing to the growth suppressive effect of Atg7 knockdown in the absence of drug treatment. Uncropped images of blots are shown in Supplementary Fig. 6.


( a , b ) BJAB and Jurkat cells stably expressing mCherry - GFP - LC3 were flow sorted for high and low autophagic flux , treated with Fas ligand ( 1 . 5 ng ml − 1 ) for 24 h and viability was assessed by MTS ( a ; percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 0046 ) . ( b ) Immunoblots following sorting of the samples in a .

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

(a,b) BJAB and Jurkat cells stably expressing mCherry-GFP-LC3 were flow sorted for high and low autophagic flux, treated with Fas ligand (1.5 ng ml−1) for 24 h and viability was assessed by MTS (a; percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0046). (b) Immunoblots following sorting of the samples in a.


( c , d ) The indicated cell lines were treated with chloroquine ( BJAB , CEM 20 μM ; SKW6 . 4 , 25 μM ; Jurkat , 10 μM ) for 16 h , followed by Fas ligand ( BJAB , 12 . 5 ng ml − 1 ; SKW6 . 4 , 50 ng ml − 1 ; Jurkat , 0 . 4 ng ml − 1 ; CEM , 40 ng ml − 1 ) . Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies ( c ) .

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

(c,d) The indicated cell lines were treated with chloroquine (BJAB, CEM 20 μM; SKW6.4, 25 μM; Jurkat, 10 μM) for 16 h, followed by Fas ligand (BJAB, 12.5 ng ml−1; SKW6.4, 50 ng ml−1; Jurkat, 0.4 ng ml−1; CEM, 40 ng ml−1). Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies (c).


( c , d ) The indicated cell lines were treated with chloroquine ( BJAB , CEM 20 μM ; SKW6 . 4 , 25 μM ; Jurkat , 10 μM ) for 16 h , followed by Fas ligand ( BJAB , 12 . 5 ng ml − 1 ; SKW6 . 4 , 50 ng ml − 1 ; Jurkat , 0 . 4 ng ml − 1 ; CEM , 40 ng ml − 1 ) . Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies ( c ) . Cell viability was assessed by MTS 24 h following Fas ligand treatment ( d ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 2 . 7 × 10 − 4 , * * P = 1 . 6 × 10 − 4 ) .

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

(c,d) The indicated cell lines were treated with chloroquine (BJAB, CEM 20 μM; SKW6.4, 25 μM; Jurkat, 10 μM) for 16 h, followed by Fas ligand (BJAB, 12.5 ng ml−1; SKW6.4, 50 ng ml−1; Jurkat, 0.4 ng ml−1; CEM, 40 ng ml−1). Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies (c). Cell viability was assessed by MTS 24 h following Fas ligand treatment (d; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 2.7×10−4, **P = 1.6×10−4).


( e , f ) BJAB and Jurkat cells were transduced with control , Atg5 , Atg7 or Vps34 shRNA lentiviruses , followed by three days of puromycin selection . Cells were then treated with Fas ligand ( 1 . 25 ng ml − 1 ) or TRAIL ( 1 . 25 ng ml − 1 ) for 24 h and viability was assessed by MTS ( e ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 1 . 4 × 10 − 6 , * * P = 2 . 3 × 10 − 5 , * * * P = 3 . 9 × 10 − 5 , § P = 9 . 2 × 10 − 6 , § § P = 3 . 4 × 10 − 6 , § § § P = 1 . 7 × 10 − 5 ) . Immunoblots demonstrate Atg5 , Atg7 and Vps34 knockdown , autophagy inhibition and altered Fap - 1 levels . The blots are separated because different exposures were required to detect the proteins in one cell line without overexposing the lanes for the other ; the Fap - 1 blot is not separated to demonstrate the lack of Fap - 1 protein in Type II Jurkat cells ( f ) . Fap - 1 blots ( b , c , f ) were run on separate gels owing to the quantity of protein required for detection ( see Methods ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

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

(e,f) BJAB and Jurkat cells were transduced with control, Atg5, Atg7 or Vps34 shRNA lentiviruses, followed by three days of puromycin selection. Cells were then treated with Fas ligand (1.25 ng ml−1) or TRAIL (1.25 ng ml−1) for 24 h and viability was assessed by MTS (e; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 1.4×10−6, **P = 2.3×10−5, ***P = 3.9×10−5, §P = 9.2×10−6, §§P = 3.4×10−6, §§§P = 1.7×10−5). Immunoblots demonstrate Atg5, Atg7 and Vps34 knockdown, autophagy inhibition and altered Fap-1 levels. The blots are separated because different exposures were required to detect the proteins in one cell line without overexposing the lanes for the other; the Fap-1 blot is not separated to demonstrate the lack of Fap-1 protein in Type II Jurkat cells (f). Fap-1 blots (b,c,f) were run on separate gels owing to the quantity of protein required for detection (see Methods). Uncropped images of blots are shown in Supplementary Fig. 6.


( a , b ) BJAB and Jurkat cells expressing the indicated Fap - 1 wild - type ( WT ) and catalytically inactive ( ΔCD ) constructs were treated with chloroquine ( BJAB , 20 μM ; Jurkat , 10 μM ) for 16 h followed by Fas ligand ( BJAB , 1 . 5 ng ml − 1 ; Jurkat 15 ng ml − 1 ) for 24 h . Cell viability was determined by MTS ( a ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 2 . 7 × 10 − 4 , * * P = 2 . 4 × 10 − 5 , * * * P = 0 . 0024 ) . Cell lysates were blotted and probed with the indicated antibodies ( b ) .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BJAB and Jurkat cells expressing the indicated Fap-1 wild-type (WT) and catalytically inactive (ΔCD) constructs were treated with chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by Fas ligand (BJAB, 1.5 ng ml−1; Jurkat 15 ng ml−1) for 24 h. Cell viability was determined by MTS (a; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 2.7×10−4, **P = 2.4×10−5, ***P = 0.0024). Cell lysates were blotted and probed with the indicated antibodies (b).


c - e ) BJAB cells expressing control or Fap - 1 shRNAs were treated with 20 μM chloroquine for 16 h followed by Fas ligand ( 12 . 5 ng ml − 1 ) or TRAIL ( 25 ng ml − 1 ) for 24 h ; cell viability was then determined by MTS ( c ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 4 . 8 × 10 − 6 , * * P = 8 . 1 × 10 − 5 , * * * P = 8 . 0 × 10 − 4 , § P = 0 . 013 , NS P > 0 . 05 ) .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-e) BJAB cells expressing control or Fap-1 shRNAs were treated with 20 μM chloroquine for 16 h followed by Fas ligand (12.5 ng ml−1) or TRAIL (25 ng ml−1) for 24 h; cell viability was then determined by MTS (c; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 4.8×10−6, **P = 8.1×10−5, ***P = 8.0×10−4, §P = 0.013, NS P>0.05).


( d ) Immunoblots of lysates collected following chloroquine treatment . Long - term growth was assessed in cells from c by re - plating them at low density and allowing them to recover for 5 days , followed by viability assay ( percentage of untreated control , mean ± s . e . m . , n = 3 wells , * P = 0 . 043 ; e ) . Fap - 1 blots ( b , d ) were run on separate gels owing to the quantity of protein required for detection ( see Methods ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

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

(d) Immunoblots of lysates collected following chloroquine treatment. Long-term growth was assessed in cells from c by re-plating them at low density and allowing them to recover for 5 days, followed by viability assay (percentage of untreated control, mean ± s.e.m., n = 3 wells, *P = 0.043; e). Fap-1 blots (b,d) were run on separate gels owing to the quantity of protein required for detection (see Methods). Uncropped images of blots are shown in Supplementary Fig. 6.


a , b ) Cell viability was determined by MTS assay following Fas ligand ( 4 ng ml − 1 ) treatment in BJAB cells expressing control or p62 lentiviral shRNAs ( a ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 3 . 2 × 10 − 5 , * * P = 1 . 8 × 10 − 6 ) . Immunoblots confirm p62 depletion and Fap - 1 levels ( b ) .

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

a,b) Cell viability was determined by MTS assay following Fas ligand (4 ng ml−1) treatment in BJAB cells expressing control or p62 lentiviral shRNAs (a; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 3.2×10−5, **P = 1.8×10−6). Immunoblots confirm p62 depletion and Fap-1 levels (b).


( c ) Co - immunoprecipitation of endogenous p62 and Fap - 1 in BJAB cells treated with 20 μM chloroquine for 16 h , followed by treatment with Fas ligand ( 50 ng ml − 1 ) for 2 h at 4 ° C .

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

(c) Co-immunoprecipitation of endogenous p62 and Fap-1 in BJAB cells treated with 20 μM chloroquine for 16 h, followed by treatment with Fas ligand (50 ng ml−1) for 2 h at 4°C.


HDAC6 KO MEF cells are defective in aggregate clearance but not in autophagosome induction or targeting . ( A ) Filter ‐ trap analysis of MG132 ‐ induced , SDS ‐ insoluble ubiquitinated aggregates generated in wild ‐ type ( WT ) , HDAC6 KO , and KO MEFs reconstituted with different HDAC6 constructs as indicated . The signal intensity from the ubiquitinimmunoblot ( bottom panel ) was quantified and presented as the average of the means from three independent experiments along with the standard deviation ( top ) . Note the significant accumulation of ubiquitin ‐ positive aggregates in HDAC6 KO MEFs and KO MEFs stably expressing HDAC6 ‐ CD and ΔBUZ mutants . WT , HDAC6 KO , and HDAC6 KO MEFs stably expressing human HDAC6 ( hHDAC6 WT ) , HDAC6 CD ( catalytically inactive mutant ) , or HDAC6 ΔBUZ ( ubiquitin ‐ binding ‐ deficient mutant ) were analysed for the level of HDAC6 using an anti ‐ human HDAC6 antibody . Mouse endogenous HDAC6 ( mHDAC6 ) and actin levels were determined by each corresponding antibody .

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

HDAC6 KO MEF cells are defective in aggregate clearance but not in autophagosome induction or targeting. (A) Filter‐trap analysis of MG132‐induced, SDS‐insoluble ubiquitinated aggregates generated in wild‐type (WT), HDAC6 KO, and KO MEFs reconstituted with different HDAC6 constructs as indicated. The signal intensity from the ubiquitinimmunoblot (bottom panel) was quantified and presented as the average of the means from three independent experiments along with the standard deviation (top). Note the significant accumulation of ubiquitin‐positive aggregates in HDAC6 KO MEFs and KO MEFs stably expressing HDAC6‐CD and ΔBUZ mutants. WT, HDAC6 KO, and HDAC6 KO MEFs stably expressing human HDAC6 (hHDAC6 WT), HDAC6 CD (catalytically inactive mutant), or HDAC6 ΔBUZ (ubiquitin‐binding‐deficient mutant) were analysed for the level of HDAC6 using an anti‐human HDAC6 antibody. Mouse endogenous HDAC6 (mHDAC6) and actin levels were determined by each corresponding antibody.


( B ) WT and HDAC6 KO MEFs were treated with MG132 and subjected to Western blot analysis for LC3 , actin , and HDAC6 .

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

(B) WT and HDAC6 KO MEFs were treated with MG132 and subjected to Western blot analysis for LC3, actin, and HDAC6.


( C ) Cells were treated with MG132 as described under Materials and methods and immunostained with antibodies to ubiquitin ( green ) and LC3 ( red ) . Arrows indicate ubiquitin ‐ positive aggregates that colocalize with LC3 ‐ positive autophagosomes . Scale bar , 10 μm .

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

(C) Cells were treated with MG132 as described under Materials and methods and immunostained with antibodies to ubiquitin (green) and LC3 (red). Arrows indicate ubiquitin‐positive aggregates that colocalize with LC3‐positive autophagosomes. Scale bar, 10 μm.


( D ) WT and ATG5 KO MEFs are treated with 2 . 5 μM MG132 for 1 day and incubated with normal growth media for 18 h . MEFs are immunostained with anti ‐ LC3 ( red ) and anti ‐ ubiquitin antibody ( green ) .

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

(D) WT and ATG5 KO MEFs are treated with 2.5 μM MG132 for 1 day and incubated with normal growth media for 18 h. MEFs are immunostained with anti‐LC3 (red) and anti‐ubiquitin antibody (green).


( A ) Wild ‐ type and HDAC6 KO MEFs were transfected with pcDNA , pcDNA ‐ HDAC6WT , HDAC6CD , or HDAC6ΔBuz , along with mCherry ‐ GFP ‐ LC3 as indicated . Yellow signals indicate non ‐ acidic autophagosomes and red signals indicate acidic autophagolysosomes . Scale bar , 10 μm .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O 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) Wild‐type and HDAC6 KO MEFs were transfected with pcDNA, pcDNA‐HDAC6WT, HDAC6CD, or HDAC6ΔBuz, along with mCherry‐GFP‐LC3 as indicated. Yellow signals indicate non‐acidic autophagosomes and red signals indicate acidic autophagolysosomes. Scale bar, 10 μm.


( B ) The total number of yellow vesicles was quantified from three independent experiments ( > 12 cells each ) and presented as the percentage of total mCherry ‐ GFP ‐ LC3 dots ( red plus yellow ) along with the standard deviation .

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(B) The total number of yellow vesicles was quantified from three independent experiments (>12 cells each) and presented as the percentage of total mCherry‐GFP‐LC3 dots (red plus yellow) along with the standard deviation.


( C ) In vitro fusion assays . Autophagosomes ( APGs ) and lysosomes ( Lys ) purified from wild type and HDAC6 − / − MEFs were subjected to heterotypic and homotypic in vitro fusion assays ( representative fields are shown in Supplementary Figure S3 ) . Values are means + s . e . of the percentages of fusion from three independent experiments ( more than 10 images per each experiment ) .

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(C)In vitro fusion assays. Autophagosomes (APGs) and lysosomes (Lys) purified from wild type and HDAC6−/− MEFs were subjected to heterotypic and homotypic in vitro fusion assays (representative fields are shown in Supplementary Figure S3). Values are means+s.e. of the percentages of fusion from three independent experiments (more than 10 images per each experiment).


( D ) EM images of wild ‐ type and HDAC6 KO MEFs in normal growth conditions . Yellow arrows , autophagosomes ; red arrows , autophagolysosomes ; green arrowheads , multilamellar bodies .

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(D) EM images of wild‐type and HDAC6 KO MEFs in normal growth conditions. Yellow arrows, autophagosomes; red arrows, autophagolysosomes; green arrowheads, multilamellar bodies.


( E ) Quantification of autophagosomes and autophagolysosomes . The error bar represents the standard error .

(E) Quantification of autophagosomes and autophagolysosomes. The error bar represents the standard error.