( F - G ) K413 in IDH2 may be a key succinylation site that is directly targeted by SIRT5 . indicated Flag - tagged IDH2 were overexpressed in HEK293T with stable SIRT5 knockdown or SIRT5 co - overexpression . Wild - type mutant IDH2 were purified by Flag beads eluted with Flag peptide , followed by measurement of IDH2 enzyme activity . results are average ± SD of 3 independent experiments * p < 0 . 05 , n . s . : not significant ( two - unpaired t - ) .

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

(F-G) K413 in IDH2 may be a key succinylation site that is directly targeted by SIRT5. The indicated Flag-tagged IDH2 proteins were overexpressed in HEK293T cells with stable SIRT5 knockdown or SIRT5 co-overexpression. Wild-type and mutant IDH2 proteins were purified by Flag beads and eluted with Flag peptide, followed by measurement of IDH2 enzyme activity. The results are average ± SD of 3 independent experiments *p<0.05, n.s.: not significant (two-tailed unpaired t-test).


( A - B ) SIRT5 expression is not affected by chemical oxidant treatment . HEK293T were treated with Paraquat ( A ) or H2O2 ( B ) for indicated periods . mRNA expression of endogenous SIRT5 were determined by quantitative real - time PCR western blot , respectively . results are average ± SD of 3 independent experiments . n . s . : not significant ( two - unpaired t - ) .

O 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 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

(A-B) SIRT5 expression is not affected by chemical oxidant treatment. HEK293T cells were treated with Paraquat (A) or H2O2 (B) for the indicated periods. The mRNA and protein expression of endogenous SIRT5 were determined by quantitative real-time PCR and western blot analysis, respectively. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).


( D ) desuccinylase activity of SIRT5 is stimulated by oxidative stimuli . Flag - SIRT5 was ectopically expressed in HEK293T . transfected were treated with increased concentrations of Paraquat or H2O2 for indicated periods . Flag - SIRT5 was purified by immunoprecipitation with Flag beads , eluted with Flag peptide , then subjected to desuccinylase activity as described in ' Materials Methods ' . SIRT5 desuccinylase activity was normalized against its level . results are average ± SD of 3 independent experiments * p < 0 . 05 , * * * p < 0 . 001 ( two - unpaired t - ) .

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 B-CONTROLLED_VAR O B-CONTROLLED_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 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

(D) The desuccinylase activity of SIRT5 is stimulated by oxidative stimuli. Flag-SIRT5 was ectopically expressed in HEK293T cells. The transfected cells were treated with increased concentrations of Paraquat or H2O2 for the indicated periods. Flag-SIRT5 was purified by immunoprecipitation with Flag beads, eluted with Flag peptide, and then subjected to the desuccinylase activity assay as described in 'Materials and Methods'. The SIRT5 desuccinylase activity was normalized against its protein level. The results are average ± SD of 3 independent experiments *p<0.05, ***p<0.001 (two-tailed unpaired t-test).


( E ) In vitro incubation with chemical oxidants does not affect desuccinylase activity of SIRT5 . Flag - tagged SIRT5 was ectopically expressed in HEK293T , then purified by immunoprecipitation with Flag - beads eluted with Flag peptide . purified Flag - SIRT5 was treated with increased concentrations of Paraquat or H2O2 at room for 1 hr , then subjected to desuccinylase activity as described in ' Materials Methods ' . SIRT5 desuccinylase activity was normalized against its level . results are average ± SD of 3 independent experiments . n . s . : not significant ( two - unpaired t - ) .

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 B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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) In vitro incubation with chemical oxidants does not affect the desuccinylase activity of SIRT5. Flag-tagged SIRT5 was ectopically expressed in HEK293T cells, and then purified by immunoprecipitation with Flag-beads and eluted with Flag peptide. The purified Flag-SIRT5 was treated with increased concentrations of Paraquat or H2O2 at room temperature for 1 hr, and then subjected to the desuccinylase activity assay as described in 'Materials and Methods'. The SIRT5 desuccinylase activity was normalized against its protein level. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).


( F ) Chemical oxidants enhance ability of SIRT5 to activate IDH2 . Flag - SIRT5 was ectopically expressed in HEK293T , transfected were treated with increased concentrations of Paraquat or H2O2 for indicated periods . Flag - SIRT5 was purified by immunoprecipitation with Flag beads , eluted with Flag peptide , then incubated with purified Flag - IDH2 in vitro . IDH2 enzyme activity was measured as described in ' Materials Methods ' , normalized by its level . results are average ± SD of 3 independent experiments * * p < 0 . 01 , * * * p < 0 . 001 ( two - unpaired t - ) .

O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Chemical oxidants enhance the ability of SIRT5 to activate IDH2. Flag-SIRT5 was ectopically expressed in HEK293T cells, and the transfected cells were treated with increased concentrations of Paraquat or H2O2 for the indicated periods. Flag-SIRT5 was purified by immunoprecipitation with Flag beads, eluted with Flag peptide, and then incubated with purified Flag-IDH2 in vitro. IDH2 enzyme activity was measured as described in 'Materials and Methods', normalized by its protein level. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test).


( A ) Quantitative of Aβ1 - 40 in TBS ( * p = 0 . 0212 ) , Triton - X ( p = 0 . 0544 ) SDS ( * * p = 0 . 0063 ) fractions of brain homogenates from ( n = 7 ) ( n = 10 ) APP23 mice . Mean ± s . e . m . , statistical : two - unpaired t - .

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

(A) Quantitative analysis of the Aβ1-40 protein in the TBS (*p=0.0212), Triton-X (p=0.0544) and SDS (**p=0.0063) fractions of brain homogenates from male (n=7) and female (n=10) APP23 mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test.


( B ) Stereological of cortical covered by 4G8 - positive plaques in ( n = 10 ) ( n = 8 ) APP23 mice ( left ) representative ( right ) , scale bar = 500 µm . Mean ± s . e . m . , statistical : two - unpaired t - , * * * p = 0 . 0004 .

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

(B) Stereological analysis of cortical area covered by 4G8-positive plaques in male (n=10) and female (n=8) APP23 mice (left) and representative images (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, ***p=0.0004.


( C ) of pFTAA - stained Aβ plaques in cortex of ( n = 10 ) ( n = 8 ) APP23 mice ( left ) representative ( right ) , scale bar = 1 mm . Mean ± s . e . m . , statistical : two - unpaired t - , * * p = 0 . 0011 .

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

(C) Fluorescence intensity based analysis of pFTAA-stained Aβ plaques in the cortex of male (n=10) and female (n=8) APP23 mice (left) and representative images (right), scale bar = 1 mm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, **p=0.0011.


( D ) Stereological of cortical covered by Congo Red - positive plaques in ( n = 10 ) ( n = 8 ) APP23 mice ( left ) representative ( right ) , scale bar = 500 µm . Mean ± s . e . m . , statistical : two - unpaired t - , * * * p = 0 . 0001 .

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

(D) Stereological analysis of cortical area covered by Congo Red-positive plaques in male (n=10) and female (n=8) APP23 mice (left) and representative images (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, ***p=0.0001.


( E ) Native filter of TBS ( p = 0 . 2453 ) , Triton - X ( p = 0 . 0604 ) , SDS ( * p = 0 . 0196 ) formic acid ( FA ) ( * * p = 0 . 0057 ) fractions from ( n = 8 ) ( n = 7 ) APP23 mouse brain homogenates . Mean ± s . e . m . , statistical : two - unpaired t - between same fractions . Data points were taken from graphs analysing APP23p40 + / + against APP23p40 - / - mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Native filter assay analysis of TBS (p=0.2453), Triton-X (p=0.0604), SDS (*p=0.0196) and formic acid (FA) (**p=0.0057) fractions from male (n=8) and female (n=7) APP23 mouse brain homogenates. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test between the same fractions. Data points were taken from graphs analysing APP23p40+/+ against APP23p40-/- mice.


( A ) Histological of plaque - associated BACE1 immunoreactivity in ( n = 10 ) ( n = 8 ) APP23 mice . BACE1 covered was normalised to 4G8 - positive covered of same image ( left ) . Right : representative , scale bar = 50 µm . Mean ± s . e . m . , statistical : two - unpaired t - , p = 0 . 3724 .

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

(A) Histological analysis of plaque-associated BACE1 immunoreactivity in male (n=10) and female (n=8) APP23 mice. BACE1 area covered was normalised to 4G8-positive area covered of the same image (left). Right: representative images, scale bar = 50 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.3724.


( B ) Stereological of number of cortical GFAP - positive astrocytes in ( n = 10 ) ( n = 8 ) APP23 mice ( left ) . Right : representative of GFAP , scale bar = 200 µm . Mean ± s . e . m . , statistical : two - unpaired t - , * p = 0 . 0134 .

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 O O O O

(B) Stereological quantification of the number of cortical GFAP-positive astrocytes in male (n=10) and female (n=8) APP23 mice (left). Right: representative images of GFAP staining, scale bar = 200 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, *p=0.0134.


( C ) of activated microglia within 30 µm from plaque borders . Top : Numbers of Iba1 - positive microglia were normalised to of nearest 4G8 - positive plaque quantified in ( n = 10 ) ( n = 8 ) APP23 mice . Mean ± s . e . m . , statistical : two - unpaired t - , p = 0 . 0576 . Bottom : Histogram representing Clec7a within plaque - associated Iba1 - positive microglia in ( n = 10 ) ( n = 8 ) APP23 mice . Mean ± s . e . m . , statistical : two - unpaired t - with Bonferroni correction for each single bin , p = N . S . . Right : representative , scale bar = 40 µm . Data points were taken from graphs analysing APP23p40 + / + against APP23p40 - / - mice .

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 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 O O O O O 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 activated microglia within 30 µm from plaque borders. Top: Numbers of Iba1-positive microglia were normalised to the size of the nearest 4G8-positive plaque and quantified in male (n=10) and female (n=8) APP23 mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.0576. Bottom: Histogram representing Clec7a staining intensity within plaque-associated Iba1-positive microglia in male (n=10) and female (n=8) APP23 mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test with Bonferroni correction for each single bin, p=N.S.. Right: representative images, scale bar = 40 µm. Data points were taken from graphs analysing APP23p40+/+ against APP23p40-/- mice.


( D ) Radial profiles of Iba1 4G8 around center of nucleus of plaque - associated Iba1 - positive microglia in ( n = 10 ) ( n = 8 ) APP23 mice . Iba1 declines until a radius of ~ 6 µm , marking periphery . 4G8 peaks inside ( ~ 4 µm ) , but stays high outside . This is very likely due to close proximity to 4G8 positive plaques . Mean ± s . e . m . , statistical : two - unpaired t - with Bonferroni correction for number of binned radii shows no significant difference between both 4G8 traces .

O O O O O O B-MEASURED_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 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

(D) Radial intensity profiles of Iba1 and 4G8 around the center of the nucleus of plaque-associated Iba1-positive microglia in male (n=10) and female (n=8) APP23 mice. Iba1 intensity declines until a radius of ~6 µm, marking the cell periphery. 4G8 intensity peaks inside the cell (~4 µm), but stays high outside the cell. This is very likely due to the close proximity to 4G8 positive plaques. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test with Bonferroni correction for the number of binned radii shows no significant difference between both 4G8 traces.


( A ) Gene expression of IL12b in whole brain ( n = 3 per gender , Il12b undetected ) , Cd11b - negative non - microglial ( n = 3 per gender , Il12b undetected ) CD11b - positive microglia ( n = 5 per gender , p = 0 . 3540 ) in APP23 mice . Gapdh expression was used as an internal reference gene . Mean ± s . e . m . , statistical : two - unpaired t - for each fraction .

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

(A) Gene expression analysis of IL12b in whole brain (n=3 per gender, Il12b undetected), Cd11b-negative non-microglial cells (n=3 per gender, Il12b undetected) and CD11b-positive microglia (n=5 per gender, p=0.3540) in male and female APP23 mice. Gapdh expression was used as an internal reference gene. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test for each fraction.


( B ) ELISA measurements of IL12p40 in TBS - soluble fraction derived from wild - type ( WT ) ( n = 5 ) , APP23p40 + / + ( n = 17 ) APP23p40 ‑ / - ( n = 16 ) mice . mice are depicted by blue squares while mice are shown as red squares . Mean ± s . e . m . , statistical : one - way ANOVA , Tukey post - hoc , * p = 0 . 0276 , * * * p = < 0 . 0001 .

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

(B) ELISA measurements of the IL12p40 concentration in the TBS-soluble protein fraction derived from wild-type (WT) (n=5), APP23p40+/+(n=17) and APP23p40‑/- (n=16) mice. Male mice are depicted by blue squares while female mice are shown as red squares. Mean ± s.e.m., statistical analysis: one-way ANOVA, Tukey post-hoc test, *p=0.0276, ***p=<0.0001.


( A ) Mesoscale for Aβ1 - 40 in TBS ( p = 0 . 2298 ) , Triton - X ( p = 0 . 1329 ) SDS ( p = 0 . 7184 ) fractions of brain homogenates from APP23p40 + / + ( n = 10 ) APP23p40 - / - ( n = 8 ) mice . Total of each sample was used as an internal reference . Mean ± s . e . m . , statistical : two - unpaired t - .

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 O O O O O O

(A) Mesoscale analysis for the Aβ1-40 protein in the TBS (p=0.2298), Triton-X (p=0.1329) and SDS (p=0.7184) fractions of brain homogenates from male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice. Total protein concentration of each sample was used as an internal reference. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test.


( B ) Stereological of cortical covered by 4G8 - positive plaques ( left ) representative of 4G8 - in APP23p40 + / + ( n = 10 ) APP23p40 - / - ( n = 8 ) mice ( right ) , scale bar = 500 µm . Mean ± s . e . m . , statistical : two - unpaired t - , * * p = 0 . 0039 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Stereological analysis of cortical area covered by 4G8-positive plaques (left) and representative images of 4G8-staining in APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, **p=0.0039.


( C ) of pFTAA - positive covered in cortex of APP23p40 + / + ( n = 10 ) APP23p40 - / - ( n = 8 ) mice ( left ) representative for each genotype ( right ) , scale bar = 1 mm . Mean ± s . e . m . , statistical : two - unpaired t - , * * p = 0 . 0051 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fluorescence intensity based analysis of pFTAA-positive area covered in the cortex of APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice (left) and representative images for each genotype (right), scale bar = 1 mm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, **p=0.0051.


( D ) Stereological of cortical covered by Congo Red - positive plaques in APP23p40 + / + ( n = 10 ) APP23p40 - / - ( n = 8 ) mice ( left ) representative ( right ) , scale bar = 500 µm . Mean ± s . e . m . , statistical : two - unpaired t - , * * p = 0 . 0070 .

(D) Stereological analysis of cortical area covered by Congo Red-positive plaques in APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice (left) and representative images (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, **p=0.0070.


( E ) Native filter of TBS ( p = 0 . 7124 ) , Triton - X ( p = 0 . 3170 ) , SDS ( p = 0 . 4833 ) formic acid ( FA ) ( p = 0 . 8144 ) fractions from APP23p40 + / + ( n = 8 ) APP23p40 - / - ( n = 8 ) mouse brain homogenates . Mean ± s . e . m . , statistical : two - unpaired t - between same fractions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Native filter assay analysis of TBS (p=0.7124), Triton-X (p=0.3170), SDS (p=0.4833) and formic acid (FA) (p=0.8144) fractions from APP23p40+/+ (n=8) and APP23p40-/- (n=8) mouse brain homogenates. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test between the same fractions.


( A ) Histological of plaque - associated BACE1 immunoreactivity in APP23p40 + / + ( n = 10 ) APP23p40 - / - ( n = 8 ) mice . BACE1 covered was normalised to 4G8 - positive covered of same image ( left ) . Right : representative , scale bar = 50 µm . Mean ± s . e . m . , statistical : two - unpaired t - , p = 0 . 1780 .

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 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

(A) Histological analysis of plaque-associated BACE1 immunoreactivity in male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice. BACE1 area covered was normalised to 4G8-positive area covered of the same image (left). Right: representative images, scale bar = 50 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.1780.


( B ) Stereological of number of cortical GFAP - positive astrocytes in APP23p40 + / + ( n = 10 ) APP23p40 - / - ( n = 8 ) mice ( left ) . Right : representative of GFAP , scale bar = 200 µm . Mean ± s . e . m . , statistical : two - unpaired t - , p = 0 . 3148 .

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 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

(B) Stereological quantification of the number of cortical GFAP-positive astrocytes in male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice (left). Right: representative images of GFAP staining, scale bar = 200 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.3148.


( C ) of activated microglia within 30 µm from plaque borders . Top : Numbers of Iba1 - positive microglia were normalised to of nearest 4G8 - positive plaque quantified in APP23p40 + / + ( n = 10 ) APP23p40 - / - ( n = 8 ) mice . Mean ± s . e . m . , statistical : two - unpaired t - , p = 0 . 1925 . Bottom : Histogram representing Clec7a within plaque - associated Iba1 - positive microglia in APP23p40 + / + ( n = 10 ) APP23p40 - / - ( n = 8 ) mice . Mean ± s . e . m . , statistical : two - unpaired t - with Bonferroni correction for each single bin , p = N . S . . Right : representative , scale bar = 40 µm .

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

(C) Quantification of activated microglia within 30 µm from plaque borders. Top: Numbers of Iba1-positive microglia were normalised to the size of the nearest 4G8-positive plaque and quantified in male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.1925. Bottom: Histogram representing Clec7a staining intensity within plaque-associated Iba1-positive microglia in male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test with Bonferroni correction for each single bin, p=N.S.. Right: representative images, scale bar = 40 µm.


( D ) Radial profiles of Iba1 4G8 around center of nucleus of plaque - associated Iba1 - positive microglia in APP23p40 + / + ( n = 10 ) APP23p40 - / - ( n = 8 ) mice . Iba1 declines until a radius of ~ 6 µm , marking periphery . 4G8 peaks inside ( ~ 4 µm ) , but stays high outside . This is very likely due to close proximity to 4G8 positive plaques . Mean ± s . e . m . , statistical : two - unpaired t - with Bonferroni correction for number of binned radii shows no significant difference between both 4G8 traces .

O O O O O O B-MEASURED_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 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

(D) Radial intensity profiles of Iba1 and 4G8 around the center of the nucleus of plaque-associated Iba1-positive microglia in male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice. Iba1 intensity declines until a radius of ~6 µm, marking the cell periphery. 4G8 intensity peaks inside the cell (~4 µm), but stays high outside the cell. This is very likely due to the close proximity to 4G8 positive plaques. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test with Bonferroni correction for the number of binned radii shows no significant difference between both 4G8 traces.


( A ) Mesoscale for Aβ1 - 40 in TBS ( * p = 0 . 0208 ) , Triton - X ( * p = 0 . 0440 ) SDS ( p = 0 . 0540 ) fractions of brain homogenates from APP23p40 + / + ( n = 7 ) APP23p40 - / - ( n = 8 ) mice . Total of each sample was used as an internal reference . Mean ± s . e . m . , statistical : two - unpaired t - .

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 O O O O O O O O

(A) Mesoscale analysis for the Aβ1-40 protein in the TBS (*p=0.0208), Triton-X (*p=0.0440) and SDS (p=0.0540) fractions of brain homogenates from APP23p40+/+(n=7) and APP23p40-/- (n=8) mice. Total protein concentration of each sample was used as an internal reference. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test.


( B ) Stereological of cortical covered by 4G8 - positive plaques ( left ) representative of 4G8 - in APP23p40 + / + ( n = 8 ) APP23p40 - / - ( n = 10 ) mice ( right ) , scale bar = 500 µm . Mean ± s . e . m . , statistical : two - unpaired t - , p = 0 . 1831 .

(B) Stereological analysis of cortical area covered by 4G8-positive plaques (left) and representative images of 4G8-staining in APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.1831.


( C ) of pFTAA - positive covered in cortex of APP23p40 + / + ( n = 8 ) APP23p40 - / - ( n = 10 ) mice ( left ) representative for each genotype ( right ) , scale bar = 1 mm . Mean ± s . e . m . , statistical : two - unpaired t - , p = 0 . 3406 .

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(C) Fluorescence intensity based analysis of pFTAA-positive area covered in the cortex of APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice (left) and representative images for each genotype (right), scale bar = 1 mm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.3406.


( D ) Stereological of cortical covered by Congo Red - positive plaques in APP23p40 + / + ( n = 8 ) APP23p40 - / - ( n = 10 ) mice ( left ) representative ( right ) , scale bar = 500 µm . Mean ± s . e . m . , statistical : two - unpaired t - , p = 0 . 4542 .

(D) Stereological analysis of cortical area covered by Congo Red-positive plaques in APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice (left) and representative images (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.4542.


( E ) Native filter of TBS ( p = 0 . 6823 ) , Triton - X ( p = 0 . 1146 ) , SDS ( p = 0 . 0508 ) formic acid ( FA ) ( p = 0 . 6603 ) fractions from APP23p40 + / + ( n = 7 ) APP23p40 - / - ( n = 7 ) mouse brain homogenates . Mean ± s . e . m . , statistical : two - unpaired t - between same fractions .

(E) Native filter assay analysis of TBS (p=0.6823), Triton-X (p=0.1146), SDS (p=0.0508) and formic acid (FA) (p=0.6603) fractions from APP23p40+/+ (n=7) and APP23p40-/- (n=7) mouse brain homogenates. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-tests between the same fractions.


( A ) Histological of plaque - associated BACE1 immunoreactivity in APP23p40 + / + ( n = 8 ) APP23p40 - / - ( n = 10 ) mice . BACE1 covered was normalised to 4G8 - positive covered of same image ( left ) . Right : representative , scale bar = 50 µm . Mean ± s . e . m . , statistical : two - unpaired t - , p = 0 . 5402 .

(A) Histological analysis of plaque-associated BACE1 immunoreactivity in female APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice. BACE1 area covered was normalised to 4G8-positive area covered of the same image (left). Right: representative images, scale bar = 50 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.5402.


( B ) Stereological of number of cortical GFAP - positive astrocytes in APP23p40 + / + ( n = 8 ) APP23p40 - / - ( n = 8 ) mice ( left ) . Right : representative of GFAP , scale bar = 200 µm . Mean ± s . e . m . , statistical : two - unpaired t - , p = 0 . 1579 .

(B) Stereological quantification of the number of cortical GFAP-positive astrocytes in female APP23p40+/+ (n=8) and APP23p40-/- (n=8) mice (left). Right: representative images of GFAP staining, scale bar = 200 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.1579.


( C ) of activated microglia within 30 µm from plaque borders . Top : Numbers of Iba1 - positive microglia were normalised to of nearest 4G8 - positive plaque quantified in APP23p40 + / + ( n = 8 ) APP23p40 - / - ( n = 10 ) mice . Mean ± s . e . m . , statistical : two - unpaired t - , p = 0 . 8240 . Bottom : Histogram representing Clec7a within plaque - associated Iba1 - positive microglia in APP23p40 + / + ( n = 8 ) APP23p40 - / - ( n = 10 ) mice . Mean ± s . e . m . , statistical : two - unpaired t - with Bonferroni correction for each single bin , p = N . S . . Right : representative , scale bar = 40 µm .

(C) Quantification of activated microglia within 30 µm from plaque borders. Top: Numbers of Iba1-positive microglia were normalised to the size of the nearest 4G8-positive plaque and quantified in female APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.8240. Bottom: Histogram representing Clec7a staining intensity within plaque-associated Iba1-positive microglia in female APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test with Bonferroni correction for each single bin, p=N.S.. Right: representative images, scale bar = 40 µm.


( D ) Radial profiles of Iba1 4G8 around center of nucleus of plaque - associated Iba1 - positive microglia in APP23p40 + / + ( n = 8 ) APP23p40 - / - ( n = 10 ) mice . Iba1 declines until a radius of ~ 6 µm , marking periphery . 4G8 peaks inside ( ~ 4 µm ) , but stays high outside . This is very likely due to close proximity to 4G8 positive plaques . Mean ± s . e . m . , statistical : two - unpaired t - with Bonferroni correction for number of binned radii shows no significant difference between both 4G8 traces .

(D) Radial intensity profiles of Iba1 and 4G8 around the center of the nucleus of plaque-associated Iba1-positive microglia in female APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice. Iba1 intensity declines until a radius of ~6 µm, marking the cell periphery. 4G8 intensity peaks inside the cell (~4 µm), but stays high outside the cell. This is very likely due to the close proximity to 4G8 positive plaques. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test with Bonferroni correction for the number of binned radii shows no significant difference between both 4G8 traces.


A Example of mapping results . Wild allele frequency in progeny of a cross of gab1 - 1 sensitive allele to YJM975 ( y - axis ) along yeast genome ( x - axis ) at permissive 26 ° C ( blue ) restrictive 34 ° C ( TS allele loss - of - function , cyan ) . allele frequency change between two temperatures is used in mapping . Labels : selected loci in cross . Blue regions : called suppressor loci . B

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A Example of the mapping results. Wild allele frequency in progeny of a cross of the gab1-1 temperature sensitive allele to the YJM975 strain (y-axis) along the yeast genome (x-axis) at the permissive 26°C (blue) and restrictive 34°C (TS allele loss-of-function, cyan). The allele frequency change between the two temperatures is used in mapping. Labels: selected loci in the cross. Blue regions: called suppressor loci. B


E Experimental validation of RPN8 as causal suppressor of rpn11 - 8 TS mutant . Cultures of indicated strains were diluted to an optical density at 600 nm of 0 . 1 a series of ten - fold dilutions was spotted on agar plates incubated for 2 - 3 days at 34 ° C . UWOPS = UWOPS87 - 2421 . See also Figure EV4 .

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E Experimental validation of RPN8 as the causal suppressor of the rpn11-8 TS mutant. Cultures of the indicated strains were diluted to an optical density at 600 nm of 0.1 and a series of ten-fold dilutions was spotted on agar plates and incubated for 2-3 days at 34°C. UWOPS = UWOPS87-2421. See also Figure EV4.


A , B Suppression of nse3 - ts4 nse4 - ts4 sensitivity ( A ) damage sensitivity ( B ) by NSE1 allele of UWOPS87 - 2421 . Cultures of indicated strains were diluted to an optical density at 600 nm of 0 . 1 a series of ten - fold dilutions was spotted on agar plates incubated for 2 - 3 days . UW = UWOPS87 - 2421 . plates shown in ( B ) were incubated at 30 ° C . C

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 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

A,B Suppression of nse3-ts4 and nse4-ts4 temperature sensitivity (A) and DNA damage sensitivity (B) by the NSE1 allele of UWOPS87-2421. Cultures of the indicated strains were diluted to an optical density at 600 nm of 0.1 and a series of ten-fold dilutions was spotted on agar plates and incubated for 2-3 days. UW = UWOPS87-2421. The plates shown in (B) were incubated at 30°C. C


E Recruitment of Smc6 - FLAG by ChIP - qPCR at two known SMC5 / 6 binding sites ( pericentromere of chromosome XIV CEN3 ) one negative control locus ( arm of chromosome III ) in G2 / M arrested strains . Relative enrichment corresponds to of signal after immunoprecipitation ( FLAG ) over beads alone , normalised to in wild - type at CEN3 . Error bars : standard error of mean of three independent experiments . Statistical significance was determined using an unpaired , two - t - ( * = p < 0 . 05 , * * = p < 0 . 005 ) . E

O O O B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Recruitment of Smc6-FLAG by ChIP-qPCR at two known SMC5/6 binding sites (pericentromere of chromosome XIV and CEN3) and one negative control locus (arm of chromosome III) in G2/M arrested strains. Relative enrichment corresponds to the ratio of the signal after immunoprecipitation (FLAG) over beads alone, normalised to the ratio in wild-type cells at CEN3. Error bars: standard error of the mean of three independent experiments. Statistical significance was determined using an unpaired, two-tailed t-test (* = p < 0.05, ** = p < 0.005). E


( A ) Representative confocal photomicrographs showing coronal slices from 8W hGFAP ‐ tTA ; H2B ‐ GFP mice with without doxycycline treatment , upon immunofluorescent with marker Nestin ( red ) . DAPI was used for counterstain . Cross indicates slice directions : M = medial , D = dorsal , V = ventral , L = lateral . Scale bar = 20 µm . ( B ) of Nestin expression in apical basal in doxycycline - treated untreated . N = 3 ( - Doxy ) , N = 4 ( + Doxy ) . Data information : N number refers to biological replicates ; for each biological replicate , at least three technical replicates were performed . Data is represented as mean ± . * # indicate significance : between apical basal ( * ) between doxycycline treatments ( # ) ; * / # p < 0 . 05 , * * p < 0 . 01 , by one - way ANOVA with Tukey ' s multiple comparisons ( B )

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

(A) Representative confocal photomicrographs showing coronal slices from 8W hGFAP‐tTA;H2B‐GFP mice with and without doxycycline treatment, upon immunofluorescent staining with the stem cell marker Nestin (red). DAPI was used for nuclear counterstain. Cross indicates slice directions: M = medial, D = dorsal, V = ventral, L = lateral. Scale bar = 20 µm. (B) Quantification of Nestin expression in apical and basal cells in doxycycline-treated and untreated animals. N = 3 (- Doxy), N = 4 (+ Doxy). Data information: N number refers to biological replicates; for each biological replicate, at least three technical replicates were performed. Data is represented as mean ± SEM. * and # indicate significance: between apical and basal (*) and between doxycycline treatments (#); */#p<0.05, **p<0.01, by one-way ANOVA with Tukey's multiple comparisons test (B)


( C ) Same as ( A ) , with addition of cilia marker AC3 ( yellow ) . Arrows indicate Nestin - positive ( ) GFP - positive ( green ) with cilium . Scale bar = 10 µm . ( D ) of ciliated GFP + NSCs in apical basal layer as of total apical / basal GFP + . N = 7 . ( E ) of cilia on in apical or basal layer according to marker ; N = 7 . Data information : N number refers to biological replicates ; for each biological replicate , at least three technical replicates were performed . Data is represented as mean ± . * # indicate significance : between apical basal ( * ) ; * / # p < 0 . 05 , * * p < 0 . 01 , by two - unpaired student ' s t - ( D ) .

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(C) Same as (A), with addition of the primary cilia marker AC3 (yellow). Arrows indicate Nestin-positive (white) and GFP-positive (green) cells with primary cilium. Scale bar = 10 µm. (D) Quantification of ciliated GFP+ NSCs in apical and basal cell layer as percentage of total apical/basal GFP+ cells. N = 7. (E) Quantification of primary cilia on cells in the apical or basal cell layer according to stem cell marker; N = 7. Data information: N number refers to biological replicates; for each biological replicate, at least three technical replicates were performed. Data is represented as mean ± SEM. * and # indicate significance: between apical and basal (*) ; */#p<0.05, **p<0.01, by two-tailed unpaired student's t-test (D).


( A ) Repesentative confocal photomicrographs of coronal sections of SVZ of 8W hGFAP - H2BGFP mice , which had been administered doxycycline ( Doxy ) as indicated . GFP expression Ki67 immunoreactivity are shown in green red , respectively . DAPI counterstaining of nuclei is blue . Scale bar indicates 20 µm .

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(A) Repesentative confocal photomicrographs of coronal sections of the SVZ of 8W hGFAP-H2BGFP mice, which had been administered doxycycline (Doxy) as indicated. Nuclear GFP expression and Ki67 immunoreactivity are shown in green and red, respectively. DAPI counterstaining of the nuclei is blue. Scale bar indicates 20 µm.


( B , C ) Quantitative analyses of number of apical basal expressing GFP ( G + ) or not ( G - ) ; N = 8 ( B ) , Ki67 immunoreactivity in these populations ; N ≥ 4 ( C ) . Data information : N number refers to biological replicates ; for each biological replicate at least three technical replicates were performed . Bars represent mean ± . * indicates significance between doxycycline treated nontreated specimens with apical or basal populations . * p < 0 . 05 , * * * p < 0 . 001 , determined by two - way ANOVA with Sidak ' s multiple comparisons .

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(B, C) Quantitative analyses of the number of apical and basal cells expressing GFP (G+) or not (G-); N = 8 (B), and Ki67 immunoreactivity in these populations; N ≥ 4 (C). Data information: N number refers to biological replicates; for each biological replicate at least three technical replicates were performed. Bars represent mean ± SEM. * indicates significance between doxycycline treated and nontreated specimens with apical or basal cell populations. * p<0.05, ***p<0.001, determined by two-way ANOVA with Sidak's multiple comparisons test.


( B ) Representative of OB of AAV - GFP - injected , at 14 days ( 14D ) 6 ( 6W ) after injection . Scale bar indicates 20 µm . ( C ) of GFP + / or DCX + in OB , as of total DAPI N ≥ 5 . Data information : N number refers to biological replicates ; for each biological replicate at least three technical replicates were performed . Bars represent mean ± . * indicates significance : * p < 0 . 05 , determined by two - way ANOVA with Sidak ' s multiple comparisons .

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(B) Representative micrographs of the OB of AAV-GFP-injected animals, at 14 days (14D) and 6 weeks (6W) after virus injection. Scale bar indicates 20 µm. (C) Quantification of GFP+ and/or DCX+ cells in the OB, as percentage of total DAPI cells N ≥ 5. Data information: N number refers to biological replicates; for each biological replicate at least three technical replicates were performed. Bars represent mean ± SEM.* indicates significance: *p<0.05, determined by two-way ANOVA with Sidak's multiple comparisons test.


Representative confocal of coronal slices of of olfactory bulb ( OB ) obtained from RiboTag mice injected with AVV - hGFAP - mycCRE particles . In ( E ) mice were analysed 16 after injection in lateral ventricle or in lateral corner as indicated . Scale bars = 20 µm . ( F ) of RiboTag - HA ( HA ) + / or DCX + in OB in N ≥ 2 . Data information : N number refers to biological replicates ; for each biological replicate at least three technical replicates were performed . Bars represent mean ± . * indicates significance : * p < 0 . 05 , determined by two - way ANOVA with Sidak ' s multiple comparisons .

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

Representative confocal images of coronal slices of the core of the olfactory bulb (OB) obtained from RiboTag mice injected with AVV-hGFAP-mycCRE particles. In (E) mice were analysed 16 weeks after injection in the lateral ventricle or in the lateral corner as indicated. Scale bars = 20 µm. (F) Quantification of RiboTag-HA (HA)+ and/or DCX+ cells in the OB in N ≥ 2. Data information: N number refers to biological replicates; for each biological replicate at least three technical replicates were performed. Bars represent mean ± SEM.* indicates significance: *p<0.05, determined by two-way ANOVA with Sidak's multiple comparisons test.


( A , B ) Representative of V - SVZ of mice injected with an AAV overexpressing GFP a shRNA against Hes1 ( AAV Hes1 - sh ) or a scrambled ( AAV sc - sh ) . Coronal sections were stained with either notch intracellular domain ( NICD , A ) or Ki67 to visualize cycling ( B ) . DAPI was used for counterstain , scale bars indicate 20 µm . Arrows indicate apical GFP + ( ) basal GFP - ( yellow ) displaying NICD . of fold change in NICD - ; N = 3 ( C ) , Ki67 + ; N = 5 ( D ) Data information : N number refers to biological replicates ; for each biological replicate at least three technical replicates were performed . Bars represent mean ± . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , determined by two - way ANOVA with Sidak ' s multiple comparisons

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(A, B) Representative micrographs of the V-SVZ of mice injected with an AAV overexpressing GFP and a shRNA against Hes1 (AAV Hes1-sh) or a scrambled sequence (AAV sc-sh). Coronal sections were stained with either notch intracellular domain (NICD, A) or Ki67 to visualize cycling cells (B). DAPI was used for nuclear counterstain, scale bars indicate 20 µm. Arrows indicate apical GFP+ (white) and basal GFP- (yellow) cells displaying NICD staining. Quantification of fold change in NICD-levels; N = 3 (C), Ki67+ cells; N = 5 (D) Data information: N number refers to biological replicates; for each biological replicate at least three technical replicates were performed. Bars represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, determined by two-way ANOVA with Sidak's multiple comparisons test


A , HeLa were treated with H2O2 ( 5 mM , 1 min ) or PR - 619 ( 10 µM , 10 min ) analysed by immunoblotting for endogenous p62 , NDP52 OPTN in either reducing ( 2 . 5 % β - mE ) or non - reducing conditions . Data information : # , non - specific band or a post - translational modification .

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A, HeLa cells were treated with H2O2 (5 mM, 1 min) or PR-619 (10 µM, 10 min) and analysed by immunoblotting for endogenous p62, NDP52 and OPTN in either reducing (2.5% β-mE) or non-reducing conditions. Data information: #, non-specific band or a post-translational modification.


B , NDP52 OPTN are reducible by thioredoxin antioxidant system . HeLa were pre - treated with TrxR inhibitors , curcumin ( 50 μM ) or auranofin ( 5 μM ) for 30 min , then treated with H2O2 ( 500 μM ) at different time points as indicated . Immunoblotting was performed to detect NDP52 , OPTN PRX - 3 ( dimer formation reflecting oxidation of ) in non - reduced conditions PRX - SO3 ( marker of oxidative stress actin in reduced conditions .

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B, NDP52 and OPTN are reducible by the thioredoxin antioxidant system. HeLa cells were pre-treated with TrxR inhibitors, curcumin (50 μM) or auranofin (5 μM) for 30 min, then treated with H2O2 (500 μM) at different time points as indicated. Immunoblotting was performed to detect NDP52, OPTN and PRX-3 (dimer formation reflecting oxidation of the protein) in non-reduced conditions and PRX-SO3 (marker of oxidative stress and actin in reduced conditions.


C , D , Immunoblotting for NDP52 in either reducing or non - reducing conditions of whole samples ( C ) mitochondrial fractions ( D ) from HeLa treated with combination of 4 µM antimycin A1 10 µM oligomycin ( AO ) in presence or absence of 400 nM bafilomycin A1 ( Baf ) for 2 h . NDP52 DLC in mitochondrial fraction was quantified ( D ) . Data are mean ± s . e . m ( D ) . P values were calculated by one - way ANOVA followed by Sidak on four independent experiments ( D ) . * * * , p < 0 . 001 ; ns ( non - significant ) . Data information : # , non - specific band or a post - translational modification .

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 I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Immunoblotting for NDP52 in either reducing or non-reducing conditions of whole cell samples (C) and mitochondrial fractions (D) from HeLa cells treated with the combination of 4 µM antimycin A1 and 10 µM oligomycin (AO) in the presence or absence of 400 nM bafilomycin A1 (Baf) for 2 h. NDP52 DLC in mitochondrial fraction was quantified (D). Data are mean ± s.e.m (D). P values were calculated by one-way ANOVA followed by Sidak test on four independent experiments (D). ***, p<0.001; ns (non-significant). Data information: #, non-specific band or a post-translational modification.


A - D , Single ( A ) , double ( B ) , triple ( C ) , quadruple ( D ) cysteine mutant NDP52 plasmids were generated transfected into HeLa PentaKO . were treated with 20 µM PR - 619 for 10 min analysed by immunoblotting for NDP52 in either reducing or non - reducing conditions . constructs highlighted in red were subjected to next round of Cys - Ala screen or further analyses .

O 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-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

A-D, Single (A), double (B), triple (C), and quadruple (D) cysteine mutant NDP52 plasmids were generated and transfected into HeLa PentaKO cells. Cells were treated with 20 µM PR-619 for 10 min and analysed by immunoblotting for NDP52 in either reducing or non-reducing conditions. The constructs highlighted in red were subjected to the next round of Cys-Ala screen or further analyses.


B , Recombinant of NDP52 WT C18 , 153 , 163 , 321S mutant ( Mut ) were exposed to indicated concentrations of H2O2 for 5 min subjected to SDS - PAGE CBB gel .

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

B, Recombinant proteins of NDP52 WT and C18,153,163,321S mutant (Mut) were exposed to the indicated concentrations of H2O2 for 5 min and subjected to SDS-PAGE and CBB gel staining.


C , Analytical - exclusion chromatography ( SEC ) of NDP52 WT Mut .

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

C, Analytical size-exclusion chromatography (SEC) of the NDP52 WT and Mut proteins.


D , Recombinant NDP52 WT Mut ( 5 ng ) were subjected to NATIVE - PAGE . S . E , short exposure ; L . E . long exposure .

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

D, Recombinant NDP52 WT and Mut proteins (5 ng) were subjected to NATIVE-PAGE analysis. S.E, short exposure; L.E. long exposure.


A , B , microscopy ( A ) ( B ) of mitophagy of HeLa wild - type ( WT ) , PentaKO + empty vector , PentaKO + NDP52 WT or NDP52 C18 , 153 , 163 , 321S ( Mut ) , stably expressing YFP - Parkin mt - mKeima were pre - treated with or without MitoQ for 22 h treated with 4 µM / 10 µM AO for indicated times . Data information Data are mean ± s . e . m . ( B or displayed as popular violin plots ( B ) . P values were calculated by one - way ANOVA followed by Sidak on three independent experiments ( B * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § , p < 0 . 05 , § § § , p < 0 . 001 ( relative to MitoQ - untreated condition ) ; ns ( non - significant ) . Scale bars : 20 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 B-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

A, B, Fluorescence microscopy images (A) and quantification (B) of mitophagy of HeLa wild-type (WT), PentaKO + empty vector, PentaKO + NDP52 WT or NDP52 C18,153,163,321S (Mut) cells, stably expressing YFP-Parkin and mt-mKeima were pre-treated with or without MitoQ for 22 h and treated with 4 µM / 10 µM AO for the indicated times. Data information Data are mean ± s.e.m. (B or displayed as cell popular violin plots (B). P values were calculated by one-way ANOVA followed by Sidak test on three independent experiments (B *, p<0.05; **, p<0.01; ***, p<0.001; §, p<0.05, §§§, p<0.001 (relative to MitoQ-untreated condition); ns (non-significant). Scale bars: 20 μm (A


C , HeLa PentaKO + NDP52 WT or NDP52 Mut stably expressing YFP - Parkin mt - mKeima were treated with 4 µM / 10 µM AO for 2 h in absence or presence of 10 μM MG132 ( MG ) 400 nM Baf , followed by a mitochondrial fractionation immunoblotting for NDP52 in non - reducing conditions . mitochondrial UQCRC2 was used as a loading control . Data information : # , non - specific band or a post - translational modification .

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

C, HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM / 10 µM AO for 2 h in the absence or presence of 10 μM MG132 (MG) and 400 nM Baf, followed by a mitochondrial fractionation and immunoblotting for NDP52 in non-reducing conditions. The mitochondrial protein UQCRC2 was used as a loading control. Data information: #, non-specific band or a post-translational modification.


D , HeLa PentaKO + NDP52 WT stably expressing YFP - Parkin mt - mKeima were treated with 1 mM DFP for 24 h ( D ) , or pre - treated with 500 nM MitoQ for 22 h treated with 4 µM / 10 µM AO for 2 h ( D were then analysed by MitoSOX ( D ) Data information : # , Data are mean ± s . e . m . D ) P values were calculated by one - way ANOVA followed by Sidak on three independent experiments D ) . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § , p < 0 . 05 , § § § , p < 0 . 001 ( relative to MitoQ - untreated condition ) ; ns ( non - significant ) . Scale bars : 20 μm D ) .

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

D, HeLa PentaKO + NDP52 WT cells stably expressing YFP-Parkin and mt-mKeima were treated with 1 mM DFP for 24 h (D), or pre-treated with 500 nM MitoQ for 22 h and treated with 4 µM / 10 µM AO for 2 h (D Cells were then analysed by MitoSOX staining (D) Data information: #, Data are mean ± s.e.m. D) P values were calculated by one-way ANOVA followed by Sidak test on three independent experiments D). *, p<0.05; **, p<0.01; ***, p<0.001; §, p<0.05, §§§, p<0.001 (relative to MitoQ-untreated condition); ns (non-significant). Scale bars: 20 μm D).


E , HeLa PentaKO + NDP52 WT stably expressing YFP - Parkin mt - mKeima were treated with 500 nM MitoQ for 22 h treated with 4 µM / 10 µM AO for 2 h E ) in presence or absence of 400 nM Baf ( E ) . immunoblotting for NDP52 ( E ) . Data information : # , non - specific band or a post - translational modification .

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

E, HeLa PentaKO + NDP52 WT cells stably expressing YFP-Parkin and mt-mKeima were treated with 500 nM MitoQ for 22 h and treated with 4 µM / 10 µM AO for 2 h E) in the presence or absence of 400 nM Baf (E). immunoblotting for NDP52 (E). Data information: #, non-specific band or a post-translational modification.


A , microscopy of mitophagy in HeLa PentaKO + NDP52 WT stably expressing YFP - Parkin mt - mKeima were treated with 4 µM rotenone ( R ) , 4 µM A , 10 µM O , or combination of RO or AO for 3h . Data information Data are mean ± s . e . m . ( A or displayed as popular violin plots ( A , P values were calculated by one - way ANOVA followed by Sidak ( for multiple groups ) or unpaired two - Student ' s t - ( for two groups ) on at least three independent experiments as indicated ( A , * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; ns ( non - significant ) . Scale bars : 20 μm (

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-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

A, Fluorescence microscopy images and quantification of mitophagy in HeLa PentaKO + NDP52 WT cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM rotenone (R), 4 µM A, 10 µM O, or the combination of RO or AO for 3h. Data information Data are mean ± s.e.m. (A or displayed as cell popular violin plots (A, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated (A, *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm (A


B , microscopy of MitoSOX in HeLa PentaKO + NDP52 WT in same conditions as ( A ) . Data information Data are mean ± s . e . m . B , or displayed as popular violin plots B , P values were calculated by one - way ANOVA followed by Sidak ( for multiple groups ) or unpaired two - Student ' s t - ( for two groups ) on at least three independent experiments as indicated B , * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; ns ( non - significant ) . Scale bars : 20 μm B ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Fluorescence microscopy images and quantification of MitoSOX staining in HeLa PentaKO + NDP52 WT cells in the same conditions as (A). Data information Data are mean ± s.e.m. B, or displayed as cell popular violin plots B, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated B, *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm B,


HeLa PentaKO + NDP52 WT stably expressing YFP - Parkin mt - mKeima ( C , were pre - treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min treated with AO for 2 h or with RO for 3 h , followed by mitophagy measurement ( C ) Data information Data are mean ± s . e . m . C , or displayed as popular violin plots C , P values were calculated by one - way ANOVA followed by Sidak ( for multiple groups ) or unpaired two - Student ' s t - ( for two groups ) on at least three independent experiments as indicated C , * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; ns ( non - significant ) . Scale bars : 20

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

HeLa PentaKO + NDP52 WT cells stably expressing YFP-Parkin and mt-mKeima (C, were pre-treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min and treated with AO for 2 h or with RO for 3 h, followed by mitophagy measurement (C) Data information Data are mean ± s.e.m. C, or displayed as cell popular violin plots C, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated C, *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm


HeLa PentaKO + NDP52 WT ( D ) were pre - treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min treated with AO for 2 h or with RO for 3 h , followed by MitoSOX ( D ) Data information Data are mean ± s . e . m . P values were calculated by one - way ANOVA followed by Sidak ( for multiple groups ) or unpaired two - Student ' s t - ( for two groups ) on at least three independent experiments as indicated * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; ns ( non - significant ) . Scale bars : 20 μm ,

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

HeLa PentaKO + NDP52 WT cells (D) were pre-treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min and treated with AO for 2 h or with RO for 3 h, followed by MitoSOX staining (D) Data information Data are mean ± s.e.m. P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm , D


HeLa PentaKO + NDP52 WT stably expressing YFP - Parkin mt - mKeima E ) were pre - treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min treated with AO for 2 h or with RO for 3 h , followed by immunoblotting for NDP52 in non - reducing conditions ( E ) . Data information : # , non - specific band or a post - translational modification .

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 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 O O O O O

HeLa PentaKO + NDP52 WT cells stably expressing YFP-Parkin and mt-mKeima E) were pre-treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min and treated with AO for 2 h or with RO for 3 h, followed by immunoblotting for NDP52 in non-reducing conditions (E). Data information: #, non-specific band or a post-translational modification.


F , HeLa PentaKO + NDP52 WT stably expressing YFP - Parkin mt - mKeima were pre - treated with or without 10 mM DTT for 30 min treated with AO for 2 h followed by mitophagy measurements ( F ) Data information Data are mean ± s . e . m . F ) or displayed as popular violin plots F ) . P values were calculated by one - way ANOVA followed by Sidak ( for multiple groups ) or unpaired two - Student ' s t - ( for two groups ) on at least three independent experiments as indicated F ) . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; ns ( non - significant ) . Scale bars : 20 μm F ) .

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 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

F, HeLa PentaKO + NDP52 WT stably expressing YFP-Parkin and mt-mKeima were pre-treated with or without 10 mM DTT for 30 min and treated with AO for 2 h followed by mitophagy measurements (F) Data information Data are mean ± s.e.m. F) or displayed as cell popular violin plots F). P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated F). *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm F).


G , HeLa PentaKO + NDP52 WT stably expressing YFP - Parkin mt - mKeima were pre - treated with or without 10 mM DTT for 30 min treated with AO for 2 h followed by immunoblotting for NDP52 in non - reducing conditions ( G ) . Data information : # , non - specific band or a post - translational modification .

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

G, HeLa PentaKO + NDP52 WT stably expressing YFP-Parkin and mt-mKeima were pre-treated with or without 10 mM DTT for 30 min and treated with AO for 2 h followed by immunoblotting for NDP52 in non-reducing conditions (G). Data information: #, non-specific band or a post-translational modification.


HeLa PentaKO + NDP52 WT or NDP52 Mut stably expressing YFP - Parkin mt - mKeima were treated with 4 µM / 10 µM AO for 2 h in presence or absence of 400 nM Baf , followed by immunofluorescence analyses ( A ) number of foci of indicated colocalised with NDP52 , or foci of NDP52 colocalised with Parkin , was quantified . Data information : Data are mean ± s . e . m . ( A , P values were calculated by one - way ANOVA followed by Sidak ( for multiple groups ) or unpaired two - Student ' s t - ( for two groups ) on three independent experiments ( A * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § § , p < 0 . 01 ; § § § , p < 0 . 001 ns ( non - significant ) . Scale bars : 20 μm ( A ,

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

HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf, followed by immunofluorescence analyses (A) The number of foci of the indicated proteins colocalised with NDP52, or foci of NDP52 colocalised with Parkin, was quantified. Data information: Data are mean ± s.e.m. (A, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments (A *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 ns (non-significant). Scale bars: 20 μm (A,


HeLa PentaKO + NDP52 WT or NDP52 Mut stably expressing YFP - Parkin mt - mKeima were treated with 4 µM / 10 µM AO for 2 h in presence or absence of 400 nM Baf , followed by immunoblotting for LC3 NDP52 in mitochondrial fraction ( B ) or whole lysate ( C ) in reducing conditions Data information : Data are mean ± s . e . m . B , C , P values were calculated by one - way ANOVA followed by Sidak ( for multiple groups ) or unpaired two - Student ' s t - ( for two groups ) on three independent experiments B , C * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § § , p < 0 . 01 ; § § § , p < 0 . 001 ns ( non - 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 O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf, followed by immunoblotting for LC3 and NDP52 in mitochondrial fraction (B) or whole cell lysate (C) in reducing conditions Data information: Data are mean ± s.e.m. B, C, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments B, C *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 ns (non-significant).


HeLa PentaKO + NDP52 WT or NDP52 Mut stably expressing YFP - Parkin mt - mKeima were treated with 4 µM / 10 µM AO for 2 h in presence or absence of 400 nM Baf , followed by co - immunoprecipitation to analyse interaction of NDP52 with FIP200 , ULK1 ATG13 ( D ) . Data information : Data are mean ± s . e . m . P values were calculated by one - way ANOVA followed by Sidak ( for multiple groups ) or unpaired two - Student ' s t - ( for two groups ) on three independent experiments D , * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § § , p < 0 . 01 ; § § § , p < 0 . 001 ns ( non - 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 O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf, followed by co-immunoprecipitation assay to analyse the interaction of NDP52 with FIP200, ULK1 and ATG13 (D). Data information: Data are mean ± s.e.m. P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments D, *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 ns (non-significant).


E , MEFs stably expressing YFP - Parkin , mt - mKeima empty , human NDP52 ( hNDP52 ) WT or hNDP52 Mut were pre - treated with or without 500 nM MitoQ for 21 h treated with AO for 3 h followed by mitophagy measurement . Data information : Data are mean ± s . e . m . E ) or displayed as popular violin plots ( E ) . P values were calculated by one - way ANOVA followed by Sidak ( for multiple groups ) or unpaired two - Student ' s t - ( for two groups ) on three independent experiments E ) . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § § , p < 0 . 01 ; § § § , p < 0 . 001 ( relative to MitoQ - untreated condition ) ; ns ( non - significant ) . Scale bars : 20 μm E ) .

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

E, MEFs stably expressing YFP-Parkin, mt-mKeima and empty, human NDP52 (hNDP52) WT or hNDP52 Mut were pre-treated with or without 500 nM MitoQ for 21 h and treated with AO for 3 h followed by mitophagy measurement. Data information: Data are mean ± s.e.m. E) or displayed as cell popular violin plots (E). P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments E). *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 (relative to MitoQ-untreated condition); ns (non-significant). Scale bars: 20 μm E).


C . Dissociation constants ( Kd ) of Ago2 - miRNA complexes ( containing an AU - rich supplementary region ) binding to target RNAs with bridging regions of various lengths . D . Dissociation constants ( Kd ) of Ago2 - miRNA complexes ( containing an GC - rich supplementary region ) binding to target RNAs with bridging regions of various lengths . Data information : plotted data are means of at least three independent replicates . Error bars indicate .

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C. Dissociation constants (Kd) of Ago2-miRNA complexes (containing an AU-rich supplementary region) binding to target RNAs with bridging regions of various lengths. D. Dissociation constants (Kd) of Ago2-miRNA complexes (containing an GC-rich supplementary region) binding to target RNAs with bridging regions of various lengths. Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.


E . Fold repression of a luciferase reporter ( relative to no target site control ) plotted versus affinity ( 1 / Kd ) values measured for same sites in vitro . Data information : plotted data are means of at least three independent replicates . Error bars indicate .

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E. Fold repression of a luciferase reporter (relative to no target site control) plotted versus affinity (1/Kd) values measured for the same sites in vitro. Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.


A . Top : schematic of miR - 122 isomiRs paired with a target complementary to seed region only . Bottom : release of seed - paired target from Ago2 - miR complexes was assessed by monitoring fraction of total target bound as a function of time . B . Top : schematic of miR - 122 isomiRs paired with a target complementary to seed supplementary ( g13 - g17 ) regions . Bottom : fraction target bound to Ago2 - miR complexes as a function of time . C . Top : schematic of miR - 122 isomiRs paired with a target complementary to seed shifted supplementary ( g15 - g19 ) regions . . Bottom : fraction target bound to Ago2 - miR complexes as a function of time . Data information : plotted data are means of at least three independent replicates . Error bars indicate .

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A. Top: schematic of miR-122 isomiRs paired with a target complementary to the seed region only. Bottom: release of the seed-paired target from Ago2-miR complexes was assessed by monitoring the fraction of the total target RNA bound as a function of time. B. Top: schematic of miR-122 isomiRs paired with a target complementary to the seed and supplementary (g13-g17) regions. Bottom: fraction target bound to the Ago2-miR complexes as a function of time. C. Top: schematic of miR-122 isomiRs paired with a target complementary to the seed and shifted supplementary (g15-g19) regions. . Bottom: fraction target bound to the Ago2-miR complexes as a function of time. Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.


E . Release half times ( t1 / 2 ) of target RNAs in panels ( A - C ) plotted against isomiR . Data information : plotted data are means of at least three independent replicates . Error bars indicate .

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E. Release half times (t1/2) of target RNAs in panels (A-C) plotted against isomiR length. Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.


C of BrdU positive cb9Δmyc , in presence or absence ( 24h after removal ) of doxycycline . Data are presented as mean ± SD ; n = 3 . Two - Student t - was used to compare between two groups expressed as p - values .

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C Percentage of BrdU positive cb9Δmyc cells, in the presence or absence (24h after removal) of doxycycline. Data are presented as mean ± SD; n = 3. Two-tailed Student t-test was used to compare between two groups and expressed as p-values.


D Immunoblot of c - myc - / - HO15 . 19 rat fibroblasts infected with retroviral vectors expressing indicated Myc . Parental TGR1 serve as control for endogenous of MycWT .

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D Immunoblot analysis of c-myc-/- HO15.19 rat fibroblasts infected with retroviral vectors expressing the indicated Myc proteins. Parental TGR1 cells serve as control for endogenous levels of the MycWT protein.


F Cumulative for same as in ( D ) . Data are presented as mean ± SD ; n = 3 ( biological replicates ) . Two - Student t - was used to compare between two groups expressed as p - values .

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F Cumulative cell counts for the same cells as in (D). Data are presented as mean ± SD; n = 3 (biological replicates). Two-tailed Student t-test was used to compare between two groups and expressed as p-values.


C ChIP - seq intensities for peaks ( promoters + distal ) containing indicated motifs in an interval of ± 100 bp around their summit . For most accurate quantitation , intensities were computed within ± 50 bp from summit . p - values were calculated using Wilcoxon ' s . central band in boxplot represents median of data , boxes lower upper quartiles ( 25 % 75 % ) whiskers minimum maximum values .

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C ChIP-seq intensities for peaks (promoters + distal) containing the indicated motifs in an interval of ±100 bp around their summit. For most accurate quantitation, intensities were computed within ± 50 bp from the summit. p-values were calculated using Wilcoxon's test. The central band in the boxplot represents the median of the data, boxes the lower and upper quartiles (25% and 75%) and whiskers the minimum and maximum values.


A 3T9 fibroblasts expressing indicated MycER control ( EV ) were treated with OHT ( 4h ) subjected to biochemical fractionation in three independent experiments . quantity of MycER in different fractions was quantified by western blotting ( shown in Fig . EV4A ) relative proportion in various fractions plotted as average standard deviation .

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A 3T9 fibroblasts expressing the indicated MycER proteins and control cells (EV) were treated with OHT (4h) and subjected to biochemical fractionation in three independent experiments. The quantity of MycER protein in the different fractions was quantified by western blotting (shown in Fig. EV4A) and the relative proportion in the various fractions plotted as average and standard deviation.


F of promoters with indicated motifs under ChIP - seq peak ( ± 100 bp from peak summit ) within each regulatory class ( no DEG , UP or DOWN ) for either MycERWT ( left ) or MycERHEA ( right ) . p - values calculated with Fisher ' s exact .

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F Percentage of promoters with the indicated DNA motifs under the ChIP-seq peak (±100 bp from the peak summit) within each regulatory class (no DEG, UP or DOWN) for either MycERWT (left) or MycERHEA (right). p-values calculated with Fisher's exact test.


A panel of 42 breast cancer lines was challenged with increasing concentrations of ATRA ( 11 nM - 10 μM ) for 3 , 6 , 9 days , growth was determined . graphs show growth - inhibitory effect exerted by indicated concentrations of ATRA in SKBR3 , HCC - 1954 , MDA - MB436 which are representative of lines characterized by a high , intermediate low ATRA score , respectively . Each point is mean ± SD of three replicate cultures . * * Significantly lower than corresponding vehicle - treated group ( P - value < 0 . 01 , Student ' s t - ) .

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A panel of 42 breast cancer cell lines was challenged with increasing concentrations of ATRA (11 nM-10 μM) for 3, 6, and 9 days, and cell growth was determined.The graphs show the growth-inhibitory effect exerted by the indicated concentrations of ATRA in SKBR3, HCC-1954, and MDA-MB436 cells which are representative of lines characterized by a high, intermediate and low ATRA score, respectively. Each point is the mean ± SD of three replicate cultures. **Significantly lower than the corresponding vehicle-treated group (P-value < 0.01, Student's t-test).


lines are ranked in ascending order according to ATRA score . plot distinguishes four separate groups of lines ( D - A ) with increasing ATRA scores , as indicated by colored scale on right . lines are also grouped in tertiles , T1 - T3 , according to an ascending ATRA score . Each calculated value is representative of at least two independent experiments .

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Cell lines are ranked in ascending order according to the ATRA score. The plot distinguishes four separate groups of cell lines (D-A) with increasing ATRA scores, as indicated by the colored scale on the right. The cell lines are also grouped in tertiles, T1-T3, according to an ascending ATRA score. Each calculated value is representative of at least two independent experiments.


SCID mice were xenografted subcutaneously with 10 × 106HCC - 1599 on both sides . One week after transplantation 10 / experimental group were treated intraperitoneally with vehicle ( DMSO ) or two doses of ATRA ( 7 . 5 15 . 0 mg / kg ) once / day , 5 days a week for a total of 24 days . At end of this period , treatment was discontinued until sacrifice . of tumors was determined with a caliper volume plotted . Each point is mean ± SE of 20 tumors . * Significantly lower than corresponding vehicle - treated group ( P - value < 0 . 05 , Student ' s t - ) . * * Significantly lower than corresponding vehicle - treated group ( P - value < 0 . 01 , Student ' s t - ) .

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SCID mice were xenografted subcutaneously with 10 × 106HCC-1599 cells on both sides. One week after transplantation 10 animals/experimental group were treated intraperitoneally with vehicle (DMSO) or two doses of ATRA (7.5 and 15.0 mg/kg) once/day, 5 days a week for a total of 24 days. At the end of this period, treatment was discontinued until sacrifice.The size of the tumors was determined with a caliper and the volume plotted. Each point is the mean ± SE of 20 tumors. *Significantly lower than the corresponding vehicle-treated group (P-value < 0.05, Student's t-test). **Significantly lower than the corresponding vehicle-treated group (P-value < 0.01, Student's t-test).


Magnetic resonance imaging ( MRI ) was performed on five / experimental group on day 24 . picture shows representative 2D of sections from one treated with vehicle one treated with 15 . 0 mg / kg ATRA . bar graph shows volume of tumors calculated after 3D reconstruction of MRI . Each point is mean ± SE of five tumors . P - value of comparisons of ATRA versus vehicle is shown .

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Magnetic resonance imaging (MRI) analysis was performed on five animals/experimental group on day 24. The picture shows representative 2D images of tumor sections from one animal treated with vehicle and one animal treated with 15.0 mg/kg ATRA. The bar graph shows the volume of the tumors calculated after 3D reconstruction of the MRI images. Each point is the mean ± SE of five tumors. The P-value of the comparisons of ATRA versus vehicle is shown.


ATRA - dependent anti - activity in short - term cultures of breast tumorsTissue slices deriving from surgical specimens were challenged with vehicle ( DMSO ) or ATRA ( 0 . 1 μM ) for 48 h . panel illustrates examples of immuno - histochemical data obtained in four representative cases : ( i ) Luminal - A ( Lum A ) ; ( ii ) Luminal - B ( Lum B ) ; ( iii ) Her2 + with RARA coamplification ( Her2 + / RARA + ) ( iv ) triple negative ( TN ) .

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ATRA-dependent anti-tumor activity in short-term cultures of primary breast tumorsTissue slices deriving from surgical specimens were challenged with vehicle (DMSO) or ATRA (0.1 μM) for 48 h.The panel illustrates examples of the immuno-histochemical data obtained in four representative cases: (i) Luminal-A (Lum A); (ii) Luminal-B (Lum B); (iii) Her2+ with RARA coamplification (Her2+/RARA+) and (iv) triple negative (TN).


of Ki67 - positive in 45 samples considered are illustrated by bar graphs . Each value represents mean ± SE of at least five separate fields for each experimental sample . * Significantly lower than corresponding vehicle - treated control ( P - value < 0 . 05 , Student ' s t - ) . * * Significantly lower than corresponding vehicle - treated control ( P - value < 0 . 01 , Student ' s t - ) .

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The percentage of Ki67-positive tumor cells in the 45 samples considered are illustrated by the bar graphs. Each value represents the mean ± SE of at least five separate fields for each experimental sample. *Significantly lower than the corresponding vehicle-treated control (P-value < 0.05, Student's t-test). **Significantly lower than the corresponding vehicle-treated control (P-value < 0.01, Student's t-test).


Associations between components of retinoid signaling pathway phenotype in breast cancer linesThe figure illustrates associations between indicated retinoid receptors / binding Luminal versus Basal phenotype ( left panels ) , ER positivity versus ER negativity ( middle panels ) as well as HER2 positivity versus HER2 negativity ( right panels ) . gene - expression microarray - seq data refer to 42 40 breast cancer lines , respectively . P - values of indicated comparisons after Student ' s t - are shown in red . L ( red ) = Luminal lines ; B ( blue ) = Basal lines ; ER + ( red ) = ER - positive lines ; ER − ( red ) = ER - negative lines ; ER − ( blue ) = ER - negative Luminal lines ; H + ( red ) = HER2 - positive lines ; H − ( red ) = HER2 - negative lines ; H + ( blue ) = HER2 - positive Luminal lines ; H − ( blue ) = HER2 - negative Luminal lines . fpkm = fragments per kilobase of exon per million fragments mapped .

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Associations between components of the retinoid signaling pathway and the phenotype in breast cancer cell linesThe figure illustrates the associations between the indicated retinoid receptors/binding proteins and the Luminal versus Basal phenotype (left panels), ER positivity versus ER negativity (middle panels) as well as HER2 positivity versus HER2 negativity (right panels). The gene-expression microarray and RNA-seq data refer to 42 and 40 breast cancer cell lines, respectively. The P-values of the indicated comparisons after Student's t-test are shown in red. L (red) = Luminal cell lines; B (blue) = Basal cell lines; ER+ (red) = ER-positive cell lines; ER− (red) = ER-negative cell lines; ER− (blue) = ER-negative Luminal cell lines; H+ (red) = HER2-positive cell lines; H− (red) = HER2-negative cell lines; H+ (blue) = HER2-positive Luminal cell lines; H− (blue) = HER2-negative Luminal cell lines. fpkm = fragments per kilobase of exon per million fragments mapped.


Associations between components of retinoid pathway breast cancer phenotypeAssociations between expression of indicated members of retinoid pathway in TCGA gene - expression database are shown . Mammary tumors are classified into Luminal - A , Luminal - B , HER2 - like , Normal - like , Basal according to PAM50 fingerprint . average expression corresponding SD values of indicated members of retinoid pathway are shown by upper box plots . For each member of retinoid pathway , significant differences between indicated groups of tumors are shown in lower table . Significant P - values for indicated comparisons ( Student ' s t - ) are shown in red .

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Associations between components of the retinoid pathway and breast cancer phenotypeAssociations between the expression of the indicated members of the retinoid pathway in the TCGA gene-expression database are shown. Mammary tumors are classified into Luminal-A, Luminal-B, HER2-like, Normal-like, and Basal according to the PAM50 fingerprint. The average expression levels and the corresponding SD values of the indicated members of the retinoid pathway are shown by the upper box plots. For each member of the retinoid pathway, significant differences between the indicated groups of tumors are shown in the lower table. Significant P-values for the indicated comparisons (Student's t-test) are shown in red.


gene - expression microarray - seq data associated with 40 of breast cancer lines were used for analyses . panels illustrate associations of RARα PPARβ / δ with ATRA sensitivity . left panels show basal average of indicated transcript in lines belonging to T1 T3 groups ( 13 lines in each of T1 T3 groups ) defined by ascending ATRA scores . intermediate right panels indicate same results after stratification for Luminal ( microarray - seq data = 7 lines in each of T1 T3 groups ) Basal ( microarray - seq data = 7 lines in each of T1 T3 groups ) phenotype , respectively . fpkm = fragments per kilobase of exon per million fragments mapped .

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The gene-expression microarray and RNA-seq data associated with 40 of the breast cancer cell lines were used for the analyses. The panels illustrate the associations of RARα and PPARβ/δ with ATRA sensitivity. The left panels show the basal average levels of the indicated transcript in the cell lines belonging to the T1 and T3 groups (13 cell lines in each of the T1 and T3 groups) defined by ascending ATRA scores. The intermediate and right panels indicate the same results after stratification for the Luminal (microarray and RNA-seq data = 7 cell lines in each of the T1 and T3 groups) and the Basal (microarray and RNA-seq data = 7 cell lines in each of the T1 and T3 groups) phenotype, respectively. fpkm = fragments per kilobase of exon per million fragments mapped.


basal expression of indicated RAR - isoform variants were determined with use of specific Taqman assays . results are associated with ATRA sensitivity before ( TOTAL ) after stratification of lines for Luminal Basal phenotype as in ( A ) . Data information : Significant P - values ( Student ' s t - ) are indicated in red .

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The basal expression levels of the indicated RAR-isoform variants were determined with the use of specific Taqman assays. The results are associated with ATRA sensitivity before (TOTAL) and after stratification of the cell lines for the Luminal and Basal phenotype as in (A).Data information: Significant P-values (Student's t-test) are indicated in red.


Basal of RARα , RARβ , RARγ mRNA splicing variants in mammary tumorsTotal was extracted from tissue slices deriving from surgical specimens of breast cancer used in Fig3 before any treatment with DMSO or ATRA . was subjected to RT - PCR to determine basal expression of indicated RAR splicing variants . Each value represents mean ± SD of two replicate measurements . table shows statistical significance of indicated comparisons . * Significantly different ( P - value < 0 . 05 , Student ' s t - ) . * * Significantly different ( P - value < 0 . 01 , Student ' s t - ) .

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Basal levels of RARα, RARβ, and RARγ mRNA splicing variants in mammary tumorsTotal RNA was extracted from the tissue slices deriving from the surgical specimens of breast cancer patients used in Fig3 before any treatment with DMSO or ATRA. RNA was subjected to RT-PCR analysis to determine the basal expression of the indicated RAR splicing variants.Each value represents the mean ± SD of two replicate measurements. The table shows the statistical significance of the indicated comparisons. *Significantly different (P-value < 0.05, Student's t-test). **Significantly different (P-value < 0.01, Student's t-test).


plots illustrate average expression of indicated mRNAs ( mean ± SD of two replicates ) in samples classified as ATRA - sensitive ( Sens ) ATRA - resistant ( Res ) according to response of Ki67 . * Significantly different ( P - value < 0 . 05 , Student ' s t - ) .

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The plots illustrate the average expression levels of the indicated mRNAs (mean ± SD of two replicates) in tumor samples classified as ATRA-sensitive (Sens) and ATRA-resistant (Res) according to the response of Ki67. *Significantly different (P-value < 0.05, Student's t-test).


RARα ATRA sensitivity in breast cancer linesTotal were extracted from logarithmically growing lines subjected to Western blot . panel illustrates representative Western blots for a number of lines . blots were sequentially developed with RARα - specific control tubulin antibodies . positions of RARα tubulin bands ( right ) along with position of a relevant molecular marker ( left ) are indicated . To normalize Western blot signals in different gels , same preparation of RARα - transfected COS - 7cell extracts ( COS - 7 ) MCF7 extracts was loaded in each gel . blots are representative of at least two independent experiments providing similar results .

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RARα protein and ATRA sensitivity in breast cancer cell linesTotal proteins were extracted from logarithmically growing cell lines and subjected to Western blot analysis.The panel illustrates representative Western blots for a number of cell lines. The blots were sequentially developed with RARα-specific and control tubulin antibodies. The positions of the RARα and tubulin bands (right) along with the position of a relevant molecular weight marker (left) are indicated. To normalize the Western blot signals in different gels, the same preparation of RARα-transfected COS-7cell extracts (COS-7) and MCF7 extracts was loaded in each gel. The blots are representative of at least two independent experiments providing similar results.


quantitative results obtained after densitometric of RARα bands are plotted against ATRA scores . lines are grouped according to ATRA score ( A - D groups T1 - T3 tertiles ) .

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The quantitative results obtained after densitometric analysis of the RARα bands are plotted against the ATRA scores. Cell lines are grouped according to the ATRA score (A-D groups and T1-T3 tertiles).