( E ) Flow cytometric analysis of cell death by AnnexinV staining in T - ALL cell lines upon treatment with indicated 3d2 concentrations . Mean values of n = 4 biological replicates ± SD are shown . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(E) Flow cytometric analysis of cell death by AnnexinV staining in T-ALL cell lines upon treatment with indicated 3d2 concentrations. Mean values of n=4 biological replicates ± SD are shown. Data information: ns = not significant, * p < 0.05, *** p < 0.001.


( F ) Flow cytometric analysis of 3d2 - induced cell death by AnnexinV staining in wild - type ( WT ) , Bcl - 2 overexpressing ( Bcl - 2 ) and Z - VAD - FMK ( zVAD ) - treated CEM - C1 . Mean values of technical triplicates of one representative experiment ( n = 3 biological replicates ) ± SD analyzed by two - way ANOVA are shown . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(F) Flow cytometric analysis of 3d2-induced cell death by AnnexinV staining in wild-type (WT), Bcl-2 overexpressing (Bcl-2) and Z-VAD-FMK (zVAD)-treated CEM-C1. Mean values of technical triplicates of one representative experiment (n=3 biological replicates) ± SD analyzed by two-way ANOVA are shown. Data information: ns = not significant, * p < 0.05, *** p < 0.001.


( G ) Immunoblots of full - length and cleaved ( cl . ) PARP , P - p38 , c - Myc , Caspase 3 ( Casp . 3 ) , cl . Casp . 3 , BimEL , BimL and BimS and Tubulin from MOLT - 4 cells treated with 40 µM 3d2 for indicated time periods . Representative results from two biological replicates are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by Ø was used as a solvent control . ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(G) Immunoblots of full-length and cleaved (cl.) PARP, P-p38, c-Myc, Caspase 3 (Casp. 3), cl. Casp. 3, BimEL, BimL and BimS and Tubulin from MOLT-4 cells treated with 40 µM 3d2 for indicated time periods. Representative results from two biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by Ø was used as a solvent control. ns = not significant, * p < 0.05, *** p < 0.001.


( A ) Immunoblots of full - length and cleaved ( cl . ) PARP , BimEL , BimL and BimS and Tubulin from MOLT - 4 cells treated for 24 h with 10 µM dexamethasone ( Dexa ) and 3d2 . Representative results from three biological replicates are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by 0 was used as a solvent control .

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(A) Immunoblots of full-length and cleaved (cl.) PARP, BimEL, BimL and BimS and Tubulin from MOLT-4 cells treated for 24 h with 10 µM dexamethasone (Dexa) and 3d2. Representative results from three biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0 was used as a solvent control.


( B ) Flow cytometric analysis of cell death by AnnexinV staining in MOLT - 4 cells treated with 3d2 , 30 µM Dexa and / or 50 µM pan caspase inhibitor Z - VAD - FMK ( zVAD ) . Individual and mean values of technical triplicates of one representative out of n = 3 biological replicates ± SD are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by 0 was used as a solvent control . Two - way ANOVA ( B ns = not significant , * * p < 0 . 01 , * * * p < 0 . 001 .

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(B) Flow cytometric analysis of cell death by AnnexinV staining in MOLT-4 cells treated with 3d2, 30 µM Dexa and/or 50 µM pan caspase inhibitor Z-VAD-FMK (zVAD). Individual and mean values of technical triplicates of one representative out of n=3 biological replicates ± SD are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0 was used as a solvent control. Two-way ANOVA (B ns = not significant, ** p < 0.01, *** p < 0.001.


( C ) Flow cytometric analysis of cell death by AnnexinV staining in control or Bim knockout ( Bim KO ) CEM - C7 cells treated with 3d2 and 30 µM Dexa . Mean values of technical triplicates of one representative of n = 4 biological replicates ± SD are shown . Data information : Two - way ANOVA C , ns = not significant , * * p < 0 . 01 , * * * p < 0 . 001 .

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(C) Flow cytometric analysis of cell death by AnnexinV staining in control or Bim knockout (Bim KO) CEM-C7 cells treated with 3d2 and 30 µM Dexa. Mean values of technical triplicates of one representative of n=4 biological replicates ± SD are shown. Data information: Two-way ANOVA C, ns = not significant, ** p < 0.01, *** p < 0.001.


( D ) Immunoblots of BimEL , BimL and BimS , Bcl - 2 and Tubulin from MOLT - 4 expressing a non - ( shcontrol ) or LRH - 1 - ( shLRH - 1 ) targeting small hairpin RNA construct .

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(D) Immunoblots of BimEL, BimL and BimS, Bcl-2 and Tubulin from MOLT-4 expressing a non- (shcontrol) or LRH-1- (shLRH-1) targeting small hairpin RNA construct.


( E ) Flow cytometric analysis of cell death by AnnexinV staining in control or glucocorticoid receptor knockout ( GR KO ) MOLT - 4 cells treated with 3d2 and 30 µM Dexa . Mean values of n = 3 biological replicates ± SD are shown . Data information : Two - way ANOVA E ) ; ns = not significant , * * p < 0 . 01 , * * * p < 0 . 001 .

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(E) Flow cytometric analysis of cell death by AnnexinV staining in control or glucocorticoid receptor knockout (GR KO) MOLT-4 cells treated with 3d2 and 30 µM Dexa. Mean values of n=3 biological replicates ± SD are shown. Data information: Two-way ANOVA E); ns = not significant, ** p < 0.01, *** p < 0.001.


( F / G ) mRNA expression levels of human GR ( F ) and Bim ( G ) in shcontrol and shLRH - 1 CEM - C1 cells were determined by real - time quantitative PCR after treatment with 1 µM Dexa for indicated times . Fold induction relative to the untreated shcontrol sample ( 0h ) was calculated after normalization to beta - actin . Individual values of technical triplicates of one representative of two biological replicates ± SD are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by 0 was used as a solvent control .

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

(F/G) mRNA expression levels of human GR (F) and Bim (G) in shcontrol and shLRH-1 CEM-C1 cells were determined by real-time quantitative PCR after treatment with 1 µM Dexa for indicated times. Fold induction relative to the untreated shcontrol sample (0h) was calculated after normalization to beta-actin. Individual values of technical triplicates of one representative of two biological replicates ± SD are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0 was used as a solvent control.


( H ) Immunoblots of full - length and cleaved ( cl . ) PARP , GR and phospho - AMPK ( P - AMPK ) , c - Myc , full - length and cl . Caspase 3 , BimEL , BimL and BimS and Tubulin from MOLT - 4 cells treated with 40 µM 3d2 and / or 10 µM Dexa . Representative results from two biological replicates are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by Ø ) was used as a solvent control .

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

(H) Immunoblots of full-length and cleaved (cl.) PARP, GR and phospho-AMPK (P-AMPK), c-Myc, full-length and cl. Caspase 3, BimEL, BimL and BimS and Tubulin from MOLT-4 cells treated with 40 µM 3d2 and/or 10 µM Dexa. Representative results from two biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by Ø) was used as a solvent control.


( A ) Ex vivo response of human T cell acute lymphoblastic leukemia ( T - ALL ) patient - derived xenografts ( PDX ) , co - cultured on hTERT - immortalized primary bone marrow MSCs , to treatment with indicated concentrations of 3d2 and / or dexamethasone ( Sample 1 , 3 and 4 : 1 nM ; Sample 2 : 0 . 1 nM ) . Cell viability was quantified by fluorescent live cell staining using CyQuant combined with automated image analysis and calculated as percentage ( % ) of the dimethyl sulfoxide treated control . Data information : All displayed experiments display one biological replicate .

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(A) Ex vivo response of human T cell acute lymphoblastic leukemia (T-ALL) patient-derived xenografts (PDX), co-cultured on hTERT-immortalized primary bone marrow MSCs, to treatment with indicated concentrations of 3d2 and/or dexamethasone (Sample 1, 3 and 4: 1 nM; Sample 2: 0.1 nM). Cell viability was quantified by fluorescent live cell staining using CyQuant combined with automated image analysis and calculated as percentage (%) of the dimethyl sulfoxide treated control. Data information: All displayed experiments display one biological replicate.


( B ) Synergy scores ( Z - scores ) between 3d2 and dexamethasone of human T - ALL patient - derived xenografts ( PDX ) were calculated from the viability curves of PDX samples treated with increasing concentrations of 3d2 and / or dexamethasone using SynergyFinder tool . Z - scores ≥ 0 indicate additivity or synergism and < 0 antagonism . Data information : All displayed experiments display one biological replicate .

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(B) Synergy scores (Z-scores) between 3d2 and dexamethasone of human T-ALL patient-derived xenografts (PDX) were calculated from the viability curves of PDX samples treated with increasing concentrations of 3d2 and/or dexamethasone using SynergyFinder tool. Z-scores ≥ 0 indicate additivity or synergism and < 0 antagonism. Data information: All displayed experiments display one biological replicate.


( C ) Expression levels of human GRα , LRH - 1 and Bim mRNA in human PDX were determined by real - time quantitative PCR , calculated as relative expression compared to beta - actin and used to determine the GR : LRH - 1 ratio ( right ) . Data information : All displayed experiments display one biological replicate .

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(C) Expression levels of human GRα, LRH-1 and Bim mRNA in human PDX were determined by real-time quantitative PCR, calculated as relative expression compared to beta-actin and used to determine the GR:LRH-1 ratio (right). Data information: All displayed experiments display one biological replicate.


( E ) In vivo leukemia progression of human T - ALL PDX transplanted into NSG mice treated for 3 weeks with vehicle ( PBS control ) , 40 mg / kg 3d2 and or 10 mg / kg Dexamethasone starting 3 days post transplantation . Engraftment was calculated as % of human vs total ( human + mouse ) CD45 positive ( CD45 + ) cells as assessed by flow cytometric analysis of peripheral blood . Data information : All displayed experiments display one biological replicate .

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(E) In vivo leukemia progression of human T-ALL PDX transplanted into NSG mice treated for 3 weeks with vehicle (PBS control), 40 mg/kg 3d2 and or 10 mg/kg Dexamethasone starting 3 days post transplantation. Engraftment was calculated as % of human vs total (human+mouse) CD45 positive (CD45+) cells as assessed by flow cytometric analysis of peripheral blood. Data information: All displayed experiments display one biological replicate.


( 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 .

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(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 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 .

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(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 ) 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

(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 ) 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

(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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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(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 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 .

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(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 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 .

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(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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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=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 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 .

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(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 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 .

(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 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 .

(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 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 .

(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 ) 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 .

(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 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 .

(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.


( B ) Quantitative PCR analysis of post - natal lungs for mitochondrial DNA ( mtDNA ) . Actin was used as a normalization control . ( mean ± SD , : p < 0 . 05 ; : p < 0 . 01 ; N = 3 ) .

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(B) Quantitative PCR analysis of post-natal lungs for mitochondrial DNA (mtDNA). Actin was used as a normalization control. (mean ± SD, : p<0.05;: p<0.01; N=3).


( C ) Quantitative analysis of oxidative stress in post - natal pups of indicated age ( in days ) using the peroxide linked oxidative stress marker DHR - 123 , as described in methods ( mean ± SD , : p < 0 . 01 ; N = 8 ) .

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(C) Quantitative analysis of oxidative stress in post-natal pups of indicated age (in days) using the peroxide linked oxidative stress marker DHR-123, as described in methods (mean ± SD, : p<0.01; N=8).


( E ) Immunofluorescence analysis of oxidative DNA damage ( left panel ) and the DNA double - strand breaks sensor kinase marker pATM ( right panel ) in post - natal murine lungs . Here anti - 8 - oxo - 7 , 8 - dihydroguanine was used for the detection of oxidative DNA modification ( 8 - oxoG , left panel ) , and anti - pATM Serine 1981 was used for the analysis of the activation of the DDR ( pATM , right panel ) . Green cytoplasmic staining represents α - Tubulin ; blue DAPI staining represents nucleus ( scale 10μm ) . No primary antibody control served as a negative control ( P14 ) of the staining .

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(E) Immunofluorescence analysis of oxidative DNA damage (left panel) and the DNA double-strand breaks sensor kinase marker pATM (right panel) in post-natal murine lungs. Here anti-8-oxo-7, 8-dihydroguanine was used for the detection of oxidative DNA modification (8-oxoG, left panel), and anti-pATM Serine 1981 was used for the analysis of the activation of the DDR (pATM, right panel). Green cytoplasmic staining represents α-Tubulin; blue DAPI staining represents nucleus (scale 10μm). No primary antibody control served as a negative control (P14) of the staining.


( F ) Mean percentage of nuclei positive for oxidative DNA damage marker 8 - oxoG in post - natal murine lungs , as determined by immunofluorescence analysis ( mean ± SEM , : p < 0 . 01 ; : p < 0 . 001 ; N = 8 ) . ( G ) Mean percentage of nuclei positive for DNA damage marker pATM in post - natal murine lungs , as determined by immunofluorescence analysis ( mean ± SEM , : p < 0 . 01 ; N = 8 ) .

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(F) Mean percentage of nuclei positive for oxidative DNA damage marker 8-oxoG in post-natal murine lungs, as determined by immunofluorescence analysis (mean ± SEM, : p<0.01; : p<0.001; N=8). (G) Mean percentage of nuclei positive for DNA damage marker pATM in post-natal murine lungs, as determined by immunofluorescence analysis (mean ± SEM, : p<0.01; N=8).


( H ) Quantitative analysis of the NAD + / NADH ratio in post - natal lungs after homogenization in extraction buffer ( mean ± SD , : p < 0 . 01 ; : p < 0 . 001 ) .

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(H) Quantitative analysis of the NAD+/NADH ratio in post-natal lungs after homogenization in extraction buffer (mean ± SD, : p<0.01; : p<0.001).


( A ) Immunofluorescence analysis of DSBs associated foci as marked by γH2AX in lung adenocarcinoma ( A549 ) cells , cultured either in low glucose ( 5 . 5mM , left panel ) or high glucose ( 30mM , right panel ) for 5 days , then treated with etoposide ( 5µM for 60 minutes ) . The resolution of DNA - DSBs foci , as marked by γH2AX was monitored over 24 hours after drug treatment , CML ( marked in green ) served as induction control ( scale 10µm ) . ( B ) Mean percentage of DSBs positive nuclei as evidenced by γH2AX positivity after pre - treatment of different reducing carbohydrates at 0 , 3 or 24 hours . More than 400 cells were analyzed for each bar ( mean ± SD , : p < 0 . 01 ; : p < 0 . 001 ) .

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(A) Immunofluorescence analysis of DSBs associated foci as marked by γH2AX in lung adenocarcinoma (A549) cells, cultured either in low glucose (5.5mM, left panel) or high glucose (30mM, right panel) for 5 days, then treated with etoposide (5µM for 60 minutes). The resolution of DNA-DSBs foci, as marked by γH2AX was monitored over 24 hours after drug treatment, CML (marked in green) served as induction control (scale 10µm). (B) Mean percentage of DSBs positive nuclei as evidenced by γH2AX positivity after pre-treatment of different reducing carbohydrates at 0, 3 or 24 hours. More than 400 cells were analyzed for each bar (mean ± SD, : p<0.01;: p<0.001).


( C ) Still images of A549 cells showing the live recruitment of hPARP - mCherry and hXRCC4 - GFP , at the site of laser - induced DNA - DSBs from the cells pre - treated with the indicated reducing sugars as described in methods .

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(C) Still images of A549 cells showing the live recruitment of hPARP-mCherry and hXRCC4-GFP, at the site of laser-induced DNA-DSBs from the cells pre-treated with the indicated reducing sugars as described in methods.


( D ) Quantitative analysis of the efficiency of NHEJ - repair in a reporter cell line , as described in Methods . Cells were cultured in low glucose ( 5 . 5mM ) or high glucose ( 30mM ) for 5 days , fructose ( 30mM ) , ribose ( 20mM ) , for 3 days ( graph bar 4 ) . I - SceI , untransfected cells served as a negative control . Shown is the average from three independent experiments ( mean ± SEM , : p < 0 . 01 : p < 0 . 001 ) .

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(D) Quantitative analysis of the efficiency of NHEJ-repair in a reporter cell line, as described in Methods. Cells were cultured in low glucose (5.5mM) or high glucose (30mM) for 5 days, fructose (30mM), ribose (20mM), for 3 days (graph bar 4). I-SceI, untransfected cells served as a negative control. Shown is the average from three independent experiments (mean ± SEM, : p<0.01 : p<0.001).


( E ) Quantitative analysis of the NAD + / NADH ratio in A549 cells , cultured in either low ( 5 . 5mM , LG ) , or high glucose ( 30mM , HG ) for 5 days , fructose ( 30mM , FR ) , ribose ( 20mM , RI ) , for 3 days ( graph bar 4 ) . Quantification was performed after homogenization in extraction buffer ( Data represent mean ± SD , : p < 0 . 01 ; : p < 0 . 001 ; N = 3 ) .

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(E) Quantitative analysis of the NAD+/NADH ratio in A549 cells, cultured in either low (5.5mM, LG), or high glucose (30mM, HG) for 5 days, fructose (30mM, FR), ribose (20mM, RI), for 3 days (graph bar 4). Quantification was performed after homogenization in extraction buffer (Data represent mean ± SD, : p<0.01;: p<0.001; N=3).


( F ) A549 cells were cultured in either low ( 5 . 5mM ; lane 1 ) or high glucose ( 30mM ; lane 2 ) for 5 days , fructose ( 30mM ; lane 3 ) , ribose ( 20mM ; lane 4 ) , for 3 days . The cell extracts were then for immunoprecipitation by using anti - PARP , or a non - specific species control antibody . The PARP , or its interacting partner DBC1 was then detected using PARP , or a DBC1 specific antibody .

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(F) A549 cells were cultured in either low (5.5mM; lane 1) or high glucose (30mM; lane 2) for 5 days, fructose (30mM; lane 3), ribose (20mM; lane 4), for 3 days. The cell extracts were then for immunoprecipitation by using anti-PARP, or a non-specific species control antibody. The PARP, or its interacting partner DBC1 was then detected using PARP, or a DBC1 specific antibody.


( A ) Representative images of lungs for nuclei positive for the DNA - DSBs marker γH2AX , versus DAPI , in age - matched 3 - , or 6 - months control , versus STZ induced diabetic mice , as determined by immunofluorescence analysis ( scale 10μm ) . ( B ) Mean percentage of nuclei positive for the DNA - DSBs marker γH2AX in lungs of age - matched control versus 6 - month STZ induced diabetic mice , as determined by immunofluorescence analysis of lungs ( mean ± SEM , : p < 0 . 05 ; : p < 0 . 01 ; : p < 0 . 001 ; N = 6 ) .

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

(A) Representative images of lungs for nuclei positive for the DNA-DSBs marker γH2AX, versus DAPI, in age-matched 3-, or 6- months control, versus STZ induced diabetic mice, as determined by immunofluorescence analysis (scale 10μm). (B) Mean percentage of nuclei positive for the DNA-DSBs marker γH2AX in lungs of age-matched control versus 6-month STZ induced diabetic mice, as determined by immunofluorescence analysis of lungs (mean ± SEM, :p<0.05;: p<0.01; : p<0.001; N=6).


( C ) Representative images of lungs from age - matched control versus 3 or 6 - month STZ mice , stained for the peroxide linked oxidative stress marker DHR - 123 ( in red ) , as determined by mean fluorescence intensity . Blue nuclear staining represents DAPI ( Scale 10µm ) . ( D ) Quantitative analysis of respectively age - matched control versus 3 or 6 - month STZ induced diabetic lungs for persistent DNA damage signaling associated inflammatory marker IL - 6 , as determined by mean fluorescence intensity of respective group lungs ( mean ± SD ; : p < 0 . 01 : p < 0 . 001 ; N = 6 ) .

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

(C) Representative images of lungs from age-matched control versus 3 or 6-month STZ mice, stained for the peroxide linked oxidative stress marker DHR-123(in red), as determined by mean fluorescence intensity. Blue nuclear staining represents DAPI (Scale 10µm). (D) Quantitative analysis of respectively age-matched control versus 3 or 6-month STZ induced diabetic lungs for persistent DNA damage signaling associated inflammatory marker IL-6, as determined by mean fluorescence intensity of respective group lungs (mean ± SD; : p<0.01: p<0.001; N=6).


( E ) Representative images of lungs from age - matched controls , versus 3 or 6 - month STZ induced diabetic mice . Sections stained for cellular senescence - associated β - galactosidase [ β - Gal ] as described in Methods and visualized by bright field and polarized light , the senescent areas are recognized by its bluish - green staining ( Scale 40µm ) .

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

(E) Representative images of lungs from age-matched controls, versus 3 or 6-month STZ induced diabetic mice. Sections stained for cellular senescence-associated β-galactosidase [β-Gal] as described in Methods and visualized by bright field and polarized light, the senescent areas are recognized by its bluish-green staining (Scale 40µm).


( A ) Pressure - volume curves were determined using the FlexiVent system in age - matched control versus 3 - month STZ induced diabetic mice . The curves represent group averages ( mean ± SD ; : p < 0 . 05 ; N = 5 ) .

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

(A) Pressure-volume curves were determined using the FlexiVent system in age-matched control versus 3-month STZ induced diabetic mice. The curves represent group averages (mean ± SD; : p<0.05; N=5).


( B ) Quantitative analysis of static pulmonary compliance in age - matched control versus 3 months STZ induced diabetic mice , as described in Figure 3a . The curves represent group averages ( mean ± SD ; : P < 0 . 05 , : p < 0 . 01 ; N = 5 ) .

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

(B) Quantitative analysis of static pulmonary compliance in age-matched control versus 3 months STZ induced diabetic mice, as described in Figure 3a. The curves represent group averages (mean ± SD; : P<0.05, : p<0.01; N=5).


( C ) Representative images of lungs from age ( 3 - or 6 - month ) matched control versus STZ induced diabetic mice , stained for Masson ' s Trichrome stain , as described in Methods and visualized by bright field and polarized light , the accumulated ECM is recognized by its blue staining ( Scale 40µm ) .

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

(C) Representative images of lungs from age (3- or 6- month) matched control versus STZ induced diabetic mice, stained for Masson's Trichrome stain, as described in Methods and visualized by bright field and polarized light, the accumulated ECM is recognized by its blue staining (Scale 40µm).


( D ) Percentage total lung capacity of controls versus patients with diabetes . The dotted line represents the cut - off decided as per the guidelines of the European Respiratory Society ( ERS ) and American Thoracic Society ( ATS ) , ( mean ± SD , : p < 0 . 05 ; triplicates from each Ncontrol = 44 , Ntype - 1 = 35 , Ntype - 2 = 110 ) . ( E ) Percentage forced vital capacity of lungs in controls versus patients with diabetes . The dotted line represents the cut - off decided as per the guidelines of ERS and ATS , ( mean ± SD ; : p < 0 . 05 ; triplicates from each Ncontrol = 44 , Ntype - 1 = 35 , Ntype - 2 = 110 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percentage total lung capacity of controls versus patients with diabetes. The dotted line represents the cut-off decided as per the guidelines of the European Respiratory Society (ERS) and American Thoracic Society (ATS), (mean ± SD, : p<0.05; triplicates from each Ncontrol=44, Ntype-1=35, Ntype-2=110). (E) Percentage forced vital capacity of lungs in controls versus patients with diabetes. The dotted line represents the cut-off decided as per the guidelines of ERS and ATS, (mean ± SD; : p<0.05; triplicates from each Ncontrol=44, Ntype-1=35, Ntype-2=110).


( F ) Quantitative analysis of the NAD + / NADH ratio in lungs harvested from age - matched 3 , or 6 months control , or STZ induced diabetic mice , after homogenization in extraction buffer ( mean ± SD ; : p < 0 . 01 , : p < 0 . 001 ; N = 5 ) .

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

(F) Quantitative analysis of the NAD+/NADH ratio in lungs harvested from age-matched 3, or 6 months control, or STZ induced diabetic mice, after homogenization in extraction buffer (mean ± SD; : p<0.01, : p<0.001; N=5).


( G ) Quantitative analysis of the NAD + / NADH ratio determined in serum from patients with diabetes , as described in methods ( N = 3 ; mean ± SD , : p < 0 . 01 , : p < 0 . 001 ) .

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

(G) Quantitative analysis of the NAD+/NADH ratio determined in serum from patients with diabetes, as described in methods (N=3; mean ± SD, : p<0.01, : p<0.001).


( A ) Representative images of γH2AX positive nuclei in lungs of 6 - month STZ induced diabetic mice , transduced with respective RAGE virions as described in Methods . The lungs were harvested 6 - weeks after viral transduction . The empty vector served as control . Red represents γH2AX foci green cytoplasmic staining represents α - tubulin ; blue nuclear staining represents DAPI ( Scale 10µm ) . No primary antibody control served as a negative control ( from control lung ) of the staining .

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

(A) Representative images of γH2AX positive nuclei in lungs of 6-month STZ induced diabetic mice, transduced with respective RAGE virions as described in Methods. The lungs were harvested 6-weeks after viral transduction. The empty vector served as control. Red represents γH2AX foci green cytoplasmic staining represents α-tubulin; blue nuclear staining represents DAPI (Scale 10µm). No primary antibody control served as a negative control (from control lung) of the staining.


( B ) Mean percentage of nuclei positive for the DNA - DSBs marker γH2AX in transduced lungs of 6 - month STZ diabetic mice as described in Figure 5a ( mean ± SEM ; : p < 0 . 01 ; N = 8 ) .

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

(B) Mean percentage of nuclei positive for the DNA-DSBs marker γH2AX in transduced lungs of 6-month STZ diabetic mice as described in Figure 5a (mean ± SEM; : p<0.01; N=8).


( C ) Representative images of lungs from 6 - month STZ induced diabetic mice , transduced with respective RAGE virions as described in Methods . Lungs were stained for cellular senescence - associated β - galactosidase as described in Figure 5a and visualized by bright field and polarized light , where the accumulated senescent areas are recognized by its blue staining ( Scale 40µm ) .

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

(C) Representative images of lungs from 6-month STZ induced diabetic mice, transduced with respective RAGE virions as described in Methods. Lungs were stained for cellular senescence-associated β-galactosidase as described in Figure 5a and visualized by bright field and polarized light, where the accumulated senescent areas are recognized by its blue staining (Scale 40µm).


( D ) Quantitative analysis of persistent DNA damage associated inflammatory marker IL - 6 , in transduced lungs from 6 - month STZ - diabetic mice , as determined by mean fluorescence intensity of respective group lungs ( mean ± SD , : p < 0 . 05 , : p < 0 . 001 ; N = 8 ) .

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

(D) Quantitative analysis of persistent DNA damage associated inflammatory marker IL-6, in transduced lungs from 6-month STZ-diabetic mice, as determined by mean fluorescence intensity of respective group lungs (mean ± SD,: p<0.05, : p<0.001; N=8).


( E ) Quantitative analysis of Masson ' s Trichrome stain for lungs of 6 - month STZ induced diabetic mice with respective RAGE virions as described in Figure 5F ( mean ± SD , : p < 0 . 001 ; N = 8 ) .

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

(E) Quantitative analysis of Masson's Trichrome stain for lungs of 6-month STZ induced diabetic mice with respective RAGE virions as described in Figure 5F (mean ± SD, : p<0.001; N=8).


( F ) Representative images of Masson ' s Trichrome stain for lungs transduced with the respective RAGE expressing virions as described in Figure 5a and visualized by bright field and polarized light , where the accumulated ECM areas are recognized by its blue staining ( Scale 40µm ) . The dotted lines represent the respective zoomed window .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of Masson's Trichrome stain for lungs transduced with the respective RAGE expressing virions as described in Figure 5a and visualized by bright field and polarized light, where the accumulated ECM areas are recognized by its blue staining (Scale 40µm). The dotted lines represent the respective zoomed window.


( G ) Pressure - volume curves of diabetic and non - diabetic mice were determined using the FlexiVent system 6 weeks after transduction using respective the RAGE virions , as described in Figure 5a . The curves represent group averages ( N = 8 ) . The lungs studied in Figure 5a , b , c and d are identical ( mean ± SD ; Vector alone vs . RAGE - EE ; : p < 0 . 05 ; N = 8 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pressure-volume curves of diabetic and non-diabetic mice were determined using the FlexiVent system 6 weeks after transduction using respective the RAGE virions, as described in Figure 5a. The curves represent group averages (N=8). The lungs studied in Figure 5a, b, c and d are identical (mean ± SD; Vector alone vs. RAGE-EE; : p<0.05; N=8).


( a ) PC12 cells stably expressing A53T mutant α - synuclein were induced for 24 h , after which expression was switched off by removing doxycycline from the medium for the next 24 h . Transgene product clearance can be inferred by assessing levels at various times after switching off expression after an initial induction period , as expression levels decay when synthesis is reduced or stopped . Cells were treated with EHNA or AMP - PNP or left untreated during the switch - off period . Blots were probed with antibody to HA to detect the transgene and antibody to actin as a loading control .

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

(a) PC12 cells stably expressing A53T mutant α-synuclein were induced for 24 h, after which expression was switched off by removing doxycycline from the medium for the next 24 h. Transgene product clearance can be inferred by assessing levels at various times after switching off expression after an initial induction period, as expression levels decay when synthesis is reduced or stopped. Cells were treated with EHNA or AMP-PNP or left untreated during the switch-off period. Blots were probed with antibody to HA to detect the transgene and antibody to actin as a loading control.


( b ) PC12 cells expressing Q74 - GFP were induced for 15 h and then treated with inhibitors for 48 h after switch - off . Band intensity quantification ( three independent experiments ) is shown .

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

(b) PC12 cells expressing Q74-GFP were induced for 15 h and then treated with inhibitors for 48 h after switch-off. Band intensity quantification (three independent experiments) is shown.


( c ) Immunofluorescence images of cells expressing Q74 - GFP after 48 h of switch - off after treatment with ( EHNA ) or without ( Control ) EHNA .

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

(c) Immunofluorescence images of cells expressing Q74-GFP after 48 h of switch-off after treatment with (EHNA) or without (Control) EHNA.


( d ) Quantification of aggregates and cell death in COS - 7 cells transiently transfected with Q74 - GFP either with ( EHNA ) or without ( Control ) EHNA ( added during the last 33 h before fixation ) .

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

(d) Quantification of aggregates and cell death in COS-7 cells transiently transfected with Q74-GFP either with (EHNA) or without (Control) EHNA (added during the last 33 h before fixation).


( e ) Western - blot analysis of HeLa cells stably expressing GFP - UbG76V treated with lactacystin ( Lac ) , EHNA or AMP - PNP or left untreated ( Control ) using antibody to GFP . * * * P 0 . 0001 ; * * P 0 . 001 .

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

(e) Western-blot analysis of HeLa cells stably expressing GFP-UbG76V treated with lactacystin (Lac), EHNA or AMP-PNP or left untreated (Control) using antibody to GFP. ***P 0.0001; **P 0.001.


( a ) Stable PC12 cells expressing A53T α - synuclein were induced for 24 h , after which expression was switched off and cells were simultaneously transfected with p50 or dynamitin ( p50 ) , CC1 , CC2 , motor domain - deleted kinesin heavy chain ( ΔM - KHC ) or the tail region of kinesin heavy chain ( DN - KHC ) for the next 24 h . Dominant - negative constructs along with pEGFP - C1 ( 3 : 1 ratio of dominant - negative construct : pEGFP - C1 ) were used to transfect cells , and FACS was used to select transfected cells , which were analyzed by western blotting as in Figure 1a . Quantification of the band intensity from multiple experiments ( three independent experiments for dynein mutants and two for kinesin mutants ) is shown . * * * P 0 . 0001 ; * P 0 . 01 ; NS , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Stable PC12 cells expressing A53T α-synuclein were induced for 24 h, after which expression was switched off and cells were simultaneously transfected with p50 or dynamitin (p50), CC1, CC2, motor domain-deleted kinesin heavy chain (ΔM-KHC) or the tail region of kinesin heavy chain (DN-KHC) for the next 24 h. Dominant-negative constructs along with pEGFP-C1 (3:1 ratio of dominant-negative construct:pEGFP-C1) were used to transfect cells, and FACS was used to select transfected cells, which were analyzed by western blotting as in Figure 1a. Quantification of the band intensity from multiple experiments (three independent experiments for dynein mutants and two for kinesin mutants) is shown. ***P 0.0001; *P 0.01; NS, not significant.


( b ) COS - 7 cells were cotransfected with HA - Q74 or GFP - Q74 with empty vector ( pEGFP or pcDNA3 . 1 ) , p50 or dynamitin ( p50 ) , p62 subunit of dynactin complex ( p62 ) , the tail region of kinesin heavy chain ( DN - KHC ) , CC1 or CC2 for 48 h , after which the cells were fixed for immunofluorescence and immunocytochemistry ( using to antibody to HA ) . The percentage of GFP - positive cells with aggregates and cell death is shown . * * * P 0 . 0001 ; * * P 0 . 001 ; NS , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) COS-7 cells were cotransfected with HA-Q74 or GFP-Q74 with empty vector (pEGFP or pcDNA3.1), p50 or dynamitin (p50), p62 subunit of dynactin complex (p62), the tail region of kinesin heavy chain (DN-KHC), CC1 or CC2 for 48 h, after which the cells were fixed for immunofluorescence and immunocytochemistry (using to antibody to HA). The percentage of GFP-positive cells with aggregates and cell death is shown. ***P 0.0001; **P 0.001; NS, not significant.


( c ) Immunocytochemistry analysis ( using antibody to HA ) of COS - 7 cells cotransfected for 48 h with HA - Q74 ( red ) and pEGFP or p50 ( green ) . Panels on left are magnifications of boxed areas in second panels . The boxes are the same size . * * * P 0 . 0001 ; * * P 0 . 001 . Examples of fragmented nuclei in cells overexpressing p50 and nuclei in cells overexpressing p50 but treated with Z - VAD - fmk ( broad - spectrum caspase inhibitor ) are shown in Supplementary Figure 4 online .

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

(c) Immunocytochemistry analysis (using antibody to HA) of COS-7 cells cotransfected for 48 h with HA-Q74 (red) and pEGFP or p50 (green). Panels on left are magnifications of boxed areas in second panels. The boxes are the same size. ***P 0.0001; **P 0.001. Examples of fragmented nuclei in cells overexpressing p50 and nuclei in cells overexpressing p50 but treated with Z-VAD-fmk (broad-spectrum caspase inhibitor) are shown in Supplementary Figure 4 online.


( a ) Immunofluorescence analysis of COS - 7 cells cotransfected for 15 h with either empty vector ( control ) or CC1 and GFP - LC3 ( green ) , in a 3 : 1 ratio . Panels on right are magnifications of boxed areas .

O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence analysis of COS-7 cells cotransfected for 15 h with either empty vector (control) or CC1 and GFP-LC3 (green), in a 3:1 ratio. Panels on right are magnifications of boxed areas.


( b ) Western - blot analysis of COS - 7 cells treated with EHNA or left untreated using antibodies to LC3 and to actin . Quantification of the band intensity from two independent experiments is shown .

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) Western-blot analysis of COS-7 cells treated with EHNA or left untreated using antibodies to LC3 and to actin. Quantification of the band intensity from two independent experiments is shown.


( c ) NRK cells transiently transfected for 15 h with either empty vector ( control ) or CC1 and GFP - LC3 ( green ) , in a 3 : 1 ratio were immunostained with antibody to lgp120 , and the numbers of GFP - LC3 and lgp120 doubly stained vesicles in individual cells were scored .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NRK cells transiently transfected for 15 h with either empty vector (control) or CC1 and GFP-LC3 (green), in a 3:1 ratio were immunostained with antibody to lgp120, and the numbers of GFP-LC3 and lgp120 doubly stained vesicles in individual cells were scored.


Frequencies of ommatidia with different numbers of rhabdomeres are shown at 3 days after eclosion in progeny of flies expressing mutant huntingtin exon 1 ( gmrQ120 ) crossed to either a control stock ( w1118 ; W ) or to Dhc64C ( dynein heavy chain mutant ; Dhc ; a ) or Roblk ( dynein light chain mutant ; Robl ; b ) flies . P ≤ 0 . 0001 ( Mann - Whitney U test ) .

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

Frequencies of ommatidia with different numbers of rhabdomeres are shown at 3 days after eclosion in progeny of flies expressing mutant huntingtin exon 1 (gmrQ120) crossed to either a control stock (w1118; W) or to Dhc64C (dynein heavy chain mutant; Dhc; a) or Roblk (dynein light chain mutant; Robl; b) flies. P ≤ 0.0001 (Mann-Whitney U test).


( c ) Ommatidia from the progeny of flies expressing mutant huntingtin exon 1 ( Q120 ) crossed with either control stock ( w1118 ; W ) or with Dhc64C ( Dhc ) flies . All fly experiments were repeated three times independently with similar significance levels . One representative experiment is shown .

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

(c) Ommatidia from the progeny of flies expressing mutant huntingtin exon 1 (Q120) crossed with either control stock (w1118; W) or with Dhc64C (Dhc) flies. All fly experiments were repeated three times independently with similar significance levels. One representative experiment is shown.


( a ) Tremor onset and severity in a mouse model of Huntington disease are enhanced by the Loa mutation . At 7 weeks : HdhHD / + Dnchc1Loa / + n = 9 , HdhHD / + Dnchc1 + / + n = 9 , P = 0 . 14 . At 9 weeks : HdhHD / + Dnchc1Loa / + n = 8 , HdhHD / + Dnchc1 + / + n = 9 , P = 0 . 027 . At 11 weeks : HdhHD / + Dnchc1Loa / + n = 6 , HdhHD / + Dnchc1 + / + n = 9 , P = 0 . 001 . At 13 weeks : HdhHD / + Dnchc1Loa / + n = 5 , HdhHD / + Dnchc1 + / + n = 8 , P = 0 . 021 . At 15 weeks : HdhHD / + Dnchc1Loa / + n = 3 , HdhHD / + Dnchc1 + / + n = 5 , P = 0 . 021 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Tremor onset and severity in a mouse model of Huntington disease are enhanced by the Loa mutation. At 7 weeks: HdhHD/+ Dnchc1Loa/+ n = 9, HdhHD/+ Dnchc1+/+ n = 9, P = 0.14. At 9 weeks: HdhHD/+ Dnchc1Loa/+ n = 8, HdhHD/+ Dnchc1+/+ n = 9, P = 0.027. At 11 weeks: HdhHD/+ Dnchc1Loa/+ n = 6, HdhHD/+ Dnchc1+/+ n = 9, P = 0.001. At 13 weeks: HdhHD/+ Dnchc1Loa/+ n = 5, HdhHD/+ Dnchc1+/+ n = 8, P = 0.021. At 15 weeks: HdhHD/+ Dnchc1Loa/+ n = 3, HdhHD/+ Dnchc1+/+ n = 5, P = 0.021.


( b , c ) Dynein mutation reduces motor coordination and muscle function in a mouse model of Huntington disease . HdhHD / + Dnchc1Loa / + , HdhHD / + Dnchc1 + / + and Hdh + / + Dnchc1Loa / + mice are shown . An average of six mice were used at every time point , with at least three mice per group after 13 weeks . ( b ) Grip strength . Overall effect for all time points between the three genotypes : P = 0 . 0024 . Overall pair comparison between HdhHD / + Dnchc1Loa / + and HdhHD / + Dnchc1 + / + : P = 0 . 001 . Overall pair comparison between HdhHD / + Dnchc1Loa / + and Hdh + / + Dnchc1Loa / + : P = 0 . 0157 . Effect at each time point between the three genotypes : 7 weeks , P = 0 . 023 ; 9 weeks , P = 0 . 001 ; 11 weeks , P 0 . 0001 ; 13 weeks , P = 0 . 004 ; 15 weeks , P = 0 . 336 ( most of the double mutants were dead by 15 weeks ) . ( c ) Accelerating rotarod . Overall effect for all time points between the three genotypes : P = 0 . 0367 . Overall pair comparison between HdhHD / + Dnchc1Loa / + and HdhHD / + Dnchc1 + / + : P = 0 . 0245 . Overall pair comparison between HdhHD / + Dnchc1Loa / + and Hdh + / + Dnchc1Loa / + : P = 0 . 0553 . Effect at each time point between the three genotypes : 8 weeks , P 0 . 001 ; 10 weeks , P = 0 . 005 ; 12 weeks , P = 0 . 001 ; 14 weeks , P = 0 . 009 ; 16 weeks , P = 0 . 039 .

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

(b,c) Dynein mutation reduces motor coordination and muscle function in a mouse model of Huntington disease. HdhHD/+ Dnchc1Loa/+, HdhHD/+ Dnchc1+/+ and Hdh+/+ Dnchc1Loa/+ mice are shown. An average of six mice were used at every time point, with at least three mice per group after 13 weeks. (b) Grip strength. Overall effect for all time points between the three genotypes: P = 0.0024. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and HdhHD/+ Dnchc1+/+: P = 0.001. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and Hdh+/+ Dnchc1Loa/+: P = 0.0157. Effect at each time point between the three genotypes: 7 weeks, P = 0.023; 9 weeks, P = 0.001; 11 weeks, P 0.0001; 13 weeks, P = 0.004; 15 weeks, P = 0.336 (most of the double mutants were dead by 15 weeks). (c) Accelerating rotarod. Overall effect for all time points between the three genotypes: P = 0.0367. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and HdhHD/+ Dnchc1+/+: P = 0.0245. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and Hdh+/+ Dnchc1Loa/+: P = 0.0553. Effect at each time point between the three genotypes: 8 weeks, P 0.001; 10 weeks, P = 0.005; 12 weeks, P = 0.001; 14 weeks, P = 0.009; 16 weeks, P = 0.039.


( d ) Reduced lifespan of HdhHD / + Dnchc1Loa / + mice . HdhHD / + Dnchc1Loa / + ( red line ) n = 13 , HdhHD / + Dnchc1 + / + ( green line ) n = 9 , P = 0 . 038 . Mice were subjected to humane end points , and so the survival curve reflects the onset of the end points rather than the complete lifespan . Asterisks ( * ) represent mice that were found dead without reaching humane end points . No mortality of Hdh + / + Dnchc1Loa / + mice was observed .

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(d) Reduced lifespan of HdhHD/+ Dnchc1Loa/+ mice. HdhHD/+ Dnchc1Loa/+ (red line) n = 13, HdhHD/+ Dnchc1+/+ (green line) n = 9, P = 0.038. Mice were subjected to humane end points, and so the survival curve reflects the onset of the end points rather than the complete lifespan. Asterisks (*) represent mice that were found dead without reaching humane end points. No mortality of Hdh+/+ Dnchc1Loa/+ mice was observed.


( a ) Brain sections from HdhHD / + Dnchc1 + / + or HdhHD / + Dnchc1Loa / + mice were stained with antibody to huntingtin ( EM48 ; green ) . No EM48 - stained aggregates were detected in brains of Hdh + / + Dnchc1Loa / + mice . Arrows indicate cells with aggregates .

(a) Brain sections from HdhHD/+ Dnchc1+/+ or HdhHD/+ Dnchc1Loa/+ mice were stained with antibody to huntingtin (EM48; green). No EM48-stained aggregates were detected in brains of Hdh+/+ Dnchc1Loa/+ mice. Arrows indicate cells with aggregates.


( b ) Western - blot analysis of mouse brain lysates from wild - type ( WT ) or Loa mutant mice using antibodies to LC3 and to actin .

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(b) Western-blot analysis of mouse brain lysates from wild-type (WT) or Loa mutant mice using antibodies to LC3 and to actin.


( A ) HeLa cells were fixed and stained for p62 . The different patterns of p62 distribution were scored in > 200 cells as cytoplasmic with small bodies , cytoplasmic with large and intense bodies , equal nuclear and cytoplasmic staining , and nuclear enriched . The percentage of cells in the respective groups is indicated .

(A) HeLa cells were fixed and stained for p62. The different patterns of p62 distribution were scored in >200 cells as cytoplasmic with small bodies, cytoplasmic with large and intense bodies, equal nuclear and cytoplasmic staining, and nuclear enriched. The percentage of cells in the respective groups is indicated.


( B ) Expression level of GFP - p62 in HeLa cells stably expressing GFP - p62 ( S - GFP - p62 ) compared with the level of endogenous p62 in the parent HeLa cells .

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(B) Expression level of GFP-p62 in HeLa cells stably expressing GFP-p62 (S-GFP-p62) compared with the level of endogenous p62 in the parent HeLa cells.


( C ) Cells stably expressing GFP - p62 display a cytoplasmic punctuate localization of GFP - tagged p62 similar to endogenous p62 . Video microscopy of live cells demonstrated that the small , faint bodies ( thin arrows ) displayed directed migration , whereas the majority of the larger , intense bodies ( thick arrows ) were nonmigratory ( Videos 1 and 2 , available at http : / / www . jcb . org / cgi / content / full / jcb . 200507002 / DC1 ) . A still image from Video 1 is shown here .

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(C) Cells stably expressing GFP-p62 display a cytoplasmic punctuate localization of GFP-tagged p62 similar to endogenous p62. Video microscopy of live cells demonstrated that the small, faint bodies (thin arrows) displayed directed migration, whereas the majority of the larger, intense bodies (thick arrows) were nonmigratory (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200507002/DC1). A still image from Video 1 is shown here.


( D ) Bodies containing either endogenous p62 or transiently or stably transfected GFP - p62 all contain polyubiquitin . HeLa cells were fixed and stained with p62 and polyubiquitin ( clone FK1 ) mAbs directly coupled with AlexaFluor555 ( red ) and AlexaFluor488 ( green ) , respectively . Cells expressing GFP - p62 were only stained for polyubiquitin ( red ) . Bars , 20 μm .

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(D) Bodies containing either endogenous p62 or transiently or stably transfected GFP-p62 all contain polyubiquitin. HeLa cells were fixed and stained with p62 and polyubiquitin (clone FK1) mAbs directly coupled with AlexaFluor555 (red) and AlexaFluor488 (green), respectively. Cells expressing GFP-p62 were only stained for polyubiquitin (red). Bars, 20 μm.


( A ) The indicated deletion constructs were fused COOH terminal to GFP and expressed in NIH3T3 fibroblasts . Asterisks indicate point mutations in the PB1 ( D69A ) and UBA ( I431A ) domains , respectively .

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(A) The indicated deletion constructs were fused COOH terminal to GFP and expressed in NIH3T3 fibroblasts. Asterisks indicate point mutations in the PB1 (D69A) and UBA (I431A) domains, respectively.


( B ) S - GFP - p62 cells were transiently transfected with the indicated myc - tagged p62 constructs , fixed , and stained for the myc tag . Bars , 20 μm .

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(B) S-GFP-p62 cells were transiently transfected with the indicated myc-tagged p62 constructs, fixed, and stained for the myc tag. Bars, 20 μm.


( A ) Localization of bodies containing endogenous p62 or transiently expressed GFP - p62 relative to EEA1 - positive early endosomes . Endogenous p62 were stained green using p62 antibodies directly labeled with AlexaFluor488 , and EEA1 was stained red with EEA1 mAbs directly labeled with AlexaFluor555 . Alternatively , transiently expressed GFP - p62 was expressed in HeLa cells , and EEA1 was stained red with EEA1 mAb ( bottom ) . The boxed area is shown to the right at a higher magnification .

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(A) Localization of bodies containing endogenous p62 or transiently expressed GFP-p62 relative to EEA1-positive early endosomes. Endogenous p62 were stained green using p62 antibodies directly labeled with AlexaFluor488, and EEA1 was stained red with EEA1 mAbs directly labeled with AlexaFluor555. Alternatively, transiently expressed GFP-p62 was expressed in HeLa cells, and EEA1 was stained red with EEA1 mAb (bottom). The boxed area is shown to the right at a higher magnification.


( B ) Colocalization of GFP - p62 and CD63 ( stained red using a mAb ) in HeLa cells stably expressing GFP - p62 .

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(B) Colocalization of GFP-p62 and CD63 (stained red using a mAb) in HeLa cells stably expressing GFP-p62.


( C ) Immunoelectron micrograph of S - GFP - p62 stained with a GFP pAb ( 10 - nm gold particle , arrows ) and monoclonal CD63 ( 15 - nm gold particle , arrowheads ) .

(C) Immunoelectron micrograph of S-GFP-p62 stained with a GFP pAb (10-nm gold particle, arrows) and monoclonal CD63 (15-nm gold particle, arrowheads).


( D ) Rapid detergent extraction of GFP - p62 from LysoTracker - positive acidic organelles . HeLa cells transiently expressing GFP - p62 were labeled with LysoTracker for 60 min . The detergent extractions were imaged in a time series with 15 - s time intervals after adding 1 % Triton X - 100 . The boxed area is shown in the bottom two images at a higher magnification before and after detergent extraction . Bars ( A , B , and D ) , 20 μm .

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(D) Rapid detergent extraction of GFP-p62 from LysoTracker-positive acidic organelles. HeLa cells transiently expressing GFP-p62 were labeled with LysoTracker for 60 min. The detergent extractions were imaged in a time series with 15-s time intervals after adding 1% Triton X-100. The boxed area is shown in the bottom two images at a higher magnification before and after detergent extraction. Bars (A, B, and D), 20 μm.


( A ) Immuno - EM of GFP - p62 . S - GFP - p62 cells were labeled with rabbit anti - GFP ( Abcam ) followed by protein A - gold ( 15 nm ) . We observed labeling in membrane - free cytosolic structures and sequestosomes ( arrows ) as well as in endosomes ( Endo ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immuno-EM of GFP-p62. S-GFP-p62 cells were labeled with rabbit anti-GFP (Abcam) followed by protein A-gold (15 nm). We observed labeling in membrane-free cytosolic structures and sequestosomes (arrows) as well as in endosomes (Endo).


( B ) Correlative immunofluorescence / EM of HeLa cells treated with 10 μM PSI for 5 h displaying typical sequestosomes . The insets show two magnifications of the sequestosome , which is labeled 1 .

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(B) Correlative immunofluorescence/EM of HeLa cells treated with 10 μM PSI for 5 h displaying typical sequestosomes. The insets show two magnifications of the sequestosome, which is labeled 1.


( C ) Representative image of a p62 - containing autophagosome . HeLa cells transfected with GFP - p62 were immunogold labeled as in A . Note the cisternal - like membrane ( arrowheads ) surrounding the GFP - positive material . The arrow indicates a fused endosome .

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(C) Representative image of a p62-containing autophagosome. HeLa cells transfected with GFP-p62 were immunogold labeled as in A. Note the cisternal-like membrane (arrowheads) surrounding the GFP-positive material. The arrow indicates a fused endosome.


( A ) Comparison of p62 degradation using pulse - chase labeling with 35S - methionine and immunoblotting after cycloheximide ( CHX ) treatment . HeLa cells were pulsed with 35S - methionine and incubated for the indicated times in nonradioactive medium . After immunopurification , the amount of radioactive p62 was determined by autoradiography , and the total amount of p62 was determined by an immunoblot of the same membrane . The p62 level in total cellular lysates after different times of 10 μg / ml cycloheximide treatment determined by immunoblotting ( bottom ) .

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

(A) Comparison of p62 degradation using pulse-chase labeling with 35S-methionine and immunoblotting after cycloheximide (CHX) treatment. HeLa cells were pulsed with 35S-methionine and incubated for the indicated times in nonradioactive medium. After immunopurification, the amount of radioactive p62 was determined by autoradiography, and the total amount of p62 was determined by an immunoblot of the same membrane. The p62 level in total cellular lysates after different times of 10 μg/ml cycloheximide treatment determined by immunoblotting (bottom).


( B ) The levels of p62 and LC3 - II change with autophagic activity . HeLa or S - GFP - p62 cells were either left untreated or rapamycin ( 10 μg / ml ) or bafilomycin A1 ( Baf . A1 ; 10 μg / ml ) was added for 18 h . Immunoblots were sequentially probed using LC3 , p62 , and actin antibodies .

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(B) The levels of p62 and LC3-II change with autophagic activity. HeLa or S-GFP-p62 cells were either left untreated or rapamycin (10 μg/ml) or bafilomycin A1 (Baf. A1; 10 μg/ml) was added for 18 h. Immunoblots were sequentially probed using LC3, p62, and actin antibodies.


( C and D ) The amount of p62 located to cytoplasmic bodies increases upon inhibition of autophagy . HeLa cells or S - GFP - p62 HeLa cells were left untreated or bafilomycin A1 was added for 18 h , the cells were fixed , and p62 was either stained red using a p62 mAb ( C ) or imaged directly ( D ) . Nuclei were visualized using the Draq5 DNA stain . The settings for imaging were identical for the treated cells and the untreated control .

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(C and D) The amount of p62 located to cytoplasmic bodies increases upon inhibition of autophagy. HeLa cells or S-GFP-p62 HeLa cells were left untreated or bafilomycin A1 was added for 18 h, the cells were fixed, and p62 was either stained red using a p62 mAb (C) or imaged directly (D). Nuclei were visualized using the Draq5 DNA stain. The settings for imaging were identical for the treated cells and the untreated control.


( E ) The majority of S - GFP - p62 cytoplasmic bodies are stained with antibodies recognizing transiently expressed LC3 . S - GFP - p62 cells were transiently transfected with myc - LC3 . Myc - LC3 was stained red using an anti - myc tag mAb . The boxed area indicates the part of the cell that is shown to the left at a higher magnification . Bars , 5 μm .

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(E) The majority of S-GFP-p62 cytoplasmic bodies are stained with antibodies recognizing transiently expressed LC3. S-GFP-p62 cells were transiently transfected with myc-LC3. Myc-LC3 was stained red using an anti-myc tag mAb. The boxed area indicates the part of the cell that is shown to the left at a higher magnification. Bars, 5 μm.


( A ) HeLa cells transiently transfected with GFP - LC3 alone or cotransfected with siRNA against p62 , HA - p62 , or HA - p62 D69A were either left in normal medium or starved for amino acids for 1 h . The cells were fixed , and p62 was stained using a p62 mAb . More than 200 randomly selected cells for each condition were scored for the cytoplasmic pattern of GFP - LC3 as either diffuse or punctuate . The frequency of GFP - LC3 - positive cells with punctuate localization are indicated to the right .

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(A) HeLa cells transiently transfected with GFP-LC3 alone or cotransfected with siRNA against p62, HA-p62, or HA-p62 D69A were either left in normal medium or starved for amino acids for 1 h. The cells were fixed, and p62 was stained using a p62 mAb. More than 200 randomly selected cells for each condition were scored for the cytoplasmic pattern of GFP-LC3 as either diffuse or punctuate. The frequency of GFP-LC3-positive cells with punctuate localization are indicated to the right.


( B ) Endogenous p62 as well as coexpressed myc - tagged wild - type p62 or a UBA deletion mutant of p62 coimmunoprecipitated with GFP - LC3 from HeLa cell extracts . GFP or GFP - LC3 was immunoprecipitated from total cellular extracts after cotransfecting the indicated constructs . The cell cultures were either left untreated or starved for amino acids for 1 h as indicated . Copurified endogenous or ectopically expressed myc - tagged p62 constructs were detected using the p62 mAb . Bars , 20 μm .

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(B) Endogenous p62 as well as coexpressed myc-tagged wild-type p62 or a UBA deletion mutant of p62 coimmunoprecipitated with GFP-LC3 from HeLa cell extracts. GFP or GFP-LC3 was immunoprecipitated from total cellular extracts after cotransfecting the indicated constructs. The cell cultures were either left untreated or starved for amino acids for 1 h as indicated. Copurified endogenous or ectopically expressed myc-tagged p62 constructs were detected using the p62 mAb. Bars, 20 μm.


( A ) HeLa cells transiently expressing a Flag - tagged NH2 - terminal fragment ( amino acids 1 - 171 ) of huntingtin containing a 68 - polyglutamine expansion , Flag - N - HttQ68 . After 48 h , the cells were fixed , and Flag - N - HttQ68 was stained green using a Flag mAb . Endogenous p62 was stained red using the p62 pAb .

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(A) HeLa cells transiently expressing a Flag-tagged NH2-terminal fragment (amino acids 1-171) of huntingtin containing a 68-polyglutamine expansion, Flag-N-HttQ68. After 48 h, the cells were fixed, and Flag-N-HttQ68 was stained green using a Flag mAb. Endogenous p62 was stained red using the p62 pAb.


( B ) Endogenous p62 forms a shell around aggregated GFP - N - HttQ68 . Transiently transfected HeLa cells were fixed 48 h after transfection , and endogenous p62 was detected using a p62 mAb ( red ) .

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(B) Endogenous p62 forms a shell around aggregated GFP-N-HttQ68. Transiently transfected HeLa cells were fixed 48 h after transfection, and endogenous p62 was detected using a p62 mAb (red).


( C and D ) The p62 shell surrounding GFP - N - HttQ68 was detected independently of the use of antibodies . ( C ) GFP - N - HttQ68 and DsRed - tagged p62 were cotransfected into HeLa cells , and images of live cells were obtained after 48 h of expression . The left panel shows one confocal plane of a representative GFP - N - HttQ68 and DsRed - p62 double - positive structure , and the right panel shows the Z - stack of planes with side views of the aggregate at the side and at the top . N , nucleus . ( A - C ) Nuclei were detected using Draq5 . ( D ) GFP - N - HttQ68 and tdTomato - tagged p62 were cotransfected into HeLa cells , and images of live cells were obtained after 24 h of expression .

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(C and D) The p62 shell surrounding GFP-N-HttQ68 was detected independently of the use of antibodies. (C) GFP-N-HttQ68 and DsRed-tagged p62 were cotransfected into HeLa cells, and images of live cells were obtained after 48 h of expression. The left panel shows one confocal plane of a representative GFP-N-HttQ68 and DsRed-p62 double-positive structure, and the right panel shows the Z-stack of planes with side views of the aggregate at the side and at the top. N, nucleus. (A-C) Nuclei were detected using Draq5. (D) GFP-N-HttQ68 and tdTomato-tagged p62 were cotransfected into HeLa cells, and images of live cells were obtained after 24 h of expression.