F Lipid ordering determined by 2H solid - state NMR analysis of liposomes containing POPC - d31 / POPS ( 4 : 1 molar ratio ) in the presence or absence of Bem1 BC motifs . Calculation of oriented - like spectra from Pake patterns ( de - Pake - ing ) and simulation of 2H solid - state NMR spectra were applied to measure individual quadrupolar splittings for POPC - d31 and determine order parameter accurately .

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F Lipid ordering determined by 2H solid-state NMR analysis of liposomes containing POPC-d31/POPS (4:1 molar ratio) in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately.


Lipid ordering determined by 2H solid - state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs . Calculation of oriented - like spectra from Pake patterns ( de - Pake - ing ) and simulation of 2H solid - state NMR spectra were applied to measure individual quadrupolar splittings for POPC - d31 and determine order parameter accurately . containing POPC - d31 / PI4P ( 19 : 1 molar ratio ) .

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Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/PI4P (19:1 molar ratio).


Lipid ordering determined by 2H solid - state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs . Calculation of oriented - like spectra from Pake patterns ( de - Pake - ing ) and simulation of 2H solid - state NMR spectra were applied to measure individual quadrupolar splittings for POPC - d31 and determine order parameter accurately . containing POPC - d31 / POPC / PI4P ( 15 : 4 : 1 molar ratio ) .

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Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/POPC/PI4P (15:4:1 molar ratio).


Lipid ordering determined by 2H solid - state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs . Calculation of oriented - like spectra from Pake patterns ( de - Pake - ing ) and simulation of 2H solid - state NMR spectra were applied to measure individual quadrupolar splittings for POPC - d31 and determine order parameter accurately . containing POPC - d31 / POPS / Yeast lipid extract / ergosterol ( 1 : 1 : 1 : 2 molar ratio ) .

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Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/POPS/Yeast lipid extract/ergosterol (1:1:1:2 molar ratio).


B Percentage of the different full - length bem1 bc mutants associated with liposomes containing 75 % PC 20 % PS and 5 % PI4P .

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B Percentage of the different full-length bem1 bc mutants associated with liposomes containing 75% PC 20% PS and 5% PI4P.


C Fluorescence intensity change associated with the nucleotide exchange of GDP - Cdc42 for mant - GTP Cdc42 . Fluorescence was measured after the addition of GDP - Cdc42 to reactions containing Mant - GTP ( 100 nM ) , GMP - PNP ( 100 µM ) and the proteins indicated .

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C Fluorescence intensity change associated with the nucleotide exchange of GDP-Cdc42 for mant-GTP Cdc42. Fluorescence was measured after the addition of GDP-Cdc42 to reactions containing Mant-GTP (100 nM), GMP-PNP (100 µM) and the proteins indicated.


D Ten - fold serial dilutions of cells and subsequent colony formation on the indicated plates , where expression of wild type GALp - CDC42 is either induced in the presence of Gal or repressed in Dex . Note how mutation of the wild type Cdc42 ( KKSKK ) to MMSMM is lethal ( see blue box ) , whereas appending the Bem1 BC - 1 motif to this cdc42 mutant restores viability ( red box ) .

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D Ten-fold serial dilutions of cells and subsequent colony formation on the indicated plates, where expression of wild type GALp-CDC42 is either induced in the presence of Gal or repressed in Dex. Note how mutation of the wild type Cdc42 (KKSKK) to MMSMM is lethal (see blue box), whereas appending the Bem1 BC-1 motif to this cdc42 mutant restores viability (red box).


E Representative images of the mEOS - cdc42 mutants signal ( cyan ) after inducing the expression of GAL1p - CDC42 in the presence of Gal or repressing it in the presence of Dex . The cells in the blue and red boxes correspond to the cells in the blue and red box in panel ( D ) . Images are average fluorescence intensity projections in which black and white pixels have been inverted .

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E Representative images of the mEOS-cdc42 mutants signal (cyan) after inducing the expression of GAL1p-CDC42 in the presence of Gal or repressing it in the presence of Dex. The cells in the blue and red boxes correspond to the cells in the blue and red box in panel (D). Images are average fluorescence intensity projections in which black and white pixels have been inverted.


A Ten - fold serial dilutions of the indicated mutant cells and subsequent colony formation at the temperatures indicated . Note how mutations on both the BC motifs and PX in bem1 compromise growth at the restrictive temperature .

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A Ten-fold serial dilutions of the indicated mutant cells and subsequent colony formation at the temperatures indicated. Note how mutations on both the BC motifs and PX in bem1 compromise growth at the restrictive temperature.


B Western blots probed with anti - HA antibody to detect the indicated bem1 constructs tagged with 3XHA ( top panel ) . Detection of Ade13 was used as loading control ( bottom panel ) .

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B Western blots probed with anti-HA antibody to detect the indicated bem1 constructs tagged with 3XHA (top panel). Detection of Ade13 was used as loading control (bottom panel).


C Representative images of the indicated bem1 mutants tagged with GFP at the BEM1 locus . The inverted fluorescence images are average intensity projections .

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C Representative images of the indicated bem1 mutants tagged with GFP at the BEM1 locus. The inverted fluorescence images are average intensity projections.


D Frequency of cells with polarized Bem1 - GFP . E Scatter dot plot showing the fluorescence levels of the indicated bem1 strains at the cell pole . The measurements were made on average intensity projections .

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D Frequency of cells with polarized Bem1-GFP. E Scatter dot plot showing the fluorescence levels of the indicated bem1 strains at the cell pole. The measurements were made on average intensity projections.


B Percentage of full - length Cdc24 and cdc24 ph domain mutant ( cdc24 ph ) associated with liposomes containing 75 % PC 20 % PS and 5 % PI4P .

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B Percentage of full-length Cdc24 and cdc24 ph domain mutant (cdc24 ph) associated with liposomes containing 75% PC 20% PS and 5% PI4P.


C Percentage of Cdc24 and cdc24 ph mutant associated with liposomes of the composition shown in ( B ) in the presence of the indicated bem1 protein . Note how additive mutations in the Bem1 lipid binding sequences reduce the percentage of Cdc24 associated with the liposomes .

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C Percentage of Cdc24 and cdc24 ph mutant associated with liposomes of the composition shown in (B) in the presence of the indicated bem1 protein. Note how additive mutations in the Bem1 lipid binding sequences reduce the percentage of Cdc24 associated with the liposomes.


D Percentage of Cdc24 and cdc24 ph associated with liposomes of the composition shown in ( B ) as a function of the indicated bem1 protein concentration . The curves denote the regression fit of the data to equation 2 in the materials and methods .

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D Percentage of Cdc24 and cdc24 ph associated with liposomes of the composition shown in (B) as a function of the indicated bem1 protein concentration. The curves denote the regression fit of the data to equation 2 in the materials and methods.


E Ten - fold serial dilutions and subsequent colony formation of the indicated mutant cells at the indicated temperatures .

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E Ten-fold serial dilutions and subsequent colony formation of the indicated mutant cells at the indicated temperatures.


F DIC images of the indicated bem1 and cdc24 mutants showing the increased morphological defects ensuing from loss of lipid tethering in the bem1 and cdc24 mutants .

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F DIC images of the indicated bem1 and cdc24 mutants showing the increased morphological defects ensuing from loss of lipid tethering in the bem1 and cdc24 mutants.


G Western blots probed with anti - HA antibody to detect Cdc24 or cdc24 ph tagged with 3XHA in the indicated mutant strains ( top panel ) . Detection of Ade13 was used as a loading control ( bottom panel ) .

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G Western blots probed with anti-HA antibody to detect Cdc24 or cdc24 ph tagged with 3XHA in the indicated mutant strains (top panel). Detection of Ade13 was used as a loading control (bottom panel).


B Global average MSD curves of mEOS - Cdc42 in the strains indicated at the Pole ( P ) and Non - Pole ( NP ) of cells . Trajectories longer than 6 frames were analyzed . Number of trajectories analyzed : BEM1 ( n = 11 cells : Non Pole : 1814 tracks ; Pole : 706 ) ; bem1 bc - 14E px ( n = 10 cells : Non Pole : 1793 tracks ; Pole : 908 ) ; bem1 bc - 14E ( n = 13 cells : Non Pole : 1714 tracks ; Pole : 975 ) . Error bars display s . e . m . C D coefficients of mEOS - Cdc42 in the strains indicated ( in box - plots displaying the median ( line ) , the 25 - 75 percentiles ( box ) and the mean ( cross ) ) , which were compared using a non - parametric , two - tailed Mann - Whitney rank sum test .

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B Global average MSD curves of mEOS-Cdc42 in the strains indicated at the Pole (P) and Non-Pole (NP) of cells. Trajectories longer than 6 frames were analyzed. Number of trajectories analyzed: BEM1 (n = 11 cells: Non Pole: 1814 tracks; Pole: 706); bem1 bc-14E px (n = 10 cells: Non Pole: 1793 tracks; Pole: 908); bem1 bc-14E (n = 13 cells: Non Pole: 1714 tracks; Pole: 975). Error bars display s.e.m. C D coefficients of mEOS-Cdc42 in the strains indicated (in box-plots displaying the median (line), the 25-75 percentiles (box) and the mean (cross)), which were compared using a non-parametric, two-tailed Mann-Whitney rank sum test.


D SR - Tesseler images of mEOS - Cdc42 nanocluster organization in BEM1 ( 5312 localizations shown in image ) and bem1 bc - 14E px cells ( 3739 localizations shown ) . Insets show mEOS - Cdc42 after 491 nm widelfield laser excitation to identify the cell pole . Scale bars : 2 μm . A zoom of the pole region shows the organization of the detected nanoclusters , circled in light blue , in the strains indicated . Scale bar in the zoom : 60 nm .

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D SR-Tesseler images of mEOS-Cdc42 nanocluster organization in BEM1 (5312 localizations shown in image) and bem1 bc-14E px cells (3739 localizations shown). Insets show mEOS-Cdc42 after 491 nm widelfield laser excitation to identify the cell pole. Scale bars: 2 μm. A zoom of the pole region shows the organization of the detected nanoclusters, circled in light blue, in the strains indicated. Scale bar in the zoom: 60 nm.


E Distribution of nanocluster area at the pole ( P ) and non - pole ( NP ) regions of BEM1 ( diameter NP : 59 nm ± 1 nm ( s . e . m ) ; P : 74 nm ± 3 . 2 nm ) ( n = 16 cells . NP : 570 clusters ; P : 162 clusters ) and bem1 bc - 14E px cells ( diameter NP : 59 nm ± 1 . 5 nm ( s . e . m ) ; P : 57 nm ± 2 . 4 nm ) ( n = 15 cells . NP : 421 clusters ; P : 141 clusters ) . Data are presented as scatter dot - plots displaying the median as a line and the 25 - 75 percentiles . Data were compared using non - parametric , two - tailed Mann - Whitney rank sum test .

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E Distribution of nanocluster area at the pole (P) and non-pole (NP) regions of BEM1 (diameter NP: 59 nm ± 1 nm (s.e.m); P: 74 nm ± 3.2 nm) (n = 16 cells. NP: 570 clusters; P: 162 clusters) and bem1 bc-14E px cells (diameter NP: 59 nm ± 1.5 nm (s.e.m); P: 57 nm ± 2.4 nm) (n = 15 cells. NP: 421 clusters; P: 141 clusters). Data are presented as scatter dot-plots displaying the median as a line and the 25-75 percentiles. Data were compared using non-parametric, two-tailed Mann-Whitney rank sum test.


F Active Cdc42 - GTP levels were quantified in the strains indicated using a gic2 ( 1 - 208 ) - yeGFP probe . Values are mean ± SD for n = 30 cells observed over 2 experiments . Data were compared using a Mann - Whitney test .

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F Active Cdc42-GTP levels were quantified in the strains indicated using a gic2(1-208)-yeGFP probe. Values are mean ± SD for n = 30 cells observed over 2 experiments. Data were compared using a Mann-Whitney test.


( c ) Substrate independent in vitro SUMOylation assay with SUMO2WT , SUMO2K11Ac or SUMO2K11Q was performed and analyzed by immunoblotting with the indicated antibodies .

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(c) Substrate independent in vitro SUMOylation assay with SUMO2WT, SUMO2K11Ac or SUMO2K11Q was performed and analyzed by immunoblotting with the indicated antibodies.


( d ) Substrate independent in vitro SUMOylation assay using either SUMO2WT or SUMO2K11Ac in the presence of increasing amounts of ZNF451 - N . Reaction products were analyzed by immunoblotting with the indicated antibodies . Note that - when compared to ( c ) assays were done at reduced concentrations of E1 and

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(d) Substrate independent in vitro SUMOylation assay using either SUMO2WT or SUMO2K11Ac in the presence of increasing amounts of ZNF451-N. Reaction products were analyzed by immunoblotting with the indicated antibodies. Note that - when compared to (c) assays were done at reduced concentrations of E1 and E2


( e ) In vitro SUMOylation assay using a fixed amount of SUMO2WT ( 220 ng ) with increasing concentrations of SUMO2K11Ac . Ratios ranged from 3 : 1 ( unmodified / K11Ac ) to 1 : 2 ( unmodified / K11Ac )

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(e) In vitro SUMOylation assay using a fixed amount of SUMO2WT (220 ng) with increasing concentrations of SUMO2K11Ac. Ratios ranged from 3:1 (unmodified/K11Ac) to 1:2 (unmodified/K11Ac)


( f ) In vitro SUMOylation assay , using SUMO2WT or SUMO2K11Ac , with p53 as a substrate . p53 was generated by in vitro translation / transcription in a rabbit reticulocyte lysate system . SUMO chain formation on p53 was analyzed by immunoblotting with α - p53 antibodies .

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(f) In vitro SUMOylation assay, using SUMO2WT or SUMO2K11Ac, with p53 as a substrate. p53 was generated by in vitro translation/transcription in a rabbit reticulocyte lysate system. SUMO chain formation on p53 was analyzed by immunoblotting with α-p53 antibodies.


( a ) In vitro deacetylation of recombinant SUMO2WT or SUMO2K11Ac ( 2 . 65 μM each ) with recombinant SIRT1 in the presence or absence of NAD + .

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(a) In vitro deacetylation of recombinant SUMO2WT or SUMO2K11Ac (2.65 μM each) with recombinant SIRT1 in the presence or absence of NAD+.


( b ) In vitro deacetylation of recombinant SUMO2WT , SUMO2K11Ac , SUMO2K33Ac and SUMO2K35Ac . Immunoblotting was performed with antibodies as indicated .

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(b) In vitro deacetylation of recombinant SUMO2WT, SUMO2K11Ac, SUMO2K33Ac and SUMO2K35Ac. Immunoblotting was performed with antibodies as indicated.


( c ) In vitro deacetylation of SUMO2K11Ac with increasing concentrations of SIRT1 or SIRT2 ( 0 . 74 µM , 1 . 5 μM , 3 µM ) , followed by immunoblotting with α - SUMO2K11Ac .

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(c) In vitro deacetylation of SUMO2K11Ac with increasing concentrations of SIRT1 or SIRT2 (0.74 µM, 1.5 μM, 3 µM), followed by immunoblotting with α-SUMO2K11Ac.


( d ) Combination of in vitro deacetylation and substrate independent in vitro SUMOylation with SUMO2WT and SUMO2K11Ac in the presence or absence of SIRT1 ( 1 . 5 μM ) .

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(d) Combination of in vitro deacetylation and substrate independent in vitro SUMOylation with SUMO2WT and SUMO2K11Ac in the presence or absence of SIRT1 (1.5 μM).


( e ) In vitro deacetylation of SUMO2WT or SUMO2K11Ac with or without addition of SIRT1 , followed by substrate dependent in vitro SUMOylation of p53 .

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(e) In vitro deacetylation of SUMO2WT or SUMO2K11Ac with or without addition of SIRT1, followed by substrate dependent in vitro SUMOylation of p53.


( f ) Enrichment of acetylated proteins after SIRT1 inhibition in HeLa cells . Western blot analysis of SUMO2K11Ac after treatment of cells with the SIRT1 inhibitior NAM ( 10 mM ) for 5h and subsequent enrichment for acetylated proteins via acetyl - lysine immunoprecipitation . As a control cells were either treated with control siRNA or siSUMO2 / 3 . The samples were analyzed by immunoblotting with α - SUMO2 / 3 and α - SUMO2K11Ac antibodies . The α - SUMO2 / 3Ac and α - SUMO2K11Ac reactive species migrating at ~ 200 kDa are marked with an asterisk .

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(f) Enrichment of acetylated proteins after SIRT1 inhibition in HeLa cells. Western blot analysis of SUMO2K11Ac after treatment of cells with the SIRT1 inhibitior NAM (10 mM) for 5h and subsequent enrichment for acetylated proteins via acetyl-lysine immunoprecipitation. As a control cells were either treated with control siRNA or siSUMO2/3. The samples were analyzed by immunoblotting with α-SUMO2/3 and α-SUMO2K11Ac antibodies. The α-SUMO2/3Ac and α-SUMO2K11Ac reactive species migrating at ~200 kDa are marked with an asterisk.


( b ) Western blot analysis with α - RGS - His and α - HA was performed to monitor poly - SUMOylation of HA - PML . The remaining samples were measured by LC - MS / MS and final data analysis was done with MaxQuant . A K11 SUMOylated SUMO2 peptide was identified and is depicted with an Andromeda score of 49 . 3 . Intensities for this SUMO2 peptide for the control ( gray ) and arsenic ( purple ) treated samples are blotted as a bar graph . The diagram shows a representative dataset derived from two replicates . The corresponding peptide is displayed below the graph . The SUMOylation site is marked with SUMO2 ( green ) and peptide fragment ions are indicated by y - ( red ) and b - ( blue ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O 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-EXP_ASSAY O O O O B-GENEPROD 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 with α-RGS-His and α-HA was performed to monitor poly-SUMOylation of HA-PML. The remaining samples were measured by LC-MS/MS and final data analysis was done with MaxQuant. A K11 SUMOylated SUMO2 peptide was identified and is depicted with an Andromeda score of 49.3. Intensities for this SUMO2 peptide for the control (gray) and arsenic (purple) treated samples are blotted as a bar graph. The diagram shows a representative dataset derived from two replicates. The corresponding peptide is displayed below the graph. The SUMOylation site is marked with SUMO2 (green) and peptide fragment ions are indicated by y- (red) and b- (blue).


( c ) The degradation of the PML reactive band ( ~ 120 kDa ) was determined by immunoblotting . HeLa cells were either mock transfected or transfected with siRNA directed against SUMO1 / 2 / 3 . The expression of RGS - His - tagged SUMO2WT or SUMO2K11Q was induced by DOX for 12 h . Prior to lysis , cells were treated with 1 μM As2O3 for 6 h . Western blot analysis for wild - type SUMO2 and SUMO2K11Q were blotted with α - RGS - His . Immunoblotting with α - Tubulin served as a loading control . Immunoblots for knockdown controls ( SUMO1 and SUMO2 / 3 ) can be

O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O

(c) The degradation of the PML reactive band (~ 120 kDa) was determined by immunoblotting. HeLa cells were either mock transfected or transfected with siRNA directed against SUMO1/2/3. The expression of RGS-His-tagged SUMO2WT or SUMO2K11Q was induced by DOX for 12 h. Prior to lysis, cells were treated with 1 μM As2O3 for 6 h. Western blot analysis for wild-type SUMO2 and SUMO2K11Q were blotted with α-RGS-His. Immunoblotting with α-Tubulin served as a loading control. Immunoblots for knockdown controls (SUMO1 and SUMO2/3) can be found


( b ) Immunoblotting of samples with α - RGS - His ( SUMO variants ) and α - PCNA as loading control .

O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O

(b) Immunoblotting of samples with α-RGS-His (SUMO variants) and α-PCNA as loading control.


( c ) Bar graph , comparing the identified SUMO - SUMO2 linkages of control and heat shock samples for SUMO2WT ( gray ) and SUMO2K11Q ( purple ) . The relative abundance ( y - axis ) of the indicated SUMO - SUMO2 linkage sites ( x - axis ) are shown . The measured intensity for K11 in the SUMO2WT under control conditions was set to 100 % and the relative abundance for the individual residues was calculated in relation to this value . One of two independent replicates is shown .

O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O 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) Bar graph, comparing the identified SUMO-SUMO2 linkages of control and heat shock samples for SUMO2WT (gray) and SUMO2K11Q (purple). The relative abundance (y-axis) of the indicated SUMO-SUMO2 linkage sites (x-axis) are shown. The measured intensity for K11 in the SUMO2WT under control conditions was set to 100% and the relative abundance for the individual residues was calculated in relation to this value. One of two independent replicates is shown.


( A ) Sirt5 deficiency leads to higher ROS levels in MEFs . ROS levels were determined in the indicated MEFs as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * * p < 0 . 01 ( two - tailed unpaired t - test ) .

O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-CELL_LINE O B-SMALL_MOLECULE O O O O O O B-CELL_LINE O O O O O O O O O O O O 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) Sirt5 deficiency leads to higher ROS levels in MEFs. ROS levels were determined in the indicated MEFs as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).


( B ) Sirt5 deficiency suppresses GSH production in MEFs . The ratio of [ GSH / GSSG ] was determined in cell extracts as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * p < 0 . 05 ( two - tailed unpaired t - test ) .

O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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) Sirt5 deficiency suppresses GSH production in MEFs. The ratio of [GSH/GSSG] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments *p<0.05 (two-tailed unpaired t-test).


( C ) Sirt5 deficiency inhibits NADPH production in MEFs . The ratio of [ NADPH / NADP + ] was determined in cell extracts as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * * * p < 0 . 001 ( two - tailed unpaired t - test ) .

O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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) Sirt5 deficiency inhibits NADPH production in MEFs. The ratio of [NADPH/NADP+] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments ***p<0.001 (two-tailed unpaired t-test).


( D ) Sirt5 deficiency decreases cell growth . MEF cells were seeded in 6 - well plates at a density of 50 , 000 cells / well , and cell growth rate was carefully monitored every 1 - 2 days by cell counting for 8 days . The results are average ± SD of 3 independent experiments * p < 0 . 05 , * * * p < 0 . 001 ( two - tailed unpaired t - test ) .

O O O B-GENEPROD O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O 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) Sirt5 deficiency decreases cell growth. MEF cells were seeded in 6-well plates at a density of 50,000 cells/well, and cell growth rate was carefully monitored every 1-2 days by cell counting for 8 days. The results are average ± SD of 3 independent experiments *p<0.05, ***p<0.001 (two-tailed unpaired t-test).


( E ) Sirt5 deficiency leads to higher sensitivity of MEFs to Paraquat . The levels of cleaved PARP and Caspase - 3 were determined by western blot analysis .

O O O B-GENEPROD O O O O O O B-CELL_LINE O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O

(E) Sirt5 deficiency leads to higher sensitivity of MEFs to Paraquat. The levels of cleaved PARP and Caspase-3 were determined by western blot analysis.


( F ) Sirt5 protects MEFs from ROS - induced cell death . Wild - type and Sirt5 KO MEFs were treated with Paraquat ( 500 μM ) for 24 hrs , and then cell viability was determined by counting the remaining adherent cells . The results are average ± SD of 3 independent experiments * * p < 0 . 01 ( two - tailed unpaired t - test ) .

O O O B-GENEPROD O B-CELL_LINE O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O 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) Sirt5 protects MEFs from ROS-induced cell death. Wild-type and Sirt5 KO MEFs were treated with Paraquat (500 μM) for 24 hrs, and then cell viability was determined by counting the remaining adherent cells. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).


( G - I ) Sirt5 deficiency inhibits the production of NADPH and GSH and increases lipid peroxide in mouse brains after Paraquat injection . Female Sirt5 KO and WT littermates ( n = 3 ~ 5 per group ) were intraperitoneally injected with saline or paraquat ( 10 mg / kg ) once a week for 3 consecutive weeks . The ratios of [ NADPH / NADP + ] and [ GSH / GSSG ] and the level of lipid peroxide were determined in tissue extracts . The results are average ± SD . * p < 0 . 05 , n . s . : not significant ( two - tailed unpaired t - test ) .

O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-ORGANISM B-TISSUE O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O 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-I) Sirt5 deficiency inhibits the production of NADPH and GSH and increases lipid peroxide in mouse brains after Paraquat injection. Female Sirt5 KO and WT littermates (n=3~5 per group) were intraperitoneally injected with saline or paraquat (10 mg/kg) once a week for 3 consecutive weeks. The ratios of [NADPH/NADP+] and [GSH/GSSG] and the level of lipid peroxide were determined in tissue extracts. The results are average ± SD. *p<0.05, n.s.: not significant (two-tailed unpaired t-test).


( J - K ) Sirt5 KO mice are more sensitive to Paraquat - induced nigrostriatal dopaminergic degeneration . Female Sirt5 KO and WT littermates were treated with paraquat as in ( G - I ) . The brain sections were stained to detect tyrosine hydroxylase ( TH ) positive dopaminergic neurons in the SNc region , as described in ' Materials and Methods ' . Representative immunohistochemistry images ( original magnification , 100 × ; scale bar , 100 μm ) and the corresponding quantifications are shown in ( J ) and ( K ) , respectively . The results are average ± SD . * p < 0 . 05 , n . s . : not significant ( two - tailed unpaired t - test ) .

O O O O O B-GENEPROD O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J-K) Sirt5 KO mice are more sensitive to Paraquat-induced nigrostriatal dopaminergic degeneration. Female Sirt5 KO and WT littermates were treated with paraquat as in (G-I). The brain sections were stained to detect tyrosine hydroxylase (TH) positive dopaminergic neurons in the SNc region, as described in 'Materials and Methods'. Representative immunohistochemistry images (original magnification, 100×; scale bar, 100 μm) and the corresponding quantifications are shown in (J) and (K), respectively. The results are average ± SD. *p<0.05, n.s.: not significant (two-tailed unpaired t-test).


( A ) Knockdown of SIRT5 inhibits NADPH production in HEK293T cells . The ratio of [ NADPH / NADP + ] was determined in cell extracts as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed unpaired t - test ) .

O O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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)Knockdown of SIRT5 inhibits NADPH production in HEK293T cells. The ratio of [NADPH/NADP+] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test).


( B ) Knockdown of SIRT5 suppresses GSH production in HEK293T cells . The ratio of [ GSH / GSSG ] was determined in cell extracts as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * * p < 0 . 01 ( two - tailed unpaired t - test ) .

O O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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) Knockdown of SIRT5 suppresses GSH production in HEK293T cells. The ratio of [GSH/GSSG] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).


( C ) Knockdown of SIRT5 leads to higher ROS levels in HEK293T cells . ROS levels were determined in the indicated stable cells as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * * * p < 0 . 001 ( two - tailed unpaired t - test ) .

O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O 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) Knockdown of SIRT5 leads to higher ROS levels in HEK293T cells. ROS levels were determined in the indicated stable cells as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments ***p<0.001 (two-tailed unpaired t-test).


( D ) Knockdown of SIRT5 leads to higher sensitivity to Paraquat . The expression levels of PARP and Caspase - 3 were determined by western blot analysis .

O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O

(D) Knockdown of SIRT5 leads to higher sensitivity to Paraquat. The expression levels of PARP and Caspase-3 were determined by western blot analysis.


( E ) SIRT5 protects HEK293T cells from ROS - induced cell death . The indicated stable HEK293T cells were treated as mentioned above in ( D ) , and then cell viability was determined by counting the remaining adherent cells . The results are average ± SD of 3 independent experiments * * p < 0 . 01 ( two - tailed unpaired t - test ) .

O O O B-GENEPROD O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O 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) SIRT5 protects HEK293T cells from ROS-induced cell death. The indicated stable HEK293T cells were treated as mentioned above in (D), and then cell viability was determined by counting the remaining adherent cells. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).


( G ) The activities of G6PD / 6PGD and IDH1 / 2 are reduced in stable HEK293T cells with SIRT5 knockdown . Activities of the indicated endogenous proteins were determined in cell extracts as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . : not significant ( two - tailed unpaired t - test ) .

O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) The activities of G6PD/6PGD and IDH1/2 are reduced in stable HEK293T cells with SIRT5 knockdown. Activities of the indicated endogenous proteins were determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments *p<0.05, **p<0.01, ***p<0.001, n.s.: not significant (two-tailed unpaired t-test).


( H ) G6PD and IDH2 are activated by SIRT5 in vitro . Upon pre - incubation with SIRT5 in vitro , the activities of the indicated ectopically expressed proteins were determined as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * * p < 0 . 01 , n . s . : not significant ( two - tailed unpaired t - test ) .

O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) G6PD and IDH2 are activated by SIRT5 in vitro. Upon pre-incubation with SIRT5 in vitro, the activities of the indicated ectopically expressed proteins were determined as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01, n.s.: not significant (two-tailed unpaired t-test).


( A ) G6PD activity is not regulated by lysine succinylation . Purified Flag - tagged G6PD was incubated with or without succinyl - CoA ( 1 mM ) at 30 ° C for 15 mins , followed by measurement of G6PD enzyme activity as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments . n . s . : not significant ( two - tailed unpaired t - test ) .

O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O 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) G6PD activity is not regulated by lysine succinylation. Purified Flag-tagged G6PD was incubated with or without succinyl-CoA (1 mM) at 30°C for 15 mins, followed by measurement of G6PD enzyme activity as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).


( B ) IDH2 activity is suppressed by lysine succinylation . Purified Flag - tagged IDH2 was incubated with or without succinyl - CoA ( 1 mM ) at 30 ° C for 15 mins , followed by measurement of IDH2 enzyme activity as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * p < 0 . 05 ( two - tailed unpaired t - test ) .

O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O 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) IDH2 activity is suppressed by lysine succinylation. Purified Flag-tagged IDH2 was incubated with or without succinyl-CoA (1 mM) at 30°C for 15 mins, followed by measurement of IDH2 enzyme activity as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments *p<0.05 (two-tailed unpaired t-test).


( C ) SIRT5 knockdown increases G6PD glutarylation and IDH2 succinylation , respectively , and decreases both enzyme activity . Flag - G6PD or Flag - IDH2 was ectopically expressed in the indicated stable HEK293T cells . G6PD / IDH2 proteins were purified by immunoprecipitated with Flag beads and eluted with Flag peptide , followed by determination of their lysine modifications and enzyme activity . The results are average ± SD of 3 independent experiments * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed unpaired t - test ) .

O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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) SIRT5 knockdown increases G6PD glutarylation and IDH2 succinylation, respectively, and decreases both enzyme activity. Flag-G6PD or Flag-IDH2 was ectopically expressed in the indicated stable HEK293T cells. G6PD/IDH2 proteins were purified by immunoprecipitated with Flag beads and eluted with Flag peptide, followed by determination of their lysine modifications and enzyme activity. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test).


( D ) SIRT5 decreases G6PD glutarylation and IDH2 succinylation , and increases both enzyme activity . Flag - G6PD or Flag - IDH2 was co - overexpressed with HA - SIRT5 in HEK293T cells . G6PD and IDH2 proteins were purified by immunoprecipitation with Flag beads and eluted with Flag peptide , followed by determination of their lysine modifications and enzyme activity . The results are average ± SD of 3 independent experiments * p < 0 . 05 , * * p < 0 . 01 ( two - tailed unpaired t - test ) .

O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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) SIRT5 decreases G6PD glutarylation and IDH2 succinylation, and increases both enzyme activity. Flag-G6PD or Flag-IDH2 was co-overexpressed with HA-SIRT5 in HEK293T cells. G6PD and IDH2 proteins were purified by immunoprecipitation with Flag beads and eluted with Flag peptide, followed by determination of their lysine modifications and enzyme activity. The results are average ± SD of 3 independent experiments *p<0.05, **p<0.01 (two-tailed unpaired t-test).


( E ) Cartoon representation of porcine mitochondrial IDH2 structure shows isocitrate binding pocket ( PDB ID : 1LWD ) [ 33 ] , made by Pymol ( www . pymol . org ) . The protein is colored in light green and isocitrate in rainbow , with Lys 360 and Lys 413 residues in IDH2 being colored in red .

O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O

(E) Cartoon representation of porcine mitochondrial IDH2 structure shows isocitrate binding pocket (PDB ID: 1LWD) [33], made by Pymol (www.pymol.org). The protein is colored in light green and isocitrate in rainbow, with Lys 360 and Lys 413 residues in IDH2 being colored in red.


( 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 ) .

O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O 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 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 ) .

O O O O O B-GENEPROD O O O O O O O O O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O 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 ) 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 ) .

O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O 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-GENEPROD O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O 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 ) 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O B-EXP_ASSAY B-SMALL_MOLECULE O O O B-TISSUE O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O 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-GENEPROD B-ORGANISM B-TISSUE O O O O O O O O O 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-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O B-GENEPROD B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-SUBCELLULAR B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O 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 ) 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 .

O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O 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-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-TISSUE O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O B-GENEPROD B-EXP_ASSAY O O O O O O O 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 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 .

O O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O 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 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 B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O 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 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 B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM B-TISSUE O O 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 B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O B-GENEPROD B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-SUBCELLULAR B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O O O O 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 ) 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 B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O 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-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O 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-EXP_ASSAY 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O O O O O O 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=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 B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O B-TISSUE O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O 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=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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY 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 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 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

O O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O

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 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 .

O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O 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 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 )

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O 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-GENEPROD B-EXP_ASSAY O O O B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-CELL_TYPE B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O O O O O O O O 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) 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 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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O B-TISSUE O O O O B-GENEPROD B-ORGANISM O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SUBCELLULAR B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O

(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 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 .

O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O B-EXP_ASSAY O O B-TISSUE O B-ORGANISM O B-GENEPROD O O O O O O O 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-GENEPROD O O O O B-GENEPROD O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Representative 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.