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

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

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

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

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

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

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

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

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 of different full - bem1 bc mutants associated with liposomes containing 75 % PC 20 % PS 5 % PI4P .

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

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


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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 of mEOS - cdc42 mutants signal ( cyan ) after inducing expression of GAL1p - CDC42 in presence of Gal or repressing it in presence of Dex . in blue red boxes correspond to in blue red box in panel ( D ) . are average projections in which black pixels have been inverted .

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

E Representative images of 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 indicated mutant subsequent colony formation at temperatures indicated . Note how mutations on both BC motifs PX in bem1 compromise growth at restrictive .

O O O O O O O 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 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 indicated bem1 constructs tagged with 3XHA ( top panel ) . Detection of Ade13 was used as loading control ( bottom panel ) .

O O O O O O O O O O O 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 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 of indicated bem1 mutants tagged with GFP at BEM1 locus . inverted are average projections .

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 with polarized Bem1 - GFP . E Scatter dot plot showing of indicated bem1 strains at pole . measurements were made on average projections .

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

D Frequency of 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 of full - Cdc24 cdc24 ph domain mutant ( cdc24 ph ) associated with liposomes containing 75 % PC 20 % PS 5 % PI4P .

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


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

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

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

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 subsequent colony formation of indicated mutant at indicated temperatures .

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


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

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 indicated mutant strains ( top panel ) . Detection of Ade13 was used as a loading control ( bottom panel ) .

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 were quantified in strains indicated using a gic2 ( 1 - 208 ) - yeGFP probe . Values are mean ± SD for n = 30 observed over 2 experiments . Data were compared using a Mann - Whitney .

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

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 with SUMO2WT , SUMO2K11Ac or SUMO2K11Q was performed analyzed by immunoblotting with indicated antibodies .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 )

O O O O O O O O O O O O O O O O O O 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 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 , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 presence or absence of NAD + .

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

(a) 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 SUMO2K35Ac . Immunoblotting was performed with antibodies as indicated .

(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 .

O O O O O O O O O O O O O O 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) 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 substrate independent in vitro SUMOylation with SUMO2WT SUMO2K11Ac in presence or absence of SIRT1 ( 1 . 5 μM ) .

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

(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 .

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

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

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

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

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

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

(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 ) α - PCNA as loading control .

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 in MEFs . ROS were determined in indicated MEFs as described in ' Materials Methods ' . results are average ± SD of 3 independent experiments * * p < 0 . 01 ( two - unpaired t - ) .

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

(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 . of [ GSH / GSSG ] was determined in extracts as described in ' Materials Methods ' . results are average ± SD of 3 independent experiments * p < 0 . 05 ( two - unpaired t - ) .

O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . of [ NADPH / NADP + ] was determined in extracts as described in ' Materials Methods ' . results are average ± SD of 3 independent experiments * * * p < 0 . 001 ( two - unpaired t - ) .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . of PARP Caspase - 3 were determined by western blot .

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

(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 . Wild - type Sirt5 KO MEFs were treated with Paraquat ( 500 μM ) for 24 hrs , then viability was determined by counting remaining adherent . results are average ± SD of 3 independent experiments * * p < 0 . 01 ( two - unpaired t - ) .

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

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

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

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

(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 . of [ NADPH / NADP + ] was determined in extracts as described in ' Materials Methods ' . results are average ± SD of 3 independent experiments * * p < 0 . 01 , * * * p < 0 . 001 ( two - unpaired t - ) .

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

(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 . of [ GSH / GSSG ] was determined in extracts as described in ' Materials Methods ' . results are average ± SD of 3 independent experiments * * p < 0 . 01 ( two - unpaired t - ) .

O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 in HEK293T . ROS were determined in indicated stable as described in ' Materials Methods ' . results are average ± SD of 3 independent experiments * * * p < 0 . 001 ( two - unpaired t - ) .

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

(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 . expression of PARP Caspase - 3 were determined by western blot .

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

(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 from ROS - induced . indicated stable HEK293T were treated as mentioned above in ( D ) , then viability was determined by counting remaining adherent . results are average ± SD of 3 independent experiments * * p < 0 . 01 ( two - unpaired t - ) .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Methods ' . results are average ± SD of 3 independent experiments . n . s . : not significant ( two - unpaired t - ) .

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

(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 Methods ' . results are average ± SD of 3 independent experiments * p < 0 . 05 ( two - unpaired t - ) .

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) . is colored in green isocitrate in rainbow , with Lys 360 Lys 413 residues in IDH2 being colored in red .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 . indicated Flag - tagged IDH2 were overexpressed in HEK293T with stable SIRT5 knockdown or SIRT5 co - overexpression . Wild - type mutant IDH2 were purified by Flag beads eluted with Flag peptide , followed by measurement of IDH2 enzyme activity . results are average ± SD of 3 independent experiments * p < 0 . 05 , n . s . : not significant ( two - unpaired t - ) .

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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