EMBO/SourceData huggingface dataset:
Figure 4(A-B) SIRT5 expression is not affected by chemical oxidant treatment. HEK293T cells were treated with Paraquat (A) or H2O2 (B) for the indicated periods. The mRNA and protein expression of endogenous SIRT5 were determined by quantitative real-time PCR and western blot analysis, respectively. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).(D) The desuccinylase activity of SIRT5 is stimulated by oxidative stimuli. Flag-SIRT5 was ectopically expressed in HEK293T cells. The transfected cells were treated with increased concentrations of Paraquat or H2O2 for the indicated periods. Flag-SIRT5 was purified by immunoprecipitation with Flag beads, eluted with Flag peptide, and then subjected to the desuccinylase activity assay as described in 'Materials and Methods'. The SIRT5 desuccinylase activity was normalized against its protein level. The results are average ± SD of 3 independent experiments *p<0.05, ***p<0.001 (two-tailed unpaired t-test).(E) In vitro incubation with chemical oxidants does not affect the desuccinylase activity of SIRT5. Flag-tagged SIRT5 was ectopically expressed in HEK293T cells, and then purified by immunoprecipitation with Flag-beads and eluted with Flag peptide. The purified Flag-SIRT5 was treated with increased concentrations of Paraquat or H2O2 at room temperature for 1 hr, and then subjected to the desuccinylase activity assay as described in 'Materials and Methods'. The SIRT5 desuccinylase activity was normalized against its protein level. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).(F) Chemical oxidants enhance the ability of SIRT5 to activate IDH2. Flag-SIRT5 was ectopically expressed in HEK293T cells, and the transfected cells were treated with increased concentrations of Paraquat or H2O2 for the indicated periods. Flag-SIRT5 was purified by immunoprecipitation with Flag beads, eluted with Flag peptide, and then incubated with purified Flag-IDH2 in vitro. IDH2 enzyme activity was measured as described in 'Materials and Methods', normalized by its protein level. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test).
Figure 1(A) Quantitative analysis of the Aβ1-40 protein in the TBS (*p=0.0212), Triton-X (p=0.0544) and SDS (**p=0.0063) fractions of brain homogenates from male (n=7) and female (n=10) APP23 mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test.(B) Stereological analysis of cortical area covered by 4G8-positive plaques in male (n=10) and female (n=8) APP23 mice (left) and representative images (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, ***p=0.0004.(C) Fluorescence intensity based analysis of pFTAA-stained Aβ plaques in the cortex of male (n=10) and female (n=8) APP23 mice (left) and representative images (right), scale bar = 1 mm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, **p=0.0011.(D) Stereological analysis of cortical area covered by Congo Red-positive plaques in male (n=10) and female (n=8) APP23 mice (left) and representative images (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, ***p=0.0001.(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.
Figure 2(A) Histological analysis of plaque-associated BACE1 immunoreactivity in male (n=10) and female (n=8) APP23 mice. BACE1 area covered was normalised to 4G8-positive area covered of the same image (left). Right: representative images, scale bar = 50 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.3724.(B) Stereological quantification of the number of cortical GFAP-positive astrocytes in male (n=10) and female (n=8) APP23 mice (left). Right: representative images of GFAP staining, scale bar = 200 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, *p=0.0134.(C) Quantification of activated microglia within 30 µm from plaque borders. Top: Numbers of Iba1-positive microglia were normalised to the size of the nearest 4G8-positive plaque and quantified in male (n=10) and female (n=8) APP23 mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.0576. Bottom: Histogram representing Clec7a staining intensity within plaque-associated Iba1-positive microglia in male (n=10) and female (n=8) APP23 mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test with Bonferroni correction for each single bin, p=N.S.. Right: representative images, scale bar = 40 µm. Data points were taken from graphs analysing APP23p40+/+ against APP23p40-/- mice.(D) Radial intensity profiles of Iba1 and 4G8 around the center of the nucleus of plaque-associated Iba1-positive microglia in male (n=10) and female (n=8) APP23 mice. Iba1 intensity declines until a radius of ~6 µm, marking the cell periphery. 4G8 intensity peaks inside the cell (~4 µm), but stays high outside the cell. This is very likely due to the close proximity to 4G8 positive plaques. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test with Bonferroni correction for the number of binned radii shows no significant difference between both 4G8 traces.
Figure 3(A) Gene expression analysis of IL12b in whole brain (n=3 per gender, Il12b undetected), Cd11b-negative non-microglial cells (n=3 per gender, Il12b undetected) and CD11b-positive microglia (n=5 per gender, p=0.3540) in male and female APP23 mice. Gapdh expression was used as an internal reference gene. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test for each fraction.(B) ELISA measurements of the IL12p40 concentration in the TBS-soluble protein fraction derived from wild-type (WT) (n=5), APP23p40+/+(n=17) and APP23p40‑/- (n=16) mice. Male mice are depicted by blue squares while female mice are shown as red squares. Mean ± s.e.m., statistical analysis: one-way ANOVA, Tukey post-hoc test, *p=0.0276, ***p=<0.0001.
Figure 4(A) Mesoscale analysis for the Aβ1-40 protein in the TBS (p=0.2298), Triton-X (p=0.1329) and SDS (p=0.7184) fractions of brain homogenates from male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice. Total protein concentration of each sample was used as an internal reference. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test.(B) Stereological analysis of cortical area covered by 4G8-positive plaques (left) and representative images of 4G8-staining in APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, **p=0.0039.(C) Fluorescence intensity based analysis of pFTAA-positive area covered in the cortex of APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice (left) and representative images for each genotype (right), scale bar = 1 mm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, **p=0.0051.(D) Stereological analysis of cortical area covered by Congo Red-positive plaques in APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice (left) and representative images (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, **p=0.0070.(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.
Figure 5(A) Histological analysis of plaque-associated BACE1 immunoreactivity in male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice. BACE1 area covered was normalised to 4G8-positive area covered of the same image (left). Right: representative images, scale bar = 50 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.1780.(B) Stereological quantification of the number of cortical GFAP-positive astrocytes in male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice (left). Right: representative images of GFAP staining, scale bar = 200 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.3148.(C) Quantification of activated microglia within 30 µm from plaque borders. Top: Numbers of Iba1-positive microglia were normalised to the size of the nearest 4G8-positive plaque and quantified in male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.1925. Bottom: Histogram representing Clec7a staining intensity within plaque-associated Iba1-positive microglia in male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test with Bonferroni correction for each single bin, p=N.S.. Right: representative images, scale bar = 40 µm.(D) Radial intensity profiles of Iba1 and 4G8 around the center of the nucleus of plaque-associated Iba1-positive microglia in male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice. Iba1 intensity declines until a radius of ~6 µm, marking the cell periphery. 4G8 intensity peaks inside the cell (~4 µm), but stays high outside the cell. This is very likely due to the close proximity to 4G8 positive plaques. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test with Bonferroni correction for the number of binned radii shows no significant difference between both 4G8 traces.
Figure 6(A) Mesoscale analysis for the Aβ1-40 protein in the TBS (*p=0.0208), Triton-X (*p=0.0440) and SDS (p=0.0540) fractions of brain homogenates from APP23p40+/+(n=7) and APP23p40-/- (n=8) mice. Total protein concentration of each sample was used as an internal reference. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test.(B) Stereological analysis of cortical area covered by 4G8-positive plaques (left) and representative images of 4G8-staining in APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.1831.(C) Fluorescence intensity based analysis of pFTAA-positive area covered in the cortex of APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice (left) and representative images for each genotype (right), scale bar = 1 mm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.3406.(D) Stereological analysis of cortical area covered by Congo Red-positive plaques in APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice (left) and representative images (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.4542.(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.
Figure 7(A) Histological analysis of plaque-associated BACE1 immunoreactivity in female APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice. BACE1 area covered was normalised to 4G8-positive area covered of the same image (left). Right: representative images, scale bar = 50 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.5402.(B) Stereological quantification of the number of cortical GFAP-positive astrocytes in female APP23p40+/+ (n=8) and APP23p40-/- (n=8) mice (left). Right: representative images of GFAP staining, scale bar = 200 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.1579.(C) Quantification of activated microglia within 30 µm from plaque borders. Top: Numbers of Iba1-positive microglia were normalised to the size of the nearest 4G8-positive plaque and quantified in female APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.8240. Bottom: Histogram representing Clec7a staining intensity within plaque-associated Iba1-positive microglia in female APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test with Bonferroni correction for each single bin, p=N.S.. Right: representative images, scale bar = 40 µm.(D) Radial intensity profiles of Iba1 and 4G8 around the center of the nucleus of plaque-associated Iba1-positive microglia in female APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice. Iba1 intensity declines until a radius of ~6 µm, marking the cell periphery. 4G8 intensity peaks inside the cell (~4 µm), but stays high outside the cell. This is very likely due to the close proximity to 4G8 positive plaques. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test with Bonferroni correction for the number of binned radii shows no significant difference between both 4G8 traces.
Figure 3A 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
Figure 4E 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.
Figure 5A,B Suppression of nse3-ts4 and nse4-ts4 temperature sensitivity (A) and DNA damage sensitivity (B) by the NSE1 allele of UWOPS87-2421. Cultures of the indicated strains were diluted to an optical density at 600 nm of 0.1 and a series of ten-fold dilutions was spotted on agar plates and incubated for 2-3 days. UW = UWOPS87-2421. The plates shown in (B) were incubated at 30°C. C E Recruitment of Smc6-FLAG by ChIP-qPCR at two known SMC5/6 binding sites (pericentromere of chromosome XIV and CEN3) and one negative control locus (arm of chromosome III) in G2/M arrested strains. Relative enrichment corresponds to the ratio of the signal after immunoprecipitation (FLAG) over beads alone, normalised to the ratio in wild-type cells at CEN3. Error bars: standard error of the mean of three independent experiments. Statistical significance was determined using an unpaired, two-tailed t-test (* = p < 0.05, ** = p < 0.005). E
Figure 2(A) Representative confocal photomicrographs showing coronal slices from 8W hGFAP‐tTA;H2B‐GFP mice with and without doxycycline treatment, upon immunofluorescent staining with the stem cell marker Nestin (red). DAPI was used for nuclear counterstain. Cross indicates slice directions: M = medial, D = dorsal, V = ventral, L = lateral. Scale bar = 20 µm. (B) Quantification of Nestin expression in apical and basal cells in doxycycline-treated and untreated animals. N = 3 (- Doxy), N = 4 (+ Doxy). Data information: N number refers to biological replicates; for each biological replicate, at least three technical replicates were performed. Data is represented as mean ± SEM. * and # indicate significance: between apical and basal (*) and between doxycycline treatments (#); */#p<0.05, **p<0.01, by one-way ANOVA with Tukey's multiple comparisons test (B)(C) Same as (A), with addition of the primary cilia marker AC3 (yellow). Arrows indicate Nestin-positive (white) and GFP-positive (green) cells with primary cilium. Scale bar = 10 µm. (D) Quantification of ciliated GFP+ NSCs in apical and basal cell layer as percentage of total apical/basal GFP+ cells. N = 7. (E) Quantification of primary cilia on cells in the apical or basal cell layer according to stem cell marker; N = 7. Data information: N number refers to biological replicates; for each biological replicate, at least three technical replicates were performed. Data is represented as mean ± SEM. * and # indicate significance: between apical and basal (*) ; */#p<0.05, **p<0.01, by two-tailed unpaired student's t-test (D).
Figure 3(A) Repesentative confocal photomicrographs of coronal sections of the SVZ of 8W hGFAP-H2BGFP mice, which had been administered doxycycline (Doxy) as indicated. Nuclear GFP expression and Ki67 immunoreactivity are shown in green and red, respectively. DAPI counterstaining of the nuclei is blue. Scale bar indicates 20 µm.(B, C) Quantitative analyses of the number of apical and basal cells expressing GFP (G+) or not (G-); N = 8 (B), and Ki67 immunoreactivity in these populations; N ≥ 4 (C). Data information: N number refers to biological replicates; for each biological replicate at least three technical replicates were performed. Bars represent mean ± SEM. * indicates significance between doxycycline treated and nontreated specimens with apical or basal cell populations. * p<0.05, ***p<0.001, determined by two-way ANOVA with Sidak's multiple comparisons test.
Figure 5(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 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.
Figure 7(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
Figure 1A, 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 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 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.
Figure 2A-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.
Figure 3B, 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 size-exclusion chromatography (SEC) of the NDP52 WT and Mut proteins.D, Recombinant NDP52 WT and Mut proteins (5 ng) were subjected to NATIVE-PAGE analysis. S.E, short exposure; L.E. long exposure.
Figure 4A, 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 (AC, 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 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 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.