EMBO/soda-nel huggingface dataset:
Commensal microbiota influence systemic autoimmune responses (2014)
Figure 3

Development of antinuclear antibodies in lymphotoxin‐deficient mice is influenced by gut microbiota

Ltbr−/− mice were treated or not with antibiotics from birth (upper panel), conventionalized or germfree wild‐type mice were treated with the LTβR‐Fc fusion protein from gestational day 18 until 6 weeks after birth (lower panel); percentage of mice with at least one ANA. ANA were tested with LIA at the age of 3 months.
16S rRNA gene analysis of luminal samples of wild‐type, ANA‐positive, and ANA‐negative Ltbr−/− mice. Left panel: Beta diversity plot using multidimensional scaling on Bray–Curtis dissimilarities distances. Right panel: Hierarchical cluster analysis using Pearson's correlation. Bacteria community compositions differ in the different groups. Please note that Candidatus arthromitus, according to Thompson et al (), should be renamed Candidatus savagella.
Real‐time PCR for SFB in luminal, mucosal, and fecal samples of multiple ANA‐positive and ANA‐negative Ltbr−/− mice. Analysis with ANOVA, P = 0.02 for differences between groups, n = 4 mice per group. Data represent means ± s.e.m.
C3H/HeN germfree pregnant mice were treated with the LTβR‐Fc fusion protein on day 16 and 18 of gestation to prevent the development of PP in their offspring. Pups were also treated in the neonatal period to prevent the development of ILF. Mice were colonized at 8 weeks of age with SFB or E. coli and were sacrificed on d20 or on d60 postcolonization; percentage of mice with at least one ANA. E denotes gestational day.
LTβR‐Fc‐treated wild‐type or IL17R−/− mice; percentage of mice with at least one ANA. E denotes gestational day. ANA were determined with LIA at the age of 3 months
Harnessing chaperone-mediated autophagy for the selective degradation of mutant huntingtin protein (2010)
figf1

(a) Schematic representation of 60Q-HSC70bm. (b) PC12 cells transfected with either 60Q or 60Q-HSC70bm. The presence of two HSC70bm motifs in 60Q-HSC70bm reduced the number of inclusions as compared to 60Q (scale bar, 20 μm). (c) Quantification of the PC12 cells with inclusions 24 h after transfection with either 60Q or 60Q-HSC70bm. 10 mM 3-methyladenine (3-MA), 10 μM pepstatin A and 10 μM leupeptin were added to inhibit macroautophagy, the proteases, cathepsins D and E, and cathepsin A, respectively. Bars in c represent relative mean values ± s.e.m. from four independent experiments, with levels of aggregation observed for 60Q normalized to a value of 1. Quantifications were performed by ArrayScan.
Harnessing chaperone-mediated autophagy for the selective degradation of mutant huntingtin protein (2010)
figf2

(a) Schematic representations of all constructs tested. R, mRFP; RH, mRFP fused to HSC70bm (VKKDQ and KFERQ); RS, mRFP fused to scrambled QBP1 (Online Methods); RHS, mRFP fused to HSC70bm and scrambled QBP1; RQ, mRFP fused to QBP1; RHQ, mRFP fused to HSC70bm and QBP1 (Sc, scrambled). (b) Confocal images of the 150Q Neuro2a cells transfected with R, RH, RS, RHS, RQ and RHQ. Molecules containing QBP1 co-localized with the tNHTT-150Q-EGFP inclusions (bar, 5 μm). Cells with inclusions were chosen to illustrate the co-localization of QBP1-containing molecules with tNHTT-150Q-EGFP. (c) The expression of RQ and RHQ in 150Q Neuro2a cells decreased the accumulation of insoluble tNHTT-150Q-EGFP on the gel top. Note the shift of tNHTT-150Q-EGFP from an insoluble to a soluble form when RQ was co-expressed. The levels of tNHTT-16Q-EGFP were not affected. Full-length western blots are presented in Supplementary Figure 13. (d) RHQ reduced the levels of aggregated and soluble forms of tNHTT-150Q-EGFP by 81.8% and 33.6%, respectively. WB, western blot. (e) Quantification of tNHTT-16Q-EGFP protein levels. (f) ArrayScan analysis revealed 28% and 56% reductions in inclusion formation in 150Q Neuro2a cells by RQ and RHQ, respectively, after 24 h of differentiation and induction. (g) In 150Q-nls Neuro2a cells, expression of RQ and RHQ reduced the number of tNHTT-150Q-nls-EGFP inclusion by 23% (RQ) and 53% (RHQ) after 48 h of differentiation and induction compared to RS and RHS, respectively. Bars in d and e represent mean values ± s.e.m. from four independent experiments. Quantifications were performed by ArrayScan.
Harnessing chaperone-mediated autophagy for the selective degradation of mutant huntingtin protein (2010)
figf3

(a) Western blot analysis of the chase experiment in 60Q Neuro2a cells. Full-length western blots are presented in Supplementary Figure 13. (b) RHQ enhanced the tNHTT-60Q-EGFP degradation during the chase period. 5 μM MG132, 10 mM 3-methyladenine and 15 mM ammonium chloride (NH4Cl) were used to inhibit proteasome, macroautophagy and lysosomal degradation, respectively. NH4Cl had no effect on proteasome activity and did not affect cell viability (Supplementary Fig. 4a,b). Bars in b represent relative mean values ± s.e.m. from three independent experiments. Protein levels at time 0 represent the control condition (arbitrarily set as 1).
Harnessing chaperone-mediated autophagy for the selective degradation of mutant huntingtin protein (2010)
figf4

(a,b) Constructs identical to those described in Figure 2a, but lacking mRFP (representations of S, HS, Q, HQ are shown in Supplementary Fig. 3a) were co-transfected with tNHTT-16Q-EGFP (a) and tNHTT-150Q-EGFP (b) constructs into Neuro2a cells (bar, 4 μm). (c) Dot blot analysis of the lumenal fraction of purified lysosomes from Neuro2a cells co-transfected with tNHTT-60Q-EGFP and R, RS, RHS, RQ or RHQ. PNS, post-nuclear supernatant. (d) Quantification of HTT translocation into lysosomes. Levels of HTT detected by EM48 (anti-huntingtin antibody) were normalized to levels of β-tubulin (in PNS) and levels of cathepsin D (in lysosomes). (e) Compound images generated by ArrayScan illustrating inclusion formation in 150Q Neuro2a cells transfected with R, RS, RHS, RQ and RHQ and shRNA specific for HSC70 and/or Lamp2a. (blue, Hoechst 33258; green, tNHTT-150Q-EGFP; red, tested molecule). (f) RNAi of HSC70, Lamp2a or both alleviated the inhibitory effect of RHQ on the formation of inclusions containing tNHTT-150Q-EGFP inclusions. (g) Western blot analysis of soluble polyQ protein in 150Q Neuro2a cells transfected with tested constructs and shRNA for HSC70 and/or Lamp2a. Full-length western blots are presented in Supplementary Figure 13. (h) Silencing of HSC70, Lamp2a or both alleviated the inhibitory effect of RHQ on levels of soluble tNHTT-150Q-EGFP. Bars in d and f represent mean values ± s.e.m. from three independent experiments. Bars in h represent relative mean values ± s.e.m. from three independent experiments. For h, values for the R construct were arbitrarily set to 1.
Harnessing chaperone-mediated autophagy for the selective degradation of mutant huntingtin protein (2010)
figf5

(a) Filter trap assay analysis of HTT-polyQ aggregation in nine R6/2 mouse brains injected with three different combinations of rAAV. (b) Quantification of the SDS-insoluble material detected by filter trap assay (*, P = 0.037; **, P = 0.0083; ***, P = 0.0015). (c) Western blot analysis of HTT aggregation (gel top, EM48 antibody), soluble HTT (1C2 antibody), and higher molecular polyQ complexes by agarose gel electrophoresis for resolving aggregates (AGERA). Full-length western blots are presented in Supplementary Figure 13. (d) Quantification of the gel top-detected aggregation (*, P = 0.093; **, P = 0.0027; ***, P = 0.0045). (e) Quantification of soluble HTT-polyQ (*, P = 0.034; **, P = 0.00003; ***, P = 0.0001). (f) Brain sections stained with anti-RFP, EM48 and anti-ubiquitin antibodies (bar, 40 μm). (g) Numbers of ubiquitin-positive inclusions in the transduced areas (*, P = 0.0026; **, P = 5.535 × 10−8; ***, P = 2.009 × 10−8). Bars in b, d, e and g represent the relative mean values ± s.e.m. from three brains injected with different rAAV in each striatum. R in R/Q and R/HQ, and Q in Q/HQ brains represent the control value of 1.
Targeted attenuation of elevated histone marks at SNCA alleviates α-synuclein in Parkinson's disease (2020)
Figure 1

Figure 1. Parkinson's disease patients harbor high levels of H3K4me3 at the α-synuclein promoter.

A Human SNCA gene contains six coding exons and two 5' non-coding exons. The exons are represented by vertical colored boxes. The region from exon 1a to exon 2 (~2.5 kb) is scaled up to show the distribution of histone PTMs. Distribution of the histone PTMs from the SN region of one donor brain sample is shown. Peaks of three different histone PTMs, H3K4me3 (green), H3K27ac (blue), and H3K27me3 (black), at the regulatory region of SNCA were adopted from Roadmap Epigenomics Database. For a detailed view, please see Appendix Figure S1 where screenshot of the original figure is shown. The TSS is indicated by a red vertical bar and distances of exon 1a and 1b from the TSS are indicated.

B ChIP gel images showing the relative enrichment by H3K4me3 in controls (n=9) and PD patients (n= 18). PCR amplified a 188-bp region of SNCA from intron 1 where H3K4me3 peak was at its optimum. Mouse IgG (mIgG) was used as control and the bands were normalized by unbiased amplification from respective inputs.

C Relative intensities calculated from the gel images. Graph shows that H3K4me3 was significantly enriched at the upstream regulatory region of SNCA in PD compared to control subjects. ****P < 0.0001. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. Two-tailed p-values were calculated for all.

Data information: Data represent mean ± standard error of the mean.
Targeted attenuation of elevated histone marks at SNCA alleviates α-synuclein in Parkinson's disease (2020)
Figure 2

Figure 2. PD patients exhibit high expression of α-synuclein.

A Western blot gel images showing α-synuclein (α-SYN) levels in SN tissues from control (n= 9) and PD subjects (n = 18). The numbers on top of each gel panel show the ID of each sample.

B The relative levels of α-synuclein between the two groups were evaluated statistically. PD subjects had moderately higher levels of α-SYN compared to controls. ID of each dot is shown in the graph.

C The normalized levels of α-synuclein in the entire cohort were divided into high (n = 12) and low levels. The cut off value for determining the threshold was set at the median from all the subjects. The high levels of α-synuclein were plotted with corresponding H3K4me3 values of those subjects. The corresponding ID of each sample is shown next to each point in the graph. There was a significant correlation between high levels of α-synuclein and H3K4me3.

Data information: Data represent mean ± standard error of the mean. Data in (B) were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections and two-tailed p-values were calculated. Spearman rank correlation test was performed in (C). Two tailed p value was calculated with Fisher's exact p.
Targeted attenuation of elevated histone marks at SNCA alleviates α-synuclein in Parkinson's disease (2020)
Figure 3

Figure 3. NeuN-positive neurons from the SN demonstrate high enrichment by H3K4me3.

A 20,000 NeuN labeled neuronal nuclei were collected by fluorescent activated nuclei sorting (FANS) from control (n = 6) and PD (n= 7) SN tissues. The NeuN- positive nuclei were labeled by anti-rabbit IgG secondary antibody tagged with Alexa fluor 488. The top and bottom panels show representative sort gating windows from an unlabeled and labeled patient sample, respectively. The left column represents size versus granularity gatings. The samples were then gated for singularity using forward scatter height (Y-axis) versus forward scatter area (X-axis), and lastly, singularly gated nuclei were sorted based on NeuN positivity (X-axis; FL1 channel) versus side scatter (Y-axis). The green rectangular quadrants on the right column of both panels represent the NeuN+ region, as unstained sample did not show any significant representation. Therefore, from each of the stained samples, 20,000 bright NeuN+ nuclei (green rectangle gate) were sorted and collected.

B Gel images from RT-PCR show purity of the sorted nuclei from a representative PD sample. Nuclear RNA was isolated, cDNA was generated and pre-amplified (see Materials and Methods for details) before target-specific PCR. Neuron specific genes (NeuN, synaptophysin) and astrocyte specific gene (GFAP), α-synuclein, and GAPDH were amplified from the isolated nuclei.

C ChIP was performed on the equal number of isolated nuclei against H3K4me3 from all samples. Gel images represent the ChIP-based PCR amplification. The same primer pair was used to amplify the target region on SNCA as Fig 1. Mouse IgG was used as a control.

D The graph represents the statistically significant difference of relative H3K4me3 enrichment between PD (n=7) and controls (n=6). Neuronal nuclei from PD brain samples show significantly higher enrichment of H3K4me3 at SNCA intron 1 (p=0.01) compared to controls. *P < 0.05. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. Two-tailed p-values were calculated for all.

Data information: Data represent mean ± standard error of the mean.
Targeted attenuation of elevated histone marks at SNCA alleviates α-synuclein in Parkinson's disease (2020)
Figure 5

Figure 5. CRISPR/dCas9 SunTag-JARID1A decreases α-synuclein expression by reducing H3K4me3 at the SNCA promoter.

The relative enrichment of H3K4me3 (A) and corresponding α-synuclein levels (B and C) were evaluated in SH-SY5Y cells expressing dCas9-5xGCN4 with (sgA-dCas9) or without scFV-sfGFP-JARID1A (sgA-dCas9-JA).

A ChIP data demonstrated a significant decrease of H3K4me3 at the SNCA promoter. The relative enrichment by H3K4me3 was normalized to the respective inputs. Four independent repeats were performed. *P < 0.05. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. Two-tailed p-values were calculated. Only representative gel is shown here, the gels for other replicates are presented in the Appendix Figure S12A.

B The levels of α-synuclein (α-SYN) in SH-SY5Y cells were evaluated using RT-PCR. The level of α-SYN was normalized to β-actin expression. A significant reduction of α-SYN in cells expressing scFV-sfGFP-JARID1A was observed. Four independent repeats were performed. *P < 0.05. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. Two-tailed p-values were calculated. Only representative gel is shown here, the gels for other replicates are presented in the Appendix Figure S12B.

C α-SYN levels in SH-SY5Y cells were also evaluated using western blot. The levels of α-SYN was normalized to β-actin. Significant reduction of α-SYN was observed in cells expressing JARID1A. Three independent repeats were performed. Percentage of reduction is shown in the graph. **P <0.01. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. Two-tailed p-values were calculated.

Data information: All data are presented as mean ± SEM.
Targeted attenuation of elevated histone marks at SNCA alleviates α-synuclein in Parkinson's disease (2020)
Figure 6

Figure 6. CRISPR/dCas9 SunTag-JARID1A ameliorates elevated α-synuclein levels in differentiated PD-iPSCs by reducing H3K4me3 from the gene promoter.

A-B Relative enrichment of H3K4me3 and H3K27me3 at the SNCA promoter/intron 1 region was evaluated from six iPSC lines of two control subjects and five iPSC lines derived from two sporadic PD (sPD) cases by ChIP. The same genomic region as shown in Fig 1 and Appendix Figure S2C was evaluated for enrichment of H3K4me3 and H3K27me3. Mouse IgG (mIgG) was used as control for the target antibody. The relative intensity of the target band was normalized by respective input.

C ChIP on H3K4me3 at the SNCA promoter between control and sPD1-1 lines revealed a marginally higher enrichment in sPD lines as compared to the controls.

D Relative comparison of H3K27me3 enrichment between control and sPD lines revealed no significant difference between the two groups.

E ChIP on H3K4me3 at the SNCA promoter between sPD1-1 lines after locus-specific epigenomic modulation. Differentiated cells were transiently transfected with either sgA-dCas9 5xGCN4-scFV-JARID1A or sg A-dCas9 5xGCN4-scFV-empty backbone vectors. A significant reduction in H3K4me3 at the SNCA promoter was observed in cells transfected with JARID1A. A representative ChIP image is shown, the gels for other replicates are presented in the Appendix Figure S12C.

F Western blot analysis of α-synuclein (α-SYN) levels in the cells under the same conditions as analyzed in (E). A significant decrease (56-66%) in α-SYN levels was observed in cells transfected with JARID1A. The normalized and relative expression of α-SYN in JARID1A transfected cells are shown as a percentage of control (transfected by empty backbone vector). A representative western blot image is shown at the top, the gels for other replicates are presented in the Appendix Figure S12D.

Data information: Three independent repeats were performed for both ChIP and western blot experiments. *P < 0.05; ****P<0.0001. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. One-tailed p-values were calculated for ChIP analysis while two-tailed p-value was calculated for western blot experiments. All data are presented as mean ± SEM.
A highly conserved pocket on PP2A-B56 is required for hSgo1 binding and cohesion protection during mitosis (2021)
Figure 1

Figure 1. hSgo1 and LxxIxE motifs compete for binding to PP2A-B56

(A) Structure of the PP2A-B56γ-hSgo1 complex (adapted from Xu et al, PDB: 3FGA). The hSgo1 coiled-coil homodimer interacts with both PP2A catalytic and B56 regulatory subunits. The model shows a LxxIxE peptide bound to B56 at its conserved binding pocket.

(B) YFP pull down from cells stably expressing YFP (control) or YFP-B56α enriches the entire PP2A-B56α holoenzyme on the beads. PP2A-A, scaffold subunit; PP2A-C, PP2A catalytic subunit.

(C) Coomassie-stained SDS-PAGE of the purified hSgo1 full length (FL) and hSgo11-155.

(D) Competition assay with the purified hSgo1 proteins shown in (C). Binding of YFP-B56α to indicated proteins was determined. Representative of 3 independent experiments.

(E) Peptide competition assay with a WT LxxIxE peptide or a mutated variant that does not bind B56 (LxxAxA). Binding of YFP-B56α to indicated proteins was determined and quantified by LiCor.

(F) A PP2A-B56γ-BubR1516-715 complex was reconstituted (blue box) and peak fractions pooled and incubated with hSgo11-155. Following incubation this complex was analyzed by size-exclusion chromatography and fractions analyzed by SDS-PAGE (black box).

(G) ﻿YFP-B56α pull down from cells stably expressing the indicated LxxIxE binding pocket variants of B56α and subsequent immunoblotting of indicated proteins. Representative of 4 independent experiments.
A highly conserved pocket on PP2A-B56 is required for hSgo1 binding and cohesion protection during mitosis (2021)
Figure 2

Figure 2. hSgo1 binding to the LxxIxE binding pocket of PP2A-B56 is required for cohesion protection

(A) Structure of the reported PP2Aγ-B56-hSgo1 binding interface (top) and residues mutated in the B56α 5A mutant are shown (bottom).

(B) IP of YFP-B56α from cells stably expressing the B56α WT, R222E, and 5A followed by immunoblotting of indicated proteins. Representative blots are shown (top). hSgo1 signals were normalized to YFP and plotted (bottom). Error bars represent SD (n=4).

(C) Reciprocal IP of (B). YFP-hSgo1 expression construct was transfected into cells stably expressing FLAG-B56α WT, R222E and 5A, followed by YFP IP and immunoblotting of indicated proteins. Representative blots are shown (top). B56α signals were normalized to YFP and plotted (bottom). Error bars represent SD (n=4).

(D) Representative images of chromosome spreads from the indicated conditions. Scale bar, 5μm.

(E) Quantification of (D). The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs and averaged for a single cell and plotted. The data are from 4 independent experiments and the mean and SD are indicated.

(F) hSgo1 RNAi and rescue with the indicated B56α variants fused to YFP and the Cenp B centromere-targeting domain (CB). Representative images of chromosome spreads are shown. CB targets all the rescue constructs (green) to the centromere. Scale bar, 5μm.

(G) Quantification of (F). The distance between the two peak intensities of YFP was measured for 5 kinetochore pairs and averaged for a single cell and plotted. The data are from 3 independent experiments and the mean and SD are indicated.
A highly conserved pocket on PP2A-B56 is required for hSgo1 binding and cohesion protection during mitosis (2021)
Figure 3

Figure 3. hSgo1 mutations in the coiled-coil domain affect PP2A-B56 binding

(A) Structure of the reported PP2A-B56γ-hSgo1 binding interfaces and residues mutated in the hSgo1 3A and 4A mutants are shown. 3A refers to Y57A, N60A, and K62A mutations at the PP2A-C binding interface. 4A refers to L83A, K87A, Y90A, and C94A mutations at the B56 binding interface of hSgo1.

(B-C) IP of YFP-hSgo1 from cells stably expressing the hSgo1 WT, 3A, and 4A using different salt conditions (50mM NaCl (B) or 150 mM (C)) followed by immunoblotting of indicated proteins and quantification by LiCor. Representative blots are shown. B56α signals were normalized to YFP and plotted. Error bars represent SD (n=3 for B and n=4 for C).

(D) Localization of B56α in cells depleted of hSgo1 and expressing the indicated YFP-hSgo1 variants. Representative immunofluorescent images are shown. Scale bar, 5μm.

(E) B56α signal intensity was quantified. B56α signal from each cell was determined from 5 kinetochore pairs and normalized to YFP-hSgo1 signal. Each circle represents an individual cell, and the average and SD are indicated.

(F) Mitotic U2OS LacO Haspin CM cells expressing hSgo1-LacI-GFP variants or LacI-GFP (control) were stained for PP2A-C. Scale bar, 5μm.

(G) PP2A-C signal intensity was quantified, normalized to GFP, and then plotted. Each circle represents an individual cell, and the mean fluorescent intensity is indicated. Representative of at least 3 independent experiments.

(H) Schematic of the TurboID-hSgo1 approach.

(I) Table summarizing the Log2 differences between hSgo1 WT, 3A and 4A.
A highly conserved pocket on PP2A-B56 is required for hSgo1 binding and cohesion protection during mitosis (2021)
Figure 4

Figure 4. Cohesion protection in hSgo1 mutants

(A) Representative images of chromosome spreads from hSgo1 RNAi treated cells stably expressing the indicated YFP-hSgo1 variants. All the YFP-hSgo1 rescue constructs (green) localize to the centromeres (CREST, red). Scale bar, 5μm.

(B) The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs from the chromosome spreads in (A) and averaged for a single cell. The data are from 3 independent experiments and the mean and SD are indicated.

(C) IP of YFP-hSgo1 from cells stably expressing hSgo1 WT, 3A, and 4A followed by immunoblotting of cohesin components, Smc1 and Smc3. Representative blots are shown (top). Signals were quantification by LiCor, normalized to YFP and plotted (bottom). Error bars represent SD (n=4 for Smc1 and n=3 for Smc3).

(D) Table summarizing the Log2 differences between hSgo1 WT, 3A and 4A from quantitative MS analysis.

(E) Experimental protocol of the live cell imaging shown in (F).

(F) hSgo1 RNAi and rescue with the indicated hSgo1 RNAi-resistant constructs was performed. Representative still images captured during the live cell imaging showing DIC and YFP-hSgo1 WT, 3A, and 4A localization during mitosis. Time (min) from nuclear envelop breakdown (NEBD) is indicated. Scale bar, 15μm.

(G) The time from NEBD to anaphase was measured from 3 independent live cell imaging experiments. Each circle represents an individual cell, and the median is indicated. Note that the WAPL KD condition is incorporated from the experiment which is shown in full in Fig. EV4C for clarity.

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