EMBO/soda-nel huggingface dataset:
NBR1-mediated p62-liquid droplets enhance the Keap1-Nrf2 system (2019)
Figure 6

Figure 6. NBR1 is induced by oxidative stress.

(A) Immunoblot analysis. Primary hepatocytes were isolated from wild-type (wt), p62f/f; Alb-Cre (p62KO) and Nrf2f/f; Alb-Cre (Nrf2KO) mice. The hepatocytes were challenged with 10 μM sodium arsenite [As (III)] for 12 hrs. Cell lysates were prepared and subjected to immunoblot analysis with anti-NBR1 and anti-Actin antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05, and **P < 0.01 as determined by Welch's t-test.

(B) Real-time PCR. Total RNAs were prepared from primary hepatocytes described in (A). Values were normalized against the amount of mRNA in each non-treated hepatocytes. The experiments were performed three times. Data are shown as means ± s.e. **P < 0.01 and ***P < 0.001 as determined by Welch's t-test.

(C) Immunofluorescence microscopy. Primary hepatocytes isolated from wild-type mice were challenged with 10 μM sodium arsenite [As (III)] for 12 hrs and then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. ***P < 0.001 as determined by Welch's t-test.
NBR1-mediated p62-liquid droplets enhance the Keap1-Nrf2 system (2019)
Figure 7

Figure 7. Deletion of NBR1 reduces p62-mediated Nrf2 activation under oxidative stress.

(A) Immunoblot analysis. Primary hepatocytes were isolated from Nbr1f/f mice and then infected with adenovirus GFP or Cre for 48 hrs. Thereafter, the hepatocytes were divided into 3 groups: 1. Cultured in regular medium (-), 2. Treated with 10 μM As (III) for 12 hr [As (III)] and 3. After treated with 10 μM As (III) for 12 hr, cultured in regular medium for another 12 hr (Wash). Cell lysates were prepared and subjected to immunoblot analysis with indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05, **P < 0.01, and ***P < 0.001 as determined by Welch's t-test.

(B) Real-time PCR. Total RNAs were prepared from primary hepatocytes described in (A). Values were normalized against the amount of mRNA in the Nbr1f/f hepatocytes expressing GFP under normal culture conditions. The experiments were performed at least three times. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.

(C) Immunofluorescence microscopy. Nbr1f/f hepatocytes were infected with LacZ or Cre adenovirus for 48 hrs and, when indicated, challenged with 10 μM As (III) for 12 hrs. The cells were then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. **P < 0.01, and ***P < 0.001 as determined by Welch's t-test.

(D) Role of NBR1 in the p62-liquid droplets and the p62-mediated Nrf2 activation. Oxidative stress up-regulates the level of NBR1, which enhances the phosphorylation and liquid-droplet formation of p62. The resulting p62-droplets are resistant to autophagic degradation and serve as signaling nodes for the activation of Nrf2. Since p62 is one of the targets of Nrf2, increased level of p62 in cooperation with NBR1 promote the formation of liquid droplets and contributes to the persistent activation of Nrf2 against oxidative stress.

Expanded View Figure Legends
Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases (2014)
f1

a, b, LDH release, IL-1&bgr; secretion (a) and immunoblots for caspase-1, caspase-11, IL-1&bgr;, IL-18 and IL-1&agr; (b) from unprimed BMDMs infected for 16 h with the indicated bacteria (grown to stationary phase). c, Time course measuring LDH release from unprimed or IFN-&ggr;-primed BMDMs infected with S. typhimurium. d, e, LDH release and IL-1&bgr; secretion from primed BMDMs infected with SPI-1-expressing logarithmic phase S. typhimurium, treated with monosodium urate, alum and nigericin or transfected with poly(dA:dT) and LPS. f, LDH release and IL-1&bgr; secretion from unprimed wild-type and Gbp2−/− BMDMs infected for 16 h with the indicated bacteria (grown to stationary phase). Graphs show mean and s.d. of quadruplicate wells and data are representative of two (b) and three (a, c–f) independent experiments. *Crossreactive band; **P  0.01; NS, not significant (two-tailed t-test).
Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases (2014)
f2

a, b, Quantification of live (mCherry-positive) and dead (mCherry-negative) S. typhimurium per cell by immunofluorescence (a) or as percent of total by flow-cytometry (b) in unprimed BMDMs at 16 h post-infection. c, Immunostaining for GBP2 and quantification of live and dead Salmonella at 4 h post-infection. Arrowheads, bacteria shown in insets. d, Quantification of GBP-positive bacteria, LDH release and IL-1&bgr; secretion at indicated time points from BMDMs infected with Salmonella, live or killed by different means. e, Immunohistochemistry for GBP2 and Salmonella on spleen tissue from Salmonella (mCherry-positive)-infected mice (representative of n = 3 per group). S. tm., S. typhimurium. f, g, Quantification of GBP-positive Salmonella in anti-IFN-&ggr;-treated or control animals (f) and live and dead bacteria among GBP2-negative/-positive Salmonella (g) (n = 3 per group). Scale bars, 10 &mgr;m (c), 1 &mgr;m (e). Graphs show mean and 5–95 percentile (box plots) or s.d. of technical triplicates, and data are representative of three independent experiments. *P  0.05, **P  0.01 (two-tailed t-test).
Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases (2014)
f3

a, b, Unprimed BMDMs infected with S. typhimurium for 4 h and immunostained for LC3 and GBP2. Arrowheads, bacteria shown in insets. Scale bars, 10 &mgr;m. c, Quantification of results from b. d–g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA). h, i, LDH release and IL-1&bgr; secretion from BMDMs infected for 16 h or transfected with LPS. Graphs show mean and s.d. of quadruplicate wells and data are representative of two (e, i) and three (a–d, f–h) independent experiments. *P  0.05, **P  0.01; NS, not significant (two-tailed t-test).
Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases (2014)
f4

a, Quantification of galectin-8-positive Salmonella in unprimed BMDMs at 4 h post-infection. b, c, Unprimed BMDMs infected with S. typhimurium for 4 h and immunostained for galectin-8, GBP2 and LC3. Arrowheads, bacteria shown in insets. Scale bars, 10 &mgr;m. d, Quantification of galectin-8/LC3-double-positive Salmonella at indicated time points post-infection. e–h, Quantification of cytosolic and vacuolar bacteria by flow cytometry in BMDMs infected with mCherry-positive S. typhimurium (e–g) or S. flexneri (h, wild-type or Dgr;T3SS) for 4 h. Graphs show mean and s.d. or 5–95 percentile (Box plots) of technical triplicates. Data are representative of 2 (g, h), 3 (a–d) and 4 (e, f) independent experiments. *P  0.05, **P  0.01 (two-tailed t-test).
Cell cycle dynamics of lamina associated DNA (2020)
Figure 2

Figure 2. pA-DamID can map NL interactions and correlates with microscopy observations.

(A) Example of raw pA-DamID data tracks from 2 million HAP-1 cells for Lamin B2, H3K27me3 and the Dam control from a single experiment. Sequenced reads are counted in 20kb bins and normalized for library size.

(B) Same pA-DamID tracks of Lamin B2 and H3K27me3 as in (A) but after normalization to the Dam-only control (to correct for accessibility and amplification biases) and log2-transformation.

(C) Correlation between the median peripheral enrichment of m6A-Tracer determined by confocal microscopy and enrichment of sequencing reads within LADs in HAP-1 cells. The LAD definition is based on conventional Lamin B1 DamID data. Every point represents a single pA-DamID experiment for which both microscopy and sequencing data were generated, colored by the antibody used. The blue line represents a linear model with a standard error confidence interval in grey. The red dashed line represents the diagonal. The Pearson correlation coefficient was converted to a t-statistic and a one-sided t-test with n-2 degrees of freedom was used to determine statistical significance.

(D) Comparisons of pA-DamID genome-wide data (bin size 20 kb) for different lamin antibodies in hTERT-RPE cells: Lamin B1 vs. Lamin B2 (left panel) and Lamin B2 vs. Lamin A/C (right panel). n denotes the number of independent biological experiments that were averaged for each data track. The blue line represents a linear model. The red dashed line represents the diagonal.
Cell cycle dynamics of lamina associated DNA (2020)
Figure 3

Figure 3. Comparison of NL interactions determined by pA-DamID and DamID.

(A) Comparison of pA-DamID and DamID data for for various lamins across a representative genomic locus in HAP-1 and hTERT-RPE cells. Arrow indicates a region with dissimilar pA-DamID and DamID scores.

(B) Genome-wide correlation of Lamin B1 pA-DamID and Lamin B1 DamID data. The blue line represents a linear model. The red dashed line represents the diagonal.

(C) Venn Diagram showing the overlap in LADs defined by a Hidden Markov Model. Results in (A-C) are based on averaged data from n independent experiments (see panel A), bin size 20 kb.

(D) Multidimensional scaling (MDS) plot (Robinson et al, 2010) showing all individual replicates of DamID and pA-DamID experiments for various lamins and cell types.
Cell cycle dynamics of lamina associated DNA (2020)
Figure 4

Figure 4. Mapping of NL interactions at various times after mitosis.

(A) Strategy for synchronization of hTERT-RPE cells. Cells were first enriched in G2 phase by 16 hours incubation with 2.0 µg/mL RO-3306, washed, and incubated for another 1.5 hours with 25 ng/mL nocodazole to arrest cells in metaphase. Metaphase cells were then collected by mitotic shake-off. A small fraction was used to determine the proportion of mitotic cells by DAPI staining and confocal microscopy (Fig. EV3A), while the remainder was replated for pA-DamID time series analysis.

(B) NL interactions in hTERT-RPE cells at indicated times after mitosis for chromosome 2, as determined by Lamin B2 pA-DamID. Log2-ratios were convered to z-scores to account for differences in dynamic range between replicates (Fig. EV3C). Average of two independent biological replicates. LADs are colored by their differential class (down:red, stable:grey, up:green, see panel C). The centromere is shown as grey box. These genome-wide data are also used for the analyses in (C-I).

(C) pA-DamID signal as a function of time after mitosis, for stable (grey, n=717) and dynamic LADs (red and green: decreasing (n=234) and increasing (n=225) NL interactions, respectively). Lines represent mean signals of the three LAD classes and the shaded area one standard deviation on both sides.

(D) Rate of changes in NL interactions (average pA-DamID z-score per hour) as function of time after mitosis for stable and dynamics LADs (see panel C). Boxplots: horizontal lines represent 25th, 50th and 75th percentiles; whiskers extend to the smallest values no further than 1.5 times distance between 25th and 75th percentiles.

(E-G) LAD size distribution in basepairs (E), density of active genes (F) and Repli-seq score distributions (G) for stable and dynamic LADs (see panel C). Statistical significance was determined using a two‐way Wilcoxin test with Benjamini-Hochberg p-value correction to account for multiple testing. Boxplot specification as in (D).

(H) Difference in pA-DamID z-scores between 1h and 21h time points, plotted against the distance to telomeres (leftpanel) or centromeres (rightpanel). Each dot represents a single LAD. Chromosomes are colored and ordered by size. Red line is a fitted loess curve. The Spearman correlation coefficient (ρ) was used to test for a significant monotonic, non-linear association.

(I) Similar as (H), but showing pA-DamID z-scores for individual time points.
Cell cycle dynamics of lamina associated DNA (2020)
Figure 5

Figure 5. LAD dynamics in other cell types.

(A) Lamin B2 pA-DamID z-scores of a representative locus in HAP-1 cells sorted for G1, mid-S and G2 phases. Arrows point to regions that lose NL interaction from G1 to G2. The red star indicates the end of the chromosome. Average of three biological replicates.

(B) Similar plot as Fig. 4H, showing the difference in pA-DamID z-scores between G2 and G1 sorted pools as function of distance to either telomere or centromere for HAP-1 (n=3), HCT116 (n=2) and K562 cells (n=2).

(C-E) LAD size distribution in basepairs (C), density of active genes (D) and Repli-seq scores (E) for stable and dynamic LADs. Repli-seq data are not available for HAP-1 cells. Statistical significance was determined using a two‐way Wilcoxin test with Benjamini-Hochberg p-value correction to account for multiple testing. Boxplots: horizontal lines represent 25th, 50th and 75th percentiles; whiskers extend to the smallest values no further than 1.5 times distance between 25th and 75th percentiles.

(F) Overlapping fractions of stable and dynamic LADs and iLADs with Hi-C subcompartments in HAP-1 and K562 cells, as imputed by SNIPER (Xiong & Ma, 2019). A permutation test with 1000 permutations (R-package regioneR (Gel et al, 2016)) was used to test for a significant enrichment of B1 subcompartments in decreasing LADs, resulting in P-values of 1e-3 and 1e-3 for HAP-1 and K562, respectively.
Cell cycle dynamics of lamina associated DNA (2020)
Figure 6

Figure 6. Mid-S chromatin has transiently increased Lamin B2 interactions.

(A) Representative K562 locus comparing Lamin B2 pA-DamID z-scores in cell cycle sorted cells with replication timing. Arrows point to regions with increased mid-S signal compared to G1 and G2, which tend to overlap with regions that exhibit intermediate replication timing. Average of two biological replicates.

(B) Difference tracks in pA-DamID z-scores between the cell cycle stages for the same locus as (A). Data were smoothed by a running mean of 5 bins (bin size 20 kb).

(C) Scatterplots of Repli-seq vs. the difference in smoothed pA-DamID z-scores between mid-S sorted cells and either G1 or G2 sorted cells for K562 and HCT116 cells (two replicates). Bins with pA-DamID differences between G1 and G2 bigger than 0.2 were filtered out to prevent artifacts from cell cycle dynamics. Blue line shows a fitted loess curve. The Spearman correlation coefficient (ρ) between the absolute Repli-seq signal and pA-DamID difference was used as a measure of the enriched signal at mid-S replicating DNA (with a Repli-seq score around zero).

(D) Scatterplots of changes in pA-DamID signal versus the pA-DamID signal in G1 (left-hand panels) or G2 cells (right-hand panels), represented in a similar way as (C). Repli-seq data are from the 4D Nucleome data repository (https://data.4dnucleome.org) (Dekker et al., 2017).

Expanded View figure legends
ALS-linked Protein Disulfide Isomerase Variants cause motor dysfunction (2016)
Figure 2

Figure 2. Motoneuron morphology upon expression of ALS-linked PDI variants.

A NSC34 cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant PDIs. After 48 h, overexpressed PDI variants was assessed under reducing conditions in an 8% SDS-PAGE. An antibody detecting total PDIA1 (endogenous mouse PDIA1 and exogenous human V5-tagged PDIA1) (first panel). A second antibody detects only human PDIA1, therefore only V5-tagged PDIA1 appears (second panel). A mouse and human specific antibody was used to detect total ERp57 (third panel). Anti-V5 was used to detect the exogenous PDI variants. Anti-β-actin was used as a loading control.

B and C NSC34 cells were transiently transfected with the indicated PDIs constructs together with a GFP expression plasmid. Cell were then differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation. Increased neurite outgrowth is indicated with white arrow heads. B Quantification of the average primary neurite lengths was performed, all cells from three independent experiments were compiled. A minimum of 100 cells per experiment was analyzed. In addition, C the percentage of cells with neurites was quantified in the three independent experiments (right panel).

D Primary rat ventral spinal cord neurons were prepared and after 4 days in vitro (DIV) transfected with GFP alone or together with the indicated PDIs constructs. Cells were fixed at 10 DIV and SMI32 staining was performed to identify motoneurons. Images were taken and the total outgrowth of GFP and SMI32 positive cells was quantified. Results are compiled from 3 independent experiments.

E Human motoneurons were differentiated from the human embryonic stem cell (ESC) HuESC 3 Hb9::GFP line. Differentiated neurons were transduced with lentivirus expressing GFP alone or together with PDIs-expressing plasmids. Transduced cells were cultured for another 10 days. To identify motoneurons after lentiviral transduction, immunocytochemistry analyses were performed. Quantification of neurite outgrowth in the different experimental conditions of GFP and HB9 positive neurons were obtained. Neurite number and assessment of the length of each neurite was performed in a similar manner as for primary rat motoneurons (see methods). Four independent experiments were performed.

F NSC34 cell lines stably knocked down for PDIA1 or ERp57 were differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation. The percentage of cells with neurites was quantified in the three independent experiments. A minimum of 100 cells per experiment was analyzed.

Statistical analyses were performed using one-way ANOVA and Bonferroni´s post hoc tests. Mean ± SEM with only statistically significant p values are shown: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
ALS-linked Protein Disulfide Isomerase Variants cause motor dysfunction (2016)
Figure 4

Figure 4. ALS-linked mutations in ERp57 lead to altered interactions with calnexin and calreticulin.

A PDI mutants form abnormal disulfide-dependent protein complexes. NSC34 cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant ERp57. After 48 h, differential disulfide-dependent interactions/aggregations of overexpressed PDI variants was assessed under reducing (+DTT) and non-reducing (-DTT) conditions in an 8% SDS-PAGE. Anti-V5 was used for detection in Western blot.

B ALS-linked ERp57 variants were generated as recombinant proteins and then analyzed by circular dichroism (CD). Averages for CD spectroscopic scans of recombinant ERp57WT and mutants are shown.

C A thermal denaturation curve of ERp57WT and mutants was performed.

D Representative electrophoresis of Proteinase K treated ERp57 recombinant proteins.

E Surface Plasmon Resonance was performed to monitor the affinity of recombinant ERp57 for recombinant CRT P-domain. KD values are expressed as percentage of ERp57WT. Values are derived from four independent experiments. For absolute KD values please see Appendix Table S2.

FHEK293T cells were transfected with expression vectors for V5-tagged wild-type and mutant ERp57, as well as empty vector. After 48 h, V5-tagged proteins were immunoprecipitated and eluted with V5 peptide. The interaction with endogenous calnexin (CNX) and calreticulin (CRT) was analyzed by Western blot. The inputs and elutions are shown. Right panel: Quantification of the degree of interaction is presented.

G The gain of an N-glycosylation site of ERp57D217N was predicted after the analysis of the protein sequence since the change of Asp217 to an Asn creates the NXT/S consensus sequence. Neuro2a cells were transfected with expression vectors for V5-tagged wild-type and mutant ERp57, as well as empty vector and treated with 1 µg/ml tunicamycin (Tm) for 20 h to inhibit N-glycosylation. Alternatively, protein extracts were digested with PNGase F and the possible removal of the N-glycosylation was analyzed by Western blot using anti-V5 antibody.

Statistical analyses were performed using Student’s t-test in panels E and F. Mean ± SEM with p values: n.s., p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
ALS-linked Protein Disulfide Isomerase Variants cause motor dysfunction (2016)
Figure 5

Figure 5. ERp57 deficiency in the nervous system leads to motor dysfunction and premature death.

A ERp57flox/flox mice were crossed with Nestin Cre transgenic mice to generate nervous system specific ERp57 deficient animals. The levels of ERp57 protein in the spinal cord were monitored by Western blot. ERp57WT (n=4), ERp57Nes+/- (n=5) and ERp57Nes-/- (n=4) mice. HSP90 levels were determined as a loading control. Right panel: Quantification of ERp57 levels was performed relative to Hsp90 levels.

B Body weight measurements were performed for indicated time points in ERp57WT (n=50), ERp57Nes+/- (n=32) and ERp57Nes-/- (n=19) mice.

C Rotarod performance was performed ERp57WT (n=20), ERp57Nes+/- (n=15) and ERp57Nes-/- (n=8) mice.

D Hanging test performance was assessed in ERp57WT (n=41), ERp57Nes+/- (n=32) and ERp57Nes-/- (n=12) mice.

E Kaplan-Meier survival curve for ERp57Nes-/- mice (N = 19) that prematurely died or had to be sacrificed because of health reasons between the ages 22 and 73 days. Mean survival of this subgroup of animals was 57 days. ERp57WT (n=50) and ERp57Nes+/- (n=32) mice are shown as a reference.

F Histological analysis of NeuN and GFAP staining was performed in spinal cord tissue from ERp57WT and ERp57Nes-/- mice in three animals per group using indirect immunofluorescence. The nucleus was stained with Hoechst. Representative images from one mouse per group are shown. Scale bar: 50 μm.

G Stereological analysis of the spinal cord from ERp57WT (n = 4), ERp57Nes+/- (n = 4) and ERp57Nes-/- (n = 4) mice. Alternate series of sections from the spinal cord of the mice were either stained for Nissl (top row images) or processed for immunohistochemistry for the cholinergic cell marker Choline Acetyl Transferase (ChAT, bottom row images). The nucleoli of the motoneurons, as stained in the Nissl series, were counted inside the motoneurons pools previously defined using the adjacent ChAT series (contours not shown). Cell densities of the three genotypes are shown on the right plots. No significant differences were found between the genotypes. Scale bar represents 200 μm and 50 μm on large and inset images, respectively.

H Representative images of the spinal cord myelination of ERp57WT and ERp57Nes-/- mice.

For statistical analysis two-way ANOVA with Bonferroni´s post hoc test was performed in B and one-way ANOVA with Bonferroni´s post hoc test was performed in A, C and D. Mean ± SEM with p values : n.s., non-significant, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.