EMBO/soda-nel huggingface dataset:
ALS-linked Protein Disulfide Isomerase Variants cause motor dysfunction (2016)
Figure 6

Figure 6. ERp57 deficiency results in loss of neuromuscular connectivity.

A Representative EMG recordings of ERp57WT (n=16), ERp57Nes-/+ (n=12) and ERp57Nes-/- (n=5) mice is presented. The presence of positive sharp waves (PSW) in ERp57Nes+/- and ERp57Nes-/- mice indicated muscle denervation.

B Analysis of NMJ and muscle morphologies were performed from ERp57WT (n=5), ERp57Nes+/- (n=5) and ERp57Nes-/- (n=4). Quantifications of endplate width, measuring the most ventral region of the diaphragms every 134 µm considering both sides of the innervation profile. Twenty to forty measurements per diaphragm were obtained per animal.

C Whole-mounted diaphragms from ERp57WT and ERp57Nes-/- mice were co-stained with anti-neurofilament (red) and α-BTX to reveal the postsynaptic densities (green). 3D reconstructions (lower panel) of higher magnification. ERp57WT NMJs are fully innervated pretzel-like, whereas ERp57Nes-/- NMJs display less complex postsynaptic densities and an incomplete or aberrantly distributed innervation pattern (Movies EV4-7).

D Representative 3D reconstructions of z-stacks showing the five categories in which NMJs morphologies were grouped: Pretzel, fragmented, O shaped, C shaped, and dismantled NMJs. Histogram of the distribution frequency of >200 different NMJ per animal is shown.

E Quantitative morphometry of >60 NMJs in the different genotypes analyzed.

For statistical analysis one-way ANOVA with Bonferroni´s post hoc test was performed in B, D, and E. Mean ± SEM with p values: n.s., non-significant, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
ALS-linked Protein Disulfide Isomerase Variants cause motor dysfunction (2016)
Figure 7

Figure 7. ERp57 deficient mice display aberrant skeletal muscle fibers.

A The levels of ERp57 protein in the muscle were monitored by Western blot. ERp57WT (n = 4), ERp57Nes+/- (n = 5) and ERp57Nes-/- (n = 4) mice. HSP90 was determined as loading control.

B Tibialis anterior muscles from ERp57WT (n = 5), ERp57Nes+/- (n = 5), and ERp57Nes-/- (n = 4) mice were dissected and cryosectioned (20 µm) for the analysis of several parameters of muscle physiology. Upper panel: hyaluronic acid (HA) staining was performed to assess the gross morphology of the muscle. Middle panel: Bismarck brown staining to monitor skeletal muscles fibrosis. Lower panel: Cryosections were then double stained with wheat germ agglutinin (WGA) to visualize plasma membrane and nuclei. The absence of central nuclei in all genotypes suggests that ERp57 deficiency does not affect degeneration/regeneration cycles. Images are representative of each genotype and where acquired in the same anatomical region of the TA muscles.

C Tibialis anterior cryosections (20 µm) from ERp57WT, ERp57Nes+/-, and ERp57Nes-/- mice were stained for NADH-TR activity to identify oxidative myofibers (upper panel). Representative micrographs of transversal cryosections show variable proportions of light, intermediate and dark positive staining in the different genotypes: (middle panel) NADH-TR positive (slow-twitch) and negative (fast-twitch) myofibers were quantified as percentage of total for every genotype (lower panel) The mean diameter of slow- and fast-twitch muscle fibers was evaluated at the light microscope using the ImageJ software. Data represent the average ± SEM of >300 fibers per animal.

Scale bars represent 50 m in B and C.

For statistical analysis one-way ANOVA with Bonferroni´s post hoc test was performed in C. Mean ± SEM with p values: n.s., non-significant, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
ALS-linked Protein Disulfide Isomerase Variants cause motor dysfunction (2016)
Figure 8

Figure 8. Loss of ERp57 in the cortex results in lower levels of the synaptic protein SV2.

A The levels of synaptic proteins SV2, NMDAR2A, PSD95 and Synaptophysin in the cortex were monitored by Western blot. ERp57WT (n = 4), ERp57Nes+/- (n = 5) and ERp57Nes-/- (n = 4) mice. HSP90 and β-Actin were determined as loading controls.

B Quantification of SV2 monomer (left panel) and SV2 high molecular weight species (right panel). β-Actin was used as the reference.
Nuclear IL-33/SMAD signaling axis promotes cancer development in chronic inflammation (2021)
Figure 1

Figure 1. IL-33 promotes skin cancer development in an ST2-independent manner.

(A-C) Colorectal tumor outcomes in WT (n=7), IL-33KO (n=4) and ST2KO (n=3) mice at the completion of the AOM/DSS carcinogenesis protocol. (A) Tumor volumes at the endpoint (each dot represents a tumor, mice with no tumor are marked as zero), (B) representative images of the exposed colonic lumen (scale bar: 1 cm), and (C) representative images of H&E-stained distal colon in each group (scale bar: 100 μm).

(D-F) Skin tumor outcomes in WT (n=7), IL-33KO (n=6) and ST2KO (n=7) mice at the completion of the DMBA/DNFB carcinogenesis protocol. (D) Tumor volumes at the endpoint (each dot represents a tumor, mice with no tumor are marked as zero), (E) representative images of the back skin (yellow circles highlight the skin tumors, scale bar: 1 cm), and (F) representative images of H&E-stained back skin of the mice in each group (scale bar: 100 μm).

(G) Representative images of IL-33 immunostaining on WT skin treated with DNFB or acetone control. Arrows in the inset highlight IL-33+ nuclei in epidermal keratinocytes (scale bar: 100 μm).

(H) Quantification of nuclear IL-33+ epidermal and dermal cells in the DNFB- versus acetone-treated WT skin. Dots represent cell counts from three randomly selected high power field (HPF) images per sample (n=5 per group).

(I) Representative images of IL-33 immunostaining on WT colon at the completion of the AOM/DSS carcinogen protocol compared with no treatment control. Arrows in the inset point to IL-33+ stromal cells and arrowheads point to lack of IL-33 nuclear stain in colonic epithelial cells (scale bar: 100 μm).

(J) Quantification of nuclear IL-33+ epithelial and stromal cells of colon treated with AOM/DSS versus no treatment. Dots represent cell counts from three randomly selected HPF images per sample (n=4 per group).

Data information: Graphs show mean + SD, unpaired t-test.
Nuclear IL-33/SMAD signaling axis promotes cancer development in chronic inflammation (2021)
Figure 2

Figure 2. Nuclear IL-33 blocks Smad6 expression via interaction with RUNX2 transcription factor.

(A) Heatmap of differentially expressed genes between IL-33KO (n=3) and WT (n=3) epidermis after topical treatment with DNFB. Representative genes in differentially affected biological pathways are listed on the heatmap (the full list of differentially expressed genes is shown in Appendix Table S1).

(B) Schematic of IL-33 full length and IL-33 cytokine domain constructs (top). Venn diagram of differential genes shared between RNA-Seq and ChIP-Seq results (bottom). For ChIP-Seq, Pam212 cells transfected with IL-33 full length versus IL-33 cytokine domain were compared. Any gene with a peak in HA-empty vector control was excluded from the final analysis.

(C) RNA-Seq result of the seven differential genes shared between RNA-Seq and ChIP-Seq results. Significance analysis comparing the two groups was performed using DESeq2 R package.

(D) Il33 and Smad6 expression levels in DNFB-treated skin relative to acetone-treated controls (n=9 in DNFB group, n=7 in acetone group for Il33, n=10 in DNFB group, and n=9 in acetone group for Smad6).

(E) Smad6 and Pdcd4 (negative control gene) expression levels upon IL-33 full length versus cytokine domain expression compared with HA-empty vector expression in Pam212 cells (n=4 in each group).

(F) Immunoblot for interaction between endogenous IL-33 and RUNX2. Lysate from PyMT and Pam212 cell lines were subjected to immunoprecipitation with an anti-IL-33 antibody followed by immunoblot analysis. Immunoprecipitation with an anti-IgG antibody on an equal amount of PyMT lysate is shown as a negative control. Data represent two independent experiments with similar results.

(G) Immunoblot for interaction between nuclear IL-33 and RUNX2. After Pam212 cells were transfected with IL-33 full length or cytokine domain for 24 hours, cell lysates were subjected to immunoprecipitation with anti-IL-33 antibody followed by immunoblot analysis.

(H) ChIP-qPCR assay for Smad6 in the presence of IL-33 full length or cytokine domain using an anti-RUNX2 antibody. After Pam212 cells were transfected with IL-33 full length or cytokine domain for 24 hours, cell lysates were subjected to chromatin-immunoprecipitation with anti-RUNX2 antibody and eluted RUNX2-bound chromatin was used for qPCR with primers against Smad6 promoter region. Anti-IgG ChIP-qPCR results are shown as negative controls (n=4 per group).

Data information: GAPDH is used as the control housekeeping protein in (F) and (G). Graphs show mean + SD, NS: not significant, unpaired t-test.
Nuclear IL-33/SMAD signaling axis promotes cancer development in chronic inflammation (2021)
Figure 3

Figure 3. Nuclear IL-33 promotes cell proliferation by upregulating TGF-β/SMAD signaling pathway.

(A) Immunoblot of p-SMAD2/3, p-SMAD1/5, SMAD6, SMAD2/3 and SMAD1 proteins in DNFB- versus acetone-treated WT skin (n=4 in each group).

(B) Quantitative PCR (qPCR) of Il33 and Smad6 mRNA in Pam212 cells treated with poly (I:C) or PBS (carrier control, n=4 in each group).

(C) ChIP-qPCR assay for Smad6 in the presence of poly (I:C) or PBS using an anti-IL-33 antibody. After Pam212 cells were transfected with poly (I:C) for 24 hours, cell lysates were subjected to chromatin-immunoprecipitation with anti-IL-33 antibody and eluted IL-33-bound chromatin was used for qPCR with Smad6 regulatory peak region (63,968k) primers and Smad6 no peak region (63,955k) primers (n=4 in each group).

(D) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins in response to TGF-β and poly (I:C) treatment. Pam212 cells were treated with poly (I:C) or PBS followed by incubation with and without TGF-β (5nM). Data represent three independent experiments with similar results.

(E) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins in response to TGF-β + poly (I:C) after knocking down of IL-33. Pam212 cells were transfected with siIl33 knockdown construct or siRNA control (siCon) for 30 hours followed by incubation with poly (I:C) and TGF-β. Data represent three independent experiments with similar results.

(F) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins upon the expression of IL-33 full length or cytokine domain. Pam212 cells were transfected with IL-33 full length or cytokine domain for 24 hours followed by incubation with and without TGF-β. Data represent three independent experiments with similar results.

(G) Impact of IL-33 knockdown on cell proliferation in response to TGF-β. Pam212 cells were treated with siIl33 or siCon followed by TGF-β treatment (n=7 in each group).

(H) Impact of IL-33 knockdown and SB431542 treatment on cell proliferation. Pam212 cells were treated with siIl33 or siCon in combination with SB431542 or DMSO (carrier control, n=7 in each group).

(I) Representative images of H&E- and Ki67-stained back skin of IL-33KO and WT mice treated with 22-day DNFB protocol in combination with SB431542 or acetone (carrier control). Note the epidermal thickness, keratinocytes proliferation and dermal inflammation in each group (scale bars: 100 μm).

(J) The epidermal thickness of DNFB-treated IL-33KO and WT skin after treatment with SB431542 or acetone. Each dot represents the average of three measurements in an HPF image. 10 random HPF images per skin sample are included (n=10 for WT with acetone, n=8 for WT with SB431542, n=6 for IL-33KO with acetone, n=5 for IL-33KO with SB431542 per group).

Data information: GAPDH is used as the control housekeeping protein in (A, D-F). Graphs show mean + SD, NS: not significant, unpaired t-test.
Nuclear IL-33/SMAD signaling axis promotes cancer development in chronic inflammation (2021)
Figure 4

Figure 4. Nuclear IL-33/SMAD signaling promotes skin cancer development.

(A) Representative images of IL-33 immunostaining on WT skin. Inset highlights the IL-33-negative nuclei in the epidermis (scale bar: 100 μm).

(B-D) Skin tumor outcomes in WT mice treated with DMBA/TPA carcinogenesis protocol in combination with SB431542 (test, n=9) or acetone (carrier control, n=5). (B) Tumor latency (log-rank test), (C) skin tumor counts per mouse over time, and (D) skin tumor volume at the completion of the DMBA/TPA carcinogenesis protocol.

(E) Representative images of IL-33 immunostaining on K14-IL33tg,ST2KO skin. Inset highlights the IL-33-positive nuclei in the epidermis (scale bar: 100 μm).

(F-H) Skin tumor outcomes in K14-IL33tg,ST2KO mice treated with DMBA/TPA carcinogenesis protocol in combination with SB431542 (test, n=6) or acetone (control, n=5). (F) Tumor latency (log-rank test), (G) skin tumor counts per mouse over time, and (H) skin tumor volume at the completion of the DMBA/TPA carcinogenesis protocol.

(I) SMAD signaling proteins levels in WT and K14-IL33tg,ST2KO skin treated with acetone (control) at the completion of the DMBA/TPA carcinogenesis protocol. Tissue lysates prepared from the whole back skin of the animals were subjected to immunoblot with p-SMAD2/3, p-SMAD1/5, SMAD6, SMAD2/3 and SMAD1 antibodies. GAPDH is used as the control housekeeping protein (n=5 in each group).

(J) Representative images of IL-33 and p-SMAD2/3 immunostaining on the adjacent sections of human squamous cell carcinoma (SCC) and normal skin (scale bar: 100 μm).

Data information: Graphs show mean + SD, NS: not significant, unpaired t-test.
Nuclear IL-33/SMAD signaling axis promotes cancer development in chronic inflammation (2021)
Figure 5

Figure 5. Nuclear IL-33 promotes pancreatic cancer development.

(A) Representative images of IL-33 and p-SMAD2/3 immunostaining on WT pancreas treated with caerulein or PBS (carrier control) to induce chronic pancreatitis (scale bar: 100 μm).

(B) Il33 and Smad6 mRNA levels in caerulein-treated WT pancreas compared with PBS-treated controls at the completion of chronic pancreatitis protocol (n=8 in caerulein and n=9 in PBS group for Il33, n=9 in caerulein and n=10 in PBS group for Smad6, unpaired t-test).

(C) Immunoblot for p-SMAD2/3, p-SMAD1/5, SMAD6, SMAD2/3 and SMAD1 proteins in lysates from caerulein (n=3) and PBS (n=4) treated WT pancreas. GAPDH is used as the control housekeeping protein.

(D) Pancreatic tumor-free survival of WT (n=4), IL-33KO (n=5) and ST2KO (n=6) mice at the completion of the DMBA/caerulein carcinogenesis protocol (log-rank test).

(E) Representative H&E images of two pancreatic tissues from WT, IL-33KO and ST2KO groups treated with DMBA/caerulein (scale bar: 100 μm).

(F) Representative images of IL-33 and p-SMAD2/3 immunostaining on adjacent sections of matched normal pancreas, chronic pancreatitis and PDAC collected from pancreatic cancer patients (scale bar: 100 μm).

(G, H) Quantification of nuclear (G) IL-33+ and (H) p-SMAD2/3+ epithelial cells per HPF in the matched human pancreatic tissues. Each dot represents the average cell counts across three randomly selected HPF images per sample (n=18 patients, paired t-test).

(I) Correlation between IL-33+ and p-SMAD2/3+ epithelial cell counts across normal pancreas, chronic pancreatitis and PDAC samples (n=54 matched samples from 18 patients, t-test for the Pearson correlation coefficient).

Data information: Graphs show mean + SD.
A human coronavirus OC43-derived polypeptide causes neuropathic pain (2022)
Figure 7

Figure 7. OC43p affinity to the Na+/K+-transporting ATPase complex

A Mass spectrometry identification of the Atp1 family of proteins captured by biotinylated OC43p peptide in rat dorsal spinal cords from female rats (n=2/groups). Negative log10P - confidence score and peptide sequence coverage (%) were calculated in PEAKS Studio™ 8.5 (BSI).

B Relationship of ATPase subunits identified in string-db.org. Lines indicate evidence-based interactions.

C ATPase gene family expression in sciatic nerves, DRG and spinal cords of naïve rats. TPM (Transcripts Per Kilobase Million) measures the transcription frequency of a specific gene by RNA-seq (n=3/group). Blue to red color intensity corresponds to Log2(TPM).

D, Affinity capture of proteins by OC43p and OC43-SCR bound magnetic beads. Specific reactivity to Atp1a1 and Atp1a2 was detected by protein immunoblotting using specific primary antibodies. OC43p-SCR-coated and uncoated beads (Beads) were used to control non-specific binding. Spinal cords were isolated from male (M) and female (F) rats (n=2/group). Input protein lysates (right side, in duplicates) served as loading controls. Approximate molecular weights (in kDa) are indicated by arrows.

Expanded View Figure Legend
Compound heterozygous variants in OTULIN are associated with fulminant atypical late-onset ORAS (2022)
Figure 1

Figure 1. Sterile abscess formation in a patient with compound-heterozygous missense variants in the OTULIN gene

(A) Blood parameters of patient are depicted. (B) Patient's skin alterations are depicted. (C) T2-weighted MR images in coronal plane show abscess formation in the left lower pulmonary lobe (upper panel) and in spleen and left axilla (lower panel). (D) Histological sections of patient biopsies stained with hematoxylin and eosin (left panel) or with an actin antibody (right panel). Scale bars: skin: left 1000µm, right 100µm; lung: left 500µm, right 250µm; spleen: left 1000µm, right 75µm. (E) Whole Exome Sequencing (WES) and targeted Sanger sequencing identified compound heterozygous variants in OTULIN at position c.258 G>A, p.M86I on the paternal allele and at position c.500 G>C, p.W167S on the maternal allele. (F) The pedigree is depicted. The patient (II-2) is the second child of non-consanguineous parents.
Compound heterozygous variants in OTULIN are associated with fulminant atypical late-onset ORAS (2022)
Figure 2

Figure 2. The compound-heterozygous missense variants p.M86I and p.W167S affect OTULIN protein expression and function

(A) Expression of indicated proteins was determined by western blot in patient-derived and control fibroblasts and B cells and A549 OTULIN WT and KO cells. (B) FLAG-tagged tandem ubiquitin binding entity (TUBE) assay was performed to pull down linear ubiquitin linkages in patient-derived and control fibroblasts and B cells. One representative (A, B) out of three independent experiments is shown.
Compound heterozygous variants in OTULIN are associated with fulminant atypical late-onset ORAS (2022)
Figure 4

Figure 4. OTULIN variants compromise binding of OTULIN to linear ubiquitin and differentially affect OTULIN's intrinsic stability and catalytic activity

Biochemical characterization of recombinant OTULIN variants by means of differential scanning fluorimetry (DSF) (A) and surface plasmon resonance (SPR) measurements (B). (A) DSF measurements with OTULINWT, OTULINM86I or OTULINW167S. Melting temperatures (Tm) are calculated from five independent experiments with standard deviations. *, P = 0.011; ****, P = 6.64x10-18, unpaired t-test. (B) SPR measurements and steady-state binding curves with calculated dissociation constants (Kd) after injection of a concentration series of OTULINC129A, OTULINC129A/M86I or OTULINC129A/W167S to CM5-immobilized di-ubiquitin chains. (C) Linear ubiquitin linkages isolated from A549 OTULIN KO cells by M1 TUBE assay were incubated with increasing concentrations of recombinant OTULINWT, OTULINM86I and OTULINW167S or catalytically inactive OTULINC129A as control for 1 hour. Afterwards, samples were subjected to analysis by western blot for the indicated proteins. Images are representative of three independent experiments.
Compound heterozygous variants in OTULIN are associated with fulminant atypical late-onset ORAS (2022)
Figure 5

Figure 5. Parental variants both contribute to defective hydrolysis of linear ubiquitin linkages in patient-derived cells, but to different extents

(A - C) A549 OTULIN KO cells were transfected with empty vector or different OTULIN constructs as indicated and analyzed by western blot. (D) FLAG-tagged tandem ubiquitin binding entity (TUBE) assay was performed in B cells from control, patient, mother and father to pull down linear ubiquitin linkages. One representative (A - D) of three independent experiments is shown.
Compound heterozygous variants in OTULIN are associated with fulminant atypical late-onset ORAS (2022)
Figure 6

Figure 6. TNF signaling is altered in patient-derived cells

(A - D) Patient-derived and control fibroblasts were used. (A) FLAG-tagged tandem ubiquitin binding entity (TUBE) assay was performed to pull down linear ubiquitin linkages upon stimulation with 500ng/ml TNF. (B) The TNFR1-SC was isolated using 500ng/ml TAP-TNF. (C) Cells were stimulated with 100ng/ml TNF for the indicated times and analyzed by western blot. One representative (A - C) of three independent experiments is shown. (D) Cells were stimulated with TNF for 24 hours and IL-6 was determined by ELISA. Data are presented as mean ± SD of 3 individual experiments performed in two technical replicates; *, P = 0.014; **, P = 0.007; multiple t-test corrected for multiple comparisons using the Holm-Sidak method. (E) B cells were incubated with 50µg/ml cycloheximide (CHX) for the indicated times and subjected to analysis by western blot. One representative (left panel) out of 4 individual experiments analyzed by densitometry (right panel) is shown. Data are presented as mean ± SD. *, P = 0.02, unpaired t-test. (F) Patient-derived and control B cells were cultivated for 24 hours and TNF was determined by ELISA. Data are presented as mean ± SD of 3 individual experiments performed in two technical replicates; *, P = 0.028; unpaired t-test. (G) Fibroblasts were incubated with 50µg/ml cycloheximide (CHX) or DMSO and stimulated with TNF as indicated. Viability was measured after 24 hours. Data are presented as mean ± SD of 5 individual experiments performed in three technical replicates; ***, P = 0.0008, mixed-effects analysis with Tukey's multiple comparisons test. (H) Intracellular TNF in CD11b-positive cells from five healthy controls, patient (before anti-TNF treatment), mother and father was determined by FACS.

Tables and their legends

Table 1. Compound heterozygous variants in the OTULIN gene in a patient with autoinflammation and sterile abscess formation

CDS, coding sequence; AA, amino acid; SIFT, Sorting Intolerant From Tolerant ( < 0.05 = deleterious); PolyPhen, Polymorphism Phenotyping ( > 0.908 "Probably Damaging", 0.446 - 0.908 "Possibly Damaging", < 0.446 "Benign")

Expanded View Figure legends
N-acetyl-L-tyrosine is an intrinsic triggering factor of mitohormesis in stressed animals (2020)
Figure 3

Fig. 3. Analysis of NAT-induced hormesis response in Drosophila larvae.

(A) Survival of Drosophila melanogaster (W1118) larvae fed with 2 µmol NAT/g-diet 2 d after heat stress at 38.5oC for 60 min (data are means ± SEM; n=10). **P<0.01 vs. zero time.

(B) Effects of NAT feeding on expression of antioxidant enzymes, catalase, SOD1, and SOD2 (data are means ± SEM; n=10). *P<0.05 vs. zero time.

(C) Representative images of nuclei (left) and FoxO (middle) in the fat body of Drosophila larvae before and after NAT feeding for 12 h. White signals in the merged image (right) show the overlap of both signals. Scale bar: 50 µm.

(D) Relative whiteness intensities before and after NAT feeding (data are means ± SEM; n=15). **P<0.01 vs. zero time.

(E) Effects of NAT feeding on FoxO expression in the fat body of Drosophila larvae (data are means ± SEM; n=8). *P<0.05, **P<0.01 vs. zero time.

(F) Effects of N-acetyltransferase (Dat) knockdown in gut enteroendocrine cells (TK-Gut-Gal4>UAS-dsDat) on survival of Drosophila larvae (data are means ± SEM; n=15). **P<0.01 vs. without NAT.

Data information: Significant difference from each control value is indicated by Tuckey's HSD.