EMBO/soda-nel huggingface dataset:
Neutralization of pro-inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis (2016)
Figure 2

Figure 2. TLR2 inhibitor peptide physically interacts with its corresponding TLR.

(A) Biochemical analyses of peptide interaction with TLR. BM-derived macrophages were incubated with 1µM fluorescently labeled rhodamine (Rho)-TLR2-p or its mutant Rho-ScrTLR2-p peptide. After 1h of incubation at 370C the cells were lysated. Soluble fraction was used for immunoprecipitation with the indicated TLR antibodies. Proteins samples were resolved by SDS-PAGE and the presence of the labeled peptide was detected with a fluorescent scanner. Subsequently, the gel was transferred to a membrane and subjected to Western blotting for TLR6,1,2 and 4 in the appropriate samples. Equal loading was measured by detecting of anti-tubulin in the cell lysate. The results are presented as the mean of fluorescent intensity from two independent experiments. Non-specific binding of the peptides to G protein beads was subtracted. The results are mean± S.E.M of at least two independent experiments.

(B) Proteins analysis of secreted pro-inflammatory cytokines, IL-6 and TNF-α from primary CD115+ cells. Cells were incubated with 20µM of TLR2-p or scrTLR2-p peptide for 0.5h, then washed and incubated with LTA (5μg/ml), PAM3CSK (0.5μg/ml) or LPS (0.2μg/ml) at 370C for 5h (for TNF-α detection) and 22h (for IL-6 detection). The results are mean± S.E.M of two independent experiments (n=8).
Neutralization of pro-inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis (2016)
Figure 3

Figure 3. TLR2-p peptide directly inhibits the dimerization of TLR2 and TLR6 after LTA treatment resulting in reduced levels of ERK1,2 signaling.

(A) A scheme showing the FRET reaction.

(B-C) Represented images of cellular interaction between TLR2 and TLR6 (A) and between F4/80 and TLR2 (B) in BM-derived macrophages with the indicated treatments were observed by Fluorescence Resonance Energy Transfer (FRET) using ImageStreamX. Scale bars 10μm. Cells were incubated with 20µM of TLR2-p or scrTLR2-p peptide for 0.5h, then washed and incubated with 500ng/ml LTA for another 0.5h at 370C. Cells were probed with anti TLR6-PE or anti F4/80-PE conjugated antibody (donor) and anti TLR2 antibody following by staining with APC-labeled secondary antibody (acceptor). PE intensity (middle panel) and FRET intensity (right panel) were measured via ImageStreanX imaging flow cytometer. The FRET intensity shown are mean ± S.E.M of two independent experiments (n=2579-15567).

(D) Represented image of ERK1,2 phosphorylation levels in CD115+ BM monocytes cells upon LTA exposure. Cells were pre-treated for 0.5h with 20µM of TLR2-p peptide, scrTLR2-p peptide or untreated, then washed and incubated with 500ng/ml LTA for the indicated times. ERK1,2 phosphorylation levels and total ERK1,2 levels were detected by western blotting. Equal loading was detected by measuring anti tubulin. Results are representative data of 2 independent experiments. The bands intensity was quantified for p-ERK levels after 15min of LTA exposure. Results are normalized to tubulin levels and are the mean of 2 experiments ± S.D.M.
Neutralization of pro-inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis (2016)
Figure 4

Figure 4. TLR2-p inhibitor peptide ameliorated DSS induced colitis.

(A) A scheme showing peptide and DSS administration regime.

(B) Representative colonoscopy images of the indicated treatments (day 7). C57BL mice were treated with 2% of dextran sulfate sodium (DSS) in drinking water for 7 days. An amount of 5mg/Kg of TLR2-p and scrTLR2-p peptides were injected IP every following day from day one.

(C) A graphical summary of endoscopic colitis grades assessed on day 7 after treated with DSS only, DSS with TLR2-p peptide, DSS with scrTLR2-p peptide and untreated mice (n= 19, 21, 22 and 19 respectively). Results represent 3 independent experiments, scale bars 100µm and 1000μm.

(D) A graphical summary of changes in body weight (day 6) of the indicated groups.

(E) Representative H&E histological images for distal colon section (day 7) of 9-10 sections from 2 independent experiments. Black asterisk indicates on large collection of blood in the lumen, arrowheads indicate on widespread mucosal collapse and ulceration and arrows indicates on edema in the sub mucosa.

(F) A graphical summary of histological severity score of the indicated treatments.
Neutralization of pro-inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis (2016)
Figure 5

Figure 5. TLR2-p inhibitor peptide down regulates pro inflammatory cytokines in the colonic mucosa.

(A-B) Proteins and RNA samples of colon tissue were extracted from mice with the indicated treatments. (A) Cytokine levels of IL-6, IL-1β and INF-λ were determined by ELISA (n=4-7) and (B) mRNA expression levels of IL-6, IL-12p35 and IL-23p19 were assessed by quantitative RT-PCR and normalized to TBP (n=4-6). Results represent two or more independent experiments.
Neutralization of pro-inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis (2016)
Figure 6

Figure 6. TLR2 inhibitor peptide do not have an effect on the recruitment of pro inflammatory monocytes: (A) Flow cytometry analysis of colonic lamina propria CD11c- CD11b+LY6Chi MHCII-monocytes from steady-state and DSS day 7 treated with TLR2 peptide or scrTLR2 peptide, showing comparable monocytes infiltrates in to the colitic colon. Plots were pre gated on live CD45+ cells. Results are representative of three independent experiments (n=3 per group). (B) Graphical summary of monocytes presented as % out of CD11b + cells.
Neutralization of pro-inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis (2016)
Figure 7

Figure 7. TLR2-p inhibitor peptide down regulates pro inflammatory cytokines expressed by pro inflammatory monocytes:

(A) A scheme showing peptide and DSS administration regime.

(B) Weight loss of DSS challenged mice on day 6.

(C) Real-time PCR analysis showing mRNA expression levels of IL-6, IL-12p35 and IL-23p19 of FACS isolated monocytes collected from mice with the indicated treatments. Data are obtained from two to three independent experiments (n=6-7 mice pooled per group).
Paternal USP26 Mutations Raise Klinefelter Syndrome Risk in the Offspring of Mice and Humans (2021)
Figure 2

Figure 2 - Usp26-deficient mice produce 41, XXY offspring

A Sequences of the WT and Usp26 mutant alleles in mice.

B The USP26 protein was absent in Usp26-/Y testes. Immunoblotting of USP26 was performed in Usp26+/Y and Usp26-/Y testes. Histone 3 served as the loading control.

C, D The fertility of Usp26-/Y mice was reduced with increasing age. The fertility assessment experiments were performed in 2-month-old Usp26+/Y, Usp26-/Y mice (n =5 independent experiments) and 6-month-old Usp26+/Y, Usp26-/Y mice (n=5 independent experiments) (C). The litter sizes were observed in 2-month-old Usp26+/Y, Usp26-/Y mice (n=14, 22 independent experiments for Usp26+/Y and Usp26-/Y, respectively) and 6-month-old Usp26+/Y, Usp26-/Y mice (n=10, 8 independent experiments for Usp26+/Y and Usp26-/Y, respectively) (D). Red dots indicate Usp26+/Y mice and green dots indicate Usp26-/Y mice.

E Genotyping of Usp26-/Y mice offspring. The upper subpanel indicates the genotyping of Usp26-/Y mice male offspring by using Usp26 and Sry primers. The lower subpanel indicates the genotyping of Usp26-/Y, Usp26+/Y male mice and Usp26+/-, Usp26+/+ female mice by using Usp26 and Sry primers.

F The wild-type (WT) and Usp26 knock out alleles in XUsp26+XUsp26-Y mice.

G The proportion of 41, XXY mice among the male offspring of 2-month-old Usp26+/Y, Usp26-/Y and 6-month-old Usp26+/Y, Usp26-/Y mice (n=14, 22, 10 and 8 independent experiments for the offspring of 2-month-old Usp26+/Y, Usp26-/Y mice, 6-month-old Usp26+/Y, and Usp26-/Y mice, respectively). Red dots indicate Usp26+/Y mice and green dots indicate Usp26-/Y mice.

H, I Two pair of X chromosomes could be detected in the 2-month-old XUsp26+XUsp26-Y spermatocytes. FISH analysis of Chr X-FISH (green) and Chr Y-FISH (red) was performed in WT and XUsp26+XUsp26-YY spermatocytes (H). Immunofluorescence analysis of Chr X-FISH (green), Chr Y-FISH (red), and SYCP3 (white) was performed in WT and XUsp26+XUsp26-YY spermatocytes (I). The arrows indicate the Y chromosome and the arrowheads indicate the X chromosome. Nuclei were stained with DAPI (blue).

J-L Three sex chromosome axes could be detected in the 2-month-old XUsp26+XUsp26-Y spermatocytes. Immunofluorescence analysis of SYCP3 (red) and SYCP1 (green) in (J), ATR (red) and SYCP3 (green) in (K) and BRCA1 (red) and SYCP3 (green) in (L) was performed in 2-month-old WT and XUsp26+XUsp26-Y spermatocytes. Nuclei were stained with DAPI (blue).The arrows indicate the Y chromosome. The arrowheads indicate the X chromosome.

Data information: In (C), (D), and (G), data are presented as mean ± SD. *P < 0.05, **P < 0.01 (Student's t-test). n.s, not significant. Numerical source data for panels C/D/G can be found in Table EV5.
Paternal USP26 Mutations Raise Klinefelter Syndrome Risk in the Offspring of Mice and Humans (2021)
Figure 3

Figure 3 - The disruption of Usp26 impairs spermatogenesis.

A The size of the testes was smaller in the 6-month-old Usp26-/Y mice.

B Quantification of body weight in 6-month-old Usp26+/Y and Usp26-/Y mice (n=6 independent experiments). P=0.2442. Red dots indicate Usp26+/Y mice and green dots indicate Usp26-/Y mice.

C Quantification of testis weight in 6-month-old Usp26+/Y and Usp26-/Y mice (n=6 independent experiments). Red dots indicate Usp26+/Y mice and green dots indicate Usp26-/Y mice.

D Histological analysis of the seminiferous tubules and caudal epididymis of the 6-month-old Usp26+/Y and Usp26-/Y mice.

E The sperm counts in the caudal epididymis were measured in 6-month-old Usp26+/Y and Usp26+/-/Y mice (n= 6 and 5 independent experiments for Usp26+/Y and Usp26-/Y, respectively). Red dots indicate Usp26+/Y mice and green dots indicate Usp26-/Y mice.

F The deletion of Usp26 causes pachytene and meiotic division arrest. Representative PAS-hematoxylin staining in 6-month-old Usp26+/Y and Usp26-/Y seminiferous tubules. Parafﬁn sections from Usp26+/Y and Usp26-/Y testes were stained with PAS-hematoxylin. P: pachytene spermatocyte, D: diplotene spermatocyte, rST: round spermatid, eST: elongating spermatid, M: meiotic spermatocyte, aP: apoptotic pachytene spermatocytes, aM: abnormal meiotic divisions.

Data information: In (B), (C), and (E), data are presented as mean ± SD. *P < 0.05, **P < 0.01 (Student's t-test). n.s, not significant. Numerical source data for panels B/C/E can be found in Table EV5.
Paternal USP26 Mutations Raise Klinefelter Syndrome Risk in the Offspring of Mice and Humans (2021)
Figure 4

Figure 4 - USP26 participates in sex chromosome pairing.

A USP26 was predominately expressed in the testis. Immunoblotting of USP26 was performed in the heart, liver, spleen, lung, kidney, intestine, brain, ovary, and testis. Histone 3 served as the loading control.

B The localization of USP26 during meiosis. Immunofluorescence analysis of SYCP3 (red) and USP26 (white) was performed in wild-type spermatocytes. Nuclei were stained with DAPI (blue). The circles indicate the XY body.

C, D The sex chromosomes failed to synapse in the 6-month-old Usp26-deficient spermatocytes. Immunofluorescence analysis of SYCP3 (green), ATR (white), SYCP1 (red) (C); SYCP3 (green), ATR (white), TRF1 (red) (D) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrows indicate the sex chromosomes.

E The X and Y chromosomes were unpaired in Usp26-/Y spermatocytes. Immunofluorescence analysis of Chr X-FISH (green), Chr Y-FISH (red), and SYCP3 (white) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. The arrowheads indicate the X chromosome.

F Quantification of unpaired X and Y chromosomes in 6-month-old Usp26+/Y and Usp26-/Y mice (n= 5 independent experiments). Red dots indicate Usp26+/Y mice and green dots indicate Usp26-/Y mice.

G-J Sex chromosome recombination was perturbed in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of SYCP3 (green), MRE11 (red) (G); SYCP3 (green), γH2AX (red) (H); SYCP3 (green), ATR (red), p-ATM (pink) (I); DMC1 (green), SYCP3 (red) (J) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrows indicate the sex chromosomes.

K Quantiﬁcation of the numbers of DMC1 foci in 6-month-old Usp26+/Y (n=50, Zygotene, n=31, Early-Pachytene) and Usp26-/Y (n=31, Zygotene, n=24, Early-Pachytene) spermatocytes. P=0.1893 for Zygotene. Red dots indicate Usp26+/Y mice and green dots indicate Usp26-/Y mice.

L Crossover on sex chromosomes was impaired in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of SYCP3 (green), MLH1 (red), and ATR (white) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue).

Data information: In (F) and (K), data are presented as mean ± SD. **P < 0.01 (Student's t-test). n.s, not significant. Numerical source data for panels F/K can be found in Table EV5.
Paternal USP26 Mutations Raise Klinefelter Syndrome Risk in the Offspring of Mice and Humans (2021)
Figure 6

Figure 6 - USP26 participates in sex chromosomes pairing by regulating the stability of TEX11.

A USP26 interacts with TEX11. pRK-FLAG-Usp26 and pEGFP-Tex11 were co-transfected into HEK293T cells. 24h after transfection, cells were collected for immunoprecipitation (IP) with anti-FLAG antibody and analyzed with anti-FLAG and anti-GFP antibodies, respectively.

B USP26 modulates the ubiquitination of TEX11. pRK-FLAG-Usp26, pEGFP-Tex11, pCMV-HA-Ub were transfected into HEK293T cells. At 24h after transfection, cells were collected for immunoprecipitation (IP) with anti-GFP antibody and analyzed with anti-HA and anti-GFP antibodies, respectively.

C The TEX11 protein was greatly reduced in the Usp26-/Y testis. Immunoblotting of TEX11 was performed in 6-month-old Usp26+/Y and Usp26-/Y testes. Actin served as the loading control.

D-F The X and Y chromosomes were unpaired in Tex11-/Y spermatocytes. Immunofluorescence analysis of SYCP3 (green), TRF1 (red), ATR (white) (D); SYCP3 (green), MLH1 (red), ATR (white) (E); Chr X-FISH (green), Chr Y-FISH (red) and SYCP3 (white) (F) was performed in in Tex11+/Y and Tex11-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrowheads indicate the X chromosome.

G Quantification of unpaired X and Y chromosomes in Tex11+/Y and Tex11-/Y mice (n= 4 independent experiments). Red dots indicate Tex11+/Y mice and green dots indicate Tex11-/Y mice.

H Laggard chromosomes were observed in the metaphase I of Tex11-/Y spermatocytes. The arrowhead indicates the laggard chromosome.

I The proportion of metaphase I spermatocytes exhibiting laggard chromosomes in Tex11+/Y and Tex11-/Y mice spermatocytes (n= 3 independent experiments). Red dots indicate Tex11+/Y mice and green dots indicate Tex11-/Y mice.

J Tex11-deficient mice produced XY aneuploid spermatozoa. FISH assay of Chr X (green) and Chr Y (red) was performed in Tex11+/Y and Tex11-/Y spermatozoa. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome.

Data information: In (G) and (I), data are presented as mean ± SD. **P < 0.01 (Student's t-test). Numerical source data for panels G/I can be found in Table EV5.
Paternal USP26 Mutations Raise Klinefelter Syndrome Risk in the Offspring of Mice and Humans (2021)
Figure 7

Figure 7 - Fertile men with USP26 mutations produce XY aneuploid spermatozoa.

A, B Quantification of different types of spermatozoa in fertile men with or without USP26 mutations. The spermatozoa from fertile men without USP26 mutations (n = 10 independent experiments); fertile men contained the USP26 c.463C>T mutation (The sample was analyzed in three repeated experiments), the USP26 c.125T>C mutation (n = 2 independent experiments, one sample was analyzed in two repeated experiments), the USP26 c.1044T>A mutation (n = 5 independent experiments), and the USP26 c.370_371insACA/494T>C/1423C>T mutation (n = 10 independent experiments) were collected to perform Chr X and Chr Y FISH. P-values are shown for comparison to fertile men without USP26 mutations.

C The fertile men with the USP26 mutated haplotype produced XY and O aneuploid spermatozoa. FISH assay of Chr X (green) and Chr Y (red) was performed in fertile men with or without the USP26 mutated haplotype. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome.

D Squares denote male family members, circles denote female family member; a solid symbol indicates affected KS member, open symbols represent unaffected KS family members. The X labeled with red color indicates the X chromosome with the USP26 mutated haplotype (c.370_371insACA/494T>C/1423C>T). All family members were Sanger sequenced.

Data information: In (A) and (B), data are presented as mean ± SD. **P < 0.01 (Student's t-test). Numerical source data for panels A/B can be found in Table EV5.

Expanded View Figure Legends
MXL-3 and HLH-30 transcriptionally link lipolysis and autophagy to nutrient availability (2013)
figf1

RNA was extracted from young fertile adults fed ad libitum or fasted for 6 h. ddCts (delta delta cycle thresholds) were calculated normalizing to ama-1 and the efficiency of the primer sets as previously described. Means+s.e.m. are depicted. lipl-1 to lipl-5,
n = 6 independent experiments. lipl-6 to lipl-8,
n = 3independent experiments. Significant
t-test derived
P-values are indicated. WT, wild type.
MXL-3 and HLH-30 transcriptionally link lipolysis and autophagy to nutrient availability (2013)
figf2

(a) Representative confocal images of LIPL-1::GFP and LIPL-3::TagRFP localization relative to the lysosomal marker PGP-2 show that LIPL-1 and LIPL-3 are localized to the lysosomal-related organelle. (b) Acid lipase activity, measured in 1-day adult whole lysates, shows that lipl-1lipl-3 double mutant animals have reduced acidic lipolytic capacity. Mean±s.d. are presented relative to wild type (WT); significant differences are indicated;
n = 4 independent experiments. (c) Oil Red O (ORO) staining and fatty acid methyl ester (FAME) analyses of wild-type and lipl-1(tm1954) lipl-3(tm4498) double mutant L3 larvae show that lysosomal lipases regulate cytosolic fat stores (see Fig. 2d for adult measurements). Means±s.e.m. are presented relative to wild type; significant differences are indicated;
n = 3 independent experiments. (d) ORO quantification of wild-type and
lipl-1(tm1954) lipl-3(tm4498) double mutant young adults fasted for 4 h shows that LIPL-1 and LIPL-3 contribute to fat mobilization following fasting. (Mean percentage ± s.e.m. relative to fed wild type). Wild-type fasted worms show 20% less ORO signal than animals fed ad libitum (P≤0.001), whereas lipl-1(tm1954) lipl-3(tm4498) double mutant worms show 7% reduction in ORO signal (a difference that is not significant to well-fed fat levels at
P≤0.01 but it is significant at
P≤0.05).
n = 3 independent experiments. (e) Representative transmission electron microscopy images of well-fed and 6 h-fasted wild-type and lipl-1lipl-3 mutant animals (11,500×) show that the lipl mutants have more lipid-droplet stores in ad libitum fed and fasted conditions. Quantification of vesicle number and size is depicted as mean+s.e.m.,
n = 4 independent experiments. (f) Representative images of well-fed transgenic animals expressing LIPL-3::TagRFP and ATGL::GFP shows that LIPL-3 does not localize to lipid droplets. (g) ORO staining and quantification of wild-type and lipl-1(tm1954) lipl-3(tm4498) double mutant young adults treated post-developmentally (from L4) with RNAi against the essential autophagy genes lgg-1 and lgg-2 or vector control show that lipl-1 lipl-3 and the autophagy genes lgg-1 lgg-2 are in the same fat regulatory pathway. (Mean percentage+s.e.m. relative to wild type on vector control is depicted.)
n = 4 independent experiments.
MXL-3 and HLH-30 transcriptionally link lipolysis and autophagy to nutrient availability (2013)
figf3

(a) Expression of lipl genes in well-fed mxl-3(ok1947) and mxl-3(tm2580) young adults normalized to same age well-fed wild-type (WT) worms shows that mxl-3 loss of function is sufficient to induce lipl-1 to 3 and lipl-5. Mean ddCts+s.e.m. are depicted.
n = 6 independent experiments for lipl-1 to lipl-5, and
n = 4 independent experiments for lipl-6 to lipl-8. (b) L4 larvae were fasted and RNA was extracted at the indicated times. Mean ddCts+s.e.m. show that the response mxl-3 orchestrates is transient. Time 0,
n = 6independent experiments; time 3–12 h,
n = 4 independent experiments; time 18–24 h,
n = 2 independent experiments. (c) Immunostainings of well-fed or 6 or 12 h fasted MXL-3::GFP young adults are presented. Quantification of GFP-positive nuclei relative to total intestinal nuclei (4,6-diamidino-2-phenylindole, DAPI) of two independent experiments shows that MXL-3 transiently delocalizes from the intestinal nuclei during fasting. (d) ChIP–quantitative PCR (ChIP-qPCR) analysis of well-fed and 6 h fasted mixed-stage worms expressing MXL-3::GFP presented as Ct in αGFP immunoprecipitated DNA normalized to input DNA and relative to a mock promoter region (CACTAT site −88 of ama-1 gene) shows that MXL-3 vacates the lipl promoters during early fasting. Three sets of primers surrounding CACGTG target sites found up to 500 bp of the ATG of the lipl-1 and lipl-3 genes were used. A representative experiment is presented; see raw data of two independent experiments in Supplementary Table S9. (e) Wild-type or lipl-1(tm1954)
lipl-3(tm4498) double mutant animals grown on control RNAi plates were transferred as L4 larvae to control or mxl-3 RNAi plates, incubated for 12 h at 20 °C, and processed for total FAMEs; as a reference, an aliquot of wild-type young adults on control RNAi bacteria was fasted for 6 h. FAME quantification (mean percentage of fed wild type ±s.e.m.) shows that acute mxl-3 inactivation, as fasting, reduces fat stores in a lipl-dependent manner.
n = 3 independent experiments. NSD, P-value>0.05.