EMBO/soda-nel huggingface dataset:
Caspase-8 in endothelial cells maintains gut homeostasis and prevents small bowel inflammation in mice (2022)
Figure 5

Fig. 5: Lymphatic ECs are the primary driver of disease in Casp8ECko mice.

A. Representative pictures of TUNEL staining together with CD31 and Lyve-1 in Peyer's Patches of Casp8WT and Casp8ECko mice. TUNEL+ LECs, but not BECs could already be seen in non-hemorrhagic Peyer's Patches (a'), and further accumulated in hemorrhagic Peyer's Patches (a''). White dotted areas outline location of the lymphatic plexus. White arrowheads point to TUNEL+ LECs (note that necroptotic LECs only weakly express Lyve-1). Scale bars: 50µm, insets: 10µm.

B. Schematic representation of Casp8 deletion in PDGFb-CreERT2 x Casp8fl/fl mice (Casp8BECko) indicating the time points for tamoxifen treatment and analysis.

C. Survival (n= 5 WT, 6 BECko; Log-Rank test) of Casp8WT and Casp8ECko mice upon tamoxifen treatment.

D. Representative pictures of intestines of Casp8WT and Casp8BECko mice.

E-G. Representative pictures of H&E staining of small intestine of Casp8WT and Casp8BECko mice (E), enteropathy score (F; n= 5 WT, 6 ECko, two tailed unpaired Student's t-test) and mucosal hemorrhages (G; n= 5 WT, 6 ECko, two tailed unpaired Student's t-test). Scale bars: 100µm, insets: 50µm.

H-J. Representative images of stainings for CD31, Lyve1 and DAPI in small intestinal wholemounts (H) and quantification of vessel density (I) and lacteal length (J) in Casp8BECko compared to Casp8WT mice (I; n= 5 WT, 6 BECko; two tailed unpaired Student's t-test, J; n= 5 WT, 5 BECko; two tailed unpaired Student's t-test with Welch's correction). Scale bars: 100µm.

K-L. Representative pictures (K) and quantification (L) of lymphatic vessel density Peyer's Patches in Casp8WT and Casp8BECko mice (n = 10 WT, 11 BECko Peyer's Patches from 5-6 mice per genotype; two tailed unpaired Student's t-test). Scale bars: 200µm.

Data information: Data shown as mean ± SEM. ns: not significant.
Caspase-8 in endothelial cells maintains gut homeostasis and prevents small bowel inflammation in mice (2022)
Figure 6

Fig. 6: Intravenous TNF-α injection in in Casp8ECko mice causes specific bleedings in the Peyer´s patches but in no other organ.

A. Schematic representation of the strategy to induce systemic inflammatory response syndrome (SIRS) via intravenous TNF-α injection.

B. Survival graph after intravenous TNF-α injection of Casp8WT vs. Casp8ECko mice within the first 24h (n=11 WT, 11 ECko; Log-Rank test).

C. Representative images of the small intestines 24h after TNF-α injection in Casp8WT and Casp8ECko mice (blue arrowheads point to Peyer's Patches).

D-E. Representative pictures of Peyer's Patches (D) and quantification of lymphatic vessel density (E) in Casp8WT, Casp8ECko and Casp8ECko/MLKLko mice injected with TNF-α (n=7 WT, 5 ECko, 15 ECko/MLKLko. Peyer's Patches are pooled from 3-5 mice per genotype). Scale bars: 200µm.

F. Representative pictures of TUNEL staining together with CD31 and Lyve-1 in Peyer's Patches of Casp8WT, Casp8ECko and Casp8ECko/MLKLko mice injected with TNF-α. TUNEL+ LECs, but not BECs could already be seen in Casp8ECko, but neither in Casp8WT nor in Casp8ECko/MLKLko mice. White dotted areas outline location of the lymphatic plexus. Yellow arrowheads point to TUNEL+ECs). Scale bars: 50µm.

G. Schematic representation of the Enbrel® treatment protocol to inhibit TNF-α signaling.

H. Survival graph of Casp8WT and Casp8ECko mice after Enbrel® treatment (n=7 WT, 5 ECko + IgG, 8 ECko + Enbrel; Log-Rank test).

I-K. Representative images of small intestines (I) and H&E staining (J) with histopathological scoring (K) of Casp8WT and Casp8ECko mice after Enbrel® treatment (n=6 WT, 4 ECko + IgG; 8 ECko + Enbrel (from which 3 mice were diseased, 5 mice healthy); one-way ANOVA with tukey's multiple comparison). Scale bars: 100µm.

Data information: All data is shown as mean ± SEM. * P<0.05, *** P <0.001, **** P <0.0001; ns: not significant.
Caspase-8 in endothelial cells maintains gut homeostasis and prevents small bowel inflammation in mice (2022)
Figure 7

Fig. 7: The presence of microbiota is required to induce small bowel inflammation in Casp8ECko mice.

A-B. Schematic representation of the strategy for antibiotic treatment of Casp8WT and Casp8ECko mice (A) and analysis of the most prominent bacterial phyla (B; n= 4 Control, 6-8 Antibiotics; multiple t-tests with Holm-Sidak method) by qPCR analysis, In B, the cycle number of 16S rDNA expression compared to the total number of cycles is shown.

C-D. Graphs showing the relative weight (C; n= 11 WT, 7 ECko, 10 ECko+ antibiotics one-way ANOVA with tukey's multiple comparison) and the survival of mice (D; n= 6 WT, 7 ECko, 7 ECko+ antibiotics; Log-Rank test) upon tamoxifen treatment in the presence of an antibiotic cocktail.

E. Representative images of the gut of Casp8WT and Casp8ECko mice with antibiotic treatment 30 days after tamoxifen.

F-H. Representative images of H&E staining (F), quantification of intestinal pathology (G) and mucosal hemorrhages (H) in Casp8WT and Casp8ECko mice treated with antibiotics compared to untreated Casp8ECko mice (G; n= 6 WT, 4 ECko+ antibiotics, 5 ECko; one-way ANOVA with tukey's multiple comparison, H; n= 6 WT, 4 ECko+ antibiotics, 4 ECko; one-way ANOVA with tukey's multiple comparison).

Data information: Data shown as mean ± SEM. ** P <0.01, *** P <0.001, **** P <0.0001; ns: not significant. Scale bars: 100μm.

Expanded View Figure Legends
Piwi/piRNAs control food intake by promoting neuropeptide F expression in locusts (2021)
Figure 1

Figure 1 - Characterization of PIWI genes in arthropod species.

A Phylogenetic analyses of PIWI genes in 174 arthropod genomes. A maximum-likelihood phylogenetic tree was generated on the basis of the protein sequences of PIWI genes using FastTree software version 2.1.10. The reliability of each tree branch was evaluated using the SH branch support test with 1000 resamples. The red dots on a branch indicate genes with an SH test result >0.5. The different colors outside of the phylogenetic tree represent different arthropod orders.

B Protein distance matrix for Piwi, Aub, and Ago3 in Drosophila (Dm) and L. migratoria (Lm).

C The numbers of Piwi-like genes in the 174 arthropod species.

D Distributions of pairwise Ka/Ks (nonsynonymous to synonymous nucleotide substitution rates) ratios between PIWI duplicates in arthropods (n = 17 in Drosophila and n = 37 in others, Mann-Whitney U test, *P<0.05). The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value.
Piwi/piRNAs control food intake by promoting neuropeptide F expression in locusts (2021)
Figure 6

Figure 6 - piRs-3-I2 and piRs-3-I3 promote feeding in locusts.

A Representative genomic loci of differentially expressed piRNAs located within the NPF1 gene (beige colored regions are exons).

B mRNA levels of differentially expressed piRNAs located within the flanking region of the NPF1 gene in dsPiwi1-treated locusts and dsGFP-treated control locusts (n=5 biological replicates).

C The mRNA expression of NPF1 was measured by qPCR after a mixture of inhibitors or mimics of piRs-3-I2 and piRs-3-I3 was injected (n=5 biological replicates).

D Effects of inhibitors (n=6 biological replicates) and mimics (n=4 biological replicates) of piRs-3-I2 and piRs-3-I3 on food intake.

E Body weight measurement of locusts administered inhibitors (NC, n=25 locusts; Inhibitors- piRs-3-I2+I3, n=23 locusts) and mimics (NC, n=37 locusts; Inhibitors-piRs-3-I2+I3, n=39 locusts) of piRs-3-I2 and piRs-3-I3. The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value.

F RNA-binding protein immunoprecipitation assay of piRs-3-I2 and piRs-3-I3 in Piwi1 immunoprecipitates from brain tissue extracts (n=6 biological replicates). Normal mouse IgG was used as a negative control.

Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).
Induction of autophagy by spermidine promotes longevity (2009)
figf2

(a) Intracellular spermidine of five-day-old Δspe1 cells, compared with wild-type cells. Data represent means ± s.e.m. (n = 3; *P 0.001). (b) Chronological ageing of wild-type (▪, o) and polyamine-depleted Δspe1 (▴, ▿) yeast cells with (open symbols) and without (closed symbols) supplementation of low doses of spermidine. Data represent means ± s.e.m. (n = 4). Cells were tested for cell death markers at day 3. (c) Fluorescence microscopy of DHE→Eth conversion in wild-type and Δspe1 cells indicating ROS production. Scale bars, 10 μm. (d) Quantification (FACS analysis) of ROS accumulation using DHE→Eth conversion of wild-type (WT) and Δspe1 cells with and without supplementation of low doses of spermidine. Data represent means ± s.e.m. (n = 4; *P 0.001). (e) Quantification (FACS analysis) of phosphatidylserine externalization and loss of membrane integrity using annexin V/PI co-staining (at day 5) and of DNA fragmentation using TUNEL staining (at day 3) of chronologically ageing wild-type (WT) and Δspe1 cells with or without supplementation of 0.1 mM spermidine as indicated. Data represent means ± s.e.m. (n = 3; *P 0.001).
Induction of autophagy by spermidine promotes longevity (2009)
figf4

(a) Relative acetylation (normalized to controls at each day) of indicated histone H3 Lys residues determined by quantification of immunoblot analysis. A representative blot using site specific antibodies is shown in b. Data represent means ± s.e.m. (n = 3). Serial dilutions of protein extracts were applied in western blots to verify linearity before quantification (examples are shown in Supplementary Information, Fig. S4). For calculation details see section on Methods. P values indicate the result of a two-factor ANOVA corrected by Bonferroni post hoc test. (b) Immunoblot of whole-cell extracts of wild-type cells chronologically aged to designated time-points with (+) or without (−) spermidine application. Blots were probed with antibodies against total histone H3 or H3 acetylation sites at the indicated Lys residues. Full scans of blots are available in Supplementary Information, Fig. S5. (c) Relative acetylation (normalized to wild-type controls at each day) of indicated histone H3 Lys residues of Δspe1 cells (open bars), compared with wild-type cells (closed bars) during chronological ageing. Data represent means ± s.e.m. (n = 3). P values indicate the result of a two-factor ANOVA corrected by Bonferroni post hoc test. (d) Relative acetylation (normalized to day 1) of histone H3 Lys 18 residue of chronologically ageing wild-type cells. Data represent means ± s.e.m. (n = 3). (e) Relative acetylation (normalized to controls) of histone H3 Lys 14 and 18 residues of cultured human PBMC after 6 days of incubation with or without 20 nM spermidine. Data represent quantification of two independent experiments performed with cells obtained from two different donors. (f) Immunoblot analysis of liver cell extracts (applied in serial dilutions) obtained from mice fed with 3 mM spermidine for 200 days and respective control mice of the same age. Blots were probed with antibodies recognizing total histone H3 (C-terminal epitope) or specific for acetylated lysine 18 residue (N-terminal epitope). Full scans of blots are available in Supplementary Information, Fig. S5.
Induction of autophagy by spermidine promotes longevity (2009)
figf5

(a) Relative acetylation of histone H3 Lys 9 and 14 residues determined by quantification of immunoblot analysis performed at day 20 of the ageing experiment shown in panel b. Data represent means ± s.e.m. (n = 3; *P 0.05). (b) Chronological ageing of wild-type (▪, o) and Δiki3Δsas3 (▴, ▿) with (open symbols) or without (closed symbols) addition of spermidine (4 mM) at day 1. Data represent means ± s.e.m. (n = 4). (c) Quantification (FACS analysis) of phosphatidylserine externalization and loss of membrane integrity (annexinV/PI costaining) as well as ROS production (DHE->Eth conversion) performed at day 20 of the experiment shown in b. Data represent means ± s.e.m. (n = 4; *P 0.001). (d) Relative inhibition of histone acetyltransferase activity (HAT-activity) by spermidine determined by an in vitro HAT-activity assay of yeast nuclear extracts of wild-type cells. Data represent means ± s.e.m. of three independent experiments (*P = 0.024). (e, f) Transcriptional change (obtained by Affymetrix array) by spermidine treatment of all autophagy-related genes categorized into GO terms of macroautophagy (GO:0016236), microautophagy (GO:0016237) or autophagy in general (GO:0006914) at day 3 (e) and day 10 (f) of chronological ageing yeast. Data represent means of two independent arrays from independent biological samples. (g) Relative change of ATG7, ATG11 and ATG15 mRNA levels by spermidine supplementation (normalised to controls) after ten days of chronological ageing as determined by reverse transcriptase real-time PCR. Data represent means ± s.e.m. (n = 3; *P 0.05 and **P 0.01). (h) Relative histone H3 Lys 18 acetylation of indicated promoter regions normalized to the acetylation of ATG7 promoter (pATG7) determined by chromatin immunoprecipitation with H3-K18Ac specific antibody and subsequent quantification of precipitated promoter DNA using quantitative real time PCR. Signals specific for indicated promoter regions were initially compared with non-immunoprecipitated samples and afterwards normalized to the pATG7 signals. Data represent means ± s.e.m. (n = 3). The P value was calculated using a two-factor ANOVA with promoter and condition (treated and non-treated) as independent factors.
Induction of autophagy by spermidine promotes longevity (2009)
figf6

(a) Fluorescence microscopy of wild-type yeast cells expressing an EGFP–Atg8p fusion protein with or without (control) treatment of 4 mM spermidine for 48 h. White arrows indicate vacuolar localization of EGFP–Atg8p indicative of autophagy. Scale bars, 5 μm. (b) Relative alkaline phosphatase activity (ALP activity) indicative of autophagy during chronological ageing of pho8ΔC60 yeast with (open bars) or without (closed bars) application of spermidine (4 mM). Data represent means ± s.e.m. (n = 3 *; P 0.01 and **P 0.001). (c) Relative alkaline phosphatase activity (ALP activity) indicative of autophagy during chronological ageing of wild-type (closed bars) and Δiki3Δsas3 (open bars) cells. Data represent means ± s.e.m. (n = 3; *P 0.01 and **P 0.001). (d) Chronological ageing of wild-type (▪, o) and Δatg7 (▴, ▿) with (open symbols) or without (closed symbols) addition of spermidine (4 mM) at day 1. Data represent means ± s.e.m. (n = 4). (e) Chronological ageing on water of wild-type (▪, o) and Δatg7 (▴, ▿) with (open symbols) or without (closed symbols) addition of spermidine (8 mM) at day 0. Data represent means ± s.e.m. (n = 4). (f) Quantification (fluorescence reader) of ROS production (DHE→Eth conversion) by wild-type and Δatg7 cells with and without supplementation of spermidine (8 mM), obtained from the ageing experiment shown in panel e. Data represent means ± s.e.m. (n = 4; *P 0.07 and **P 0.01).
Induction of autophagy by spermidine promotes longevity (2009)
figf7

(a) Fluorescence microscopy of Hoechst-counterstained HeLa cells transiently transfected with LC3–GFP subjected to 100 μM spermidine for 6 h. Representative pictures are shown. (b) Percentage of adherent cells exhibiting a clear LC3–GFP relocalization into cytoplasmic vacuoles. Numbers were determined using micrographs of Hoechst-counterstained HeLa cells as representatively shown in a. Data represent means ± s.d. (n = 3). (c) LysoTracker Red staining of vacuoles indicative of autophagy in oesophagus tissue from flies fed with 1 mM spermidine for two days, compared with controls (without spermidine). Nuclei were visualized by Hoechst staining. Scale bars, 10 μm. (d) Quantification of autophagic vesicles per nucleus in LysoTracker Red stained muscle tissue of female flies fed with supplementation of 1 mM spermidine or with 10% glucose (starved) for 48 h, compared with normal food (control). Data represent means ± s.e.m. of at least 20 flies for each group (*P 0.01). (e, f) Survival of Drosophila during ageing without (control) and with supplementation of food at various concentrations of spermidine (as indicated). Autophagy-deficient flies (f) homozygous mutant for Atg7 (Δatg7) were compared with flies capable of autophagy (e) and heterozygous for Atg7 (control). For details of strains and additional wild-type controls, see Supplementary Information Methods and Fig. S6h, i. (g) Fluorescence microscopy of C. elegans transgenic embryos expressing a full-length plgg-1DsRED::LGG-1 fusion protein indicative of autophagic activity. Shown are two representative pictures of embryos untreated (control) or treated with spermidine (0.2 mM) supplementation of food. (h) Quantification of autophagic activity through measurement of DsRED::LGG-1 pixel intensity from images of wild-type animals shown in g, and bec-1 RNAi knockdown animals (bec-1 RNAi). Data represent means ± s.e.m. (n = 3) with at least 25 images processed for each trial. (i) Survival of C. elegans during ageing with and without (control) supplementation of food (UV-killed E. coli) with spermidine (0.2 mM). Wild-type (N2) animals were compared with bec-1 RNAi animals deficient in autophagy induction. For mean life spans see Supplementary Information, Table S3.
The RAS-related GTPase RHOB confers resistance to EGFR-tyrosine kinase inhibitors in non-small cell lung cancer via an AKT-dependent mechanism (2016)
Figure 1

Figure 1. RHOB expression predicts the response to EGFR-TKI in patients harboring EGFR-activating mutations.

A. Four representative images of RHOB immunostaining in human non-small-cell lung cancers. Figures correspond to the percentage of tumor analyzed. B. Characteristics of 96 patients with an EGFR-activating mutation who were treated with EGFR-TKI. Each column corresponds to one patient. Data include general characteristics (stage at diagnosis, age, sex, tobacco usage, position of EGFR mutation, TKI type), progression-free survival (PFS; lower graph) and intensity of RHOB staining as determined by immunohistochemistry (null: 0, weak: +, moderate: ++ and high: +++). C. Representative scans of patients with low or high RHOB-expressing lung tumors, before and after erlotinib treatment. Red arrows indicate lung tumors. D. Progression-free survival of erlotinib-treated patients with EGFR-mutated lung tumors, according to RHOB expression, assessed by immunohistochemistry (low RHOB group = negative + weak staining; high RHOB group = moderate + high staining). E. Progression-free survival of patients who received EGFR-TKi as first-line therapy (n=63). F. Progression-free survival of patients who received EGFR-TKi as 2nd-line (n=28), 3rd-line (n=3) or 4th-line (n=2) therapy. G. RHOB immunostaining score evolution in EGFR-mutated lung tumors before treatment and after EGFR-TKI relapse.
The RAS-related GTPase RHOB confers resistance to EGFR-tyrosine kinase inhibitors in non-small cell lung cancer via an AKT-dependent mechanism (2016)
Figure 2

Figure 2. RHOB loss of expression increases sensitivity to erlotinib in mice with EGFRL858R-driven lung tumors.

A. Representative H&E staining of whole lungs from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days. Scale bar: 5 mm. B. Quantification of the tumor/lung ratio. n=7 for each group except for EGFRL858R/Rhob+/- placebo (n=6). C. Representative Ki67 immunostaining of EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days (scale bar: 50 µm), and the corresponding quantification (D). Three independent zones/mice lung were used for quantification. n=21 (7 mice) for each group except for EGFRL858R/Rhob+/- placebo (n=18; 6 mice). E. Immunostaining of cleaved caspase-3 in lung tumors from EGFRL858R/Rhob+/+ or EGFRL858R/Rhob-/- mice treated for 24 h with erlotinib at 12.5 mg/kg. Scale bar: 50 µm. (**: p<0.001 vs. placebo; ***: p<0.0001 vs. placebo).
The RAS-related GTPase RHOB confers resistance to EGFR-tyrosine kinase inhibitors in non-small cell lung cancer via an AKT-dependent mechanism (2016)
Figure 3

Figure 3. The modulation of RHOB expression determines the level of resistance to erlotinib in EGFR-mutated lung cancer cell lines.

HCC4006 cells were either (A) transfected with two siRNA against RHOB (siB1, siB2), (B) transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB), or (C) both transfected with siRNA and transduced by adenoviruses; then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells. Erlotinib IC50 values were quantified in RHOB-overexpressing or RHOB-depleted (D) HCC827 cells, (E) HCC2935 cells and (F) H3255 cells, as determined by an MTS assay after 72 h treatment. RHOB overexpression or inhibition was monitored by western blotting for each condition. G. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) at an increasing multiplicity of infection (MOI), then erlotinib IC50 values were determined after 72 h by an MTS assay and a correlation analysis was performed. RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.
The RAS-related GTPase RHOB confers resistance to EGFR-tyrosine kinase inhibitors in non-small cell lung cancer via an AKT-dependent mechanism (2016)
Figure 4

Figure 4. RHOB induces resistance to erlotinib through the AKT pathway.

A. HCC4006, HCC827, HCC2935 and H3255 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for four hours with erlotinib at concentrations corresponding to the respective IC50 values determined for each control cell line. The phosphorylation status of AKT, ERK1/2 and EGFR was assessed by western blotting and normalized according to total protein levels. RHOB overexpression was also monitored by western blotting. B. Representative immunostaining of phospho-AKT (Ser473), phospho ERK1/2 and their total protein amounts in lung tumors from EGFRL858R/Rhob-/- or EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days. The remaining hyperplastic areas were selected in erlotinib-treated mice to efficiently characterize the effect of erlotinib on ERK and AKT pathways in both Rhob genotypes. Scale bar: 100 µm. C. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKTmyr, myristoylated) or an empty vector (ø), and treated for 72 h with increasing concentrations of erlotinib. The surviving cell fraction was determined by an MTS assay and AKT overexpression and phosphorylation at Ser473 were assessed by western blotting. Data are representative of at least three independent experiments.