EMBO/soda-nel huggingface dataset:
Gut colonization by Bacteroides requires translation by an EF-G paralog lacking GTPase activity (2022)
Figure 6

Figure 6. Differential expression of the EF-G1 and EF-G2 protein in laboratory medium and mouse cecum

A-D. Western blot analysis of B. thetaiotaomicron strains expressing C-terminally FLAG-tagged EF-G1 (WH405) (A), or EF-G2 (GT1301) (B), from their normal promoters and chromosomal locations, grown in Tryptone Yeast Extract Glucose liquid medium (TYG) and sampled at the indicated times. Bacterial growth (OD600) is shown in (C) and Western blot quantifications (FLAG signal normalized to loading control GroEL) are show in (D). Blots were developed with anti-FLAG antibodies; anti-GroEL antibodies were used as loading controls. Shown is a representative from at least two independent experiments.

E-F. Western blot of crude extracts from wild-type B. thetaiotaomicron harvested from the ceca of mice (representative blot for 2 out of 4 mice tested) or corresponding to stationary phase cultures harvested following growth in TYG for 24 h or purified EF-G1 and EF-G2 proteins at the indicated amounts (E). Blots were developed with anti-EF-G1 or EF-G2 polyclonal antibodies; anti-GroEL antibodies were used as loading controls. Western blot quantifications of all four biological replicates and their average are shown in (F), where EF-G1 and EF-G2 abundances were estimated by normalizing the signal of bacterial sample to the closest signal of known amount of purified protein, and the derived absolute amount (pmol) was normalized to the signal of GroEL. Error bars represent SD. Please note log scale of y axis.
TCF/LEF dependent and independent transcriptional regulation of Wnt/β-catenin target genes (2018)
Figure 1

Fig.1 Quadruple TCF/LEF knockout (K.O.) HEK 293T cells are viable and proliferative but do not respond to Wnt pathway activation.

a Schematic representation of the TCF/LEF protein structure. Below, the guideRNAs (gRNAs) used for each specific gene are annotated. Grey boxes indicate the DNA-binding HMG box.

b Western blots for detection of TCF7, LEF1, TCF7L1, TCF7L2 and β-catenin (B') proteins in wild-type (WT) and knockout (K.O.) cells. Uncropped version of the same western blots is shown in Figure EV 1.

c TCF-reporter TOP-flash assay performed on the indicated cell lines treated with 10 μM CHIR, for 24 hrs. The control FOP-flash reporter is not activated upon CHIR administration. The data represent the mean ± s.e.m. of averages of nine independent experiments (N = 9). dBcat, β-catenin knockout cells; d4TCF, TCF/LEF quadruple knockout cells.

d Quantitative RT-PCR analyses of Axin2 transcripts, performed on the different cell lines treated with 10 μM CHIR or DMSO (control) for 24 hrs. Error bars show that standard deviation obtained from 3 independent experiments.

e, f Transfection of individual TCF-expressing plasmids could restore the ability of d4TCF cells to respond to Wnt3a (E), as measured in a TOP-flash assay, or of CHIR (F), as measured by Axin2 mRNA abundance. The data represent the mean ± s.e.m. obtained from three independent experiments (N = 3). Samples were compared using Student's t-test. Asterisks (*) indicate a p-value<0.05.
TCF/LEF dependent and independent transcriptional regulation of Wnt/β-catenin target genes (2018)
Figure 4

Fig.4 β-catenin genome-wide occupancy in the presence or in the absence of TCF/LEF transcription factors.

A-C (A, C) Genome browser-based schematic representation of β-catenin peaks in control wild-type (WT), d4TCF or dBcat cells. (A, B) β-catenin occupancy on regulatory regions of the prototypical Wnt target genes, AXIN2 and LEF1 (A), and at the promoter regions of HOXC4 and ZNF503 (B). (C) β-catenin ChIP-seq enrichment over ENCODE TCF7L2 (also known as TCF4) ChIP peaks in HEK293T cells. The data represent the mean ± 95% C.I. obtained from three independent experiments (N = 3).

D, e Motif analysis of β-catenin peaks in WT- (D) and d4TCF cells (E).

f Fraction of peaks containing FOX- ("TGAGWARAMWCC"), TCF- ("CCTTTGAAST") of both motifs, allowing maximum 1 mismatch.
TCF/LEF dependent and independent transcriptional regulation of Wnt/β-catenin target genes (2018)
Figure 5

Fig.5 β-catenin drives a GHOST response in the absence of TCF/LEF transcription factors.

a Differential gene expression analysis shows a set of 196 genes being regulated by CHIR in d4TCF cells; several of these changes only appear when TCF/LEF proteins are not present (dark green set). Of the 134 genes that are not regulated by CHIR in dBcat cells (putative β-catenin-dependent genes), 107 require the removal of TCF/LEF factors, in that their regulation only occurs in d4TCF cells. The other 27/134 β-catenin-dependent transcriptional changes match the TCF/LEF-independent target genes previously described in Figure 3a. Their regulation occurs both in the presence and in the absence of TCF/LEF factors. 90/107 of the putative β-catenin-dependent genes, stop being regulated by CHIR in pentaKO cells (β-catenin-GHOST, blue box).

b RT-qPCR validation of the β-catenin-GHOST response. A selection of 7 genes (colour coded) that are activated upon CHIR treatment in d4TCF, or in wild-type (WT) in combination with ICAT overexpression, but not in pentaKO cells - indicating that their regulation is dependent on β-catenin but can only occur in the absence of TCF/LEF-β-catenin interaction. The genotype and the treatment for each condition is indicated in the x-axis. Error bars show that standard deviation obtained from 3 independent experiments.

c FOXO4 but not FOXO3 overexpression upregulates the β-catenin-GHOST target GADD45 both in CHIR-treated WT (black bars) or d4TCF (grey bars) cells. Samples were compared using Student's t-test. Asterisks (*) indicate a p-value<0.05.

d FOXO4 overexpression induced GADD45 transcription in WT (black bars) but not in dBcat (grey bars) cells. Samples were compared using Student's t-test. Asterisks (*) indicate a p-value<0.05.

e Endogenous FOXO4, when immunoprecipitated (IP) pulls down β-catenin. Overexpressed FOXO4 increases the amount of β-catenin detected in the immunoprecipitated, indicating physical association.

f GADD45 is upregulated by CHIR in d4TCF cell; this positive transcriptional regulation is blocked by FOXO4-specific siRNA but not control scrambled siRNA. All experiments were done at least three times (N=3). Samples were compared using Student's t-test. Asterisks (**) indicate a p-value<0.02; ns, non-statistically significant change was observed.
Adipocyte autophagy limits gut inflammation by controlling oxylipin-IL10 (2023)
Figure 1

Figure 1: Intestinal inflammation promotes lipolysis and autophagy in adipose tissues.

(A) Schematic of experimental design. Sex-matched and age-matched wild-type mice were treated for 5 days with 1.5-2% DSS in drinking water, before switched to water for two more days. Mice were sacrificed at day 7 post-DSS induction.

(B) Body weight development upon DSS treatment (n = 13/group).

(C) Tissue weights measured in mesenteric (mWAT) and collective visceral white adipose tissue (visWAT), consisting of gonadal (gWAT), retroperitoneal and omental white adipose tissue at day 7 after start of DSS regime (n = 8/group).

(D) Circulating serum levels of FFA during DSS-induced colitis at day 7 (n = 15/group).

(E) Representative immunoblot for key lipolytic enzymes HSL and ATGL protein expression and quantification (n = 5-6/group).

(F) Immunoblot analysis of autophagic flux in mWAT (upper panel) and gWAT (lower panel) adipose tissue stimulated ex vivo with lysosomal inhibitors 100nM Bafilomycin A1 and 20mM NH4Cl for 4 hours or DMSO (Vehicle) (n = 3-4/group).

(G) Representative transmission electron microscopy images from mesenteric adipose tissue 7 days post DSS-induced colitis induction. Lower panel is showing magnification of selected area. White arrows show autophagosomal structures.

(H) Atg8 homologues expression was measured by qPCR in visceral adipocytes fraction (right) and stromal vascular fraction (left panel) during DSS-induced colitis (n = 7-8/group).

(I) Representative immunoblot for LC3-I/-II protein expression and quantification of autophagic flux in gWAT via ex vivo lysosomal inhibition using 100nM Bafilomycin A1 and 20mM NH4Cl for 4 hours or DMSO (Vehicle). Mice were initially treated with 500µg anti-TNF antibody or isotype control, before administrating DSS in drinking water for 5 days. Mice were sacrificed at day 7 post-DSS induction. (n = 5-6/group).

(J) Representative immunoblot for LC3-I/-II and ACTIN protein expression and quantification of autophagic flux in creeping fat tissues (CrF) and adjacent mesenteric adipose tissues (Ad. MAT) of Crohn's disease patients (n = 3/group). Additionally, autophagic flux was determined in the mesenteric adipose tissue (MAT) of a colorectal cancer patient as control (dotted line).

Data are represented as mean ± s.e.m. (B) Two-Way repeated measures ANOVA. (C, D, E, G) Unpaired Student's t-test. (I) Two-Way ANOVA. (J) Paired Student's t-test.
Adipocyte autophagy limits gut inflammation by controlling oxylipin-IL10 (2023)
Figure 2

Figure 2: Loss of adipocyte autophagy exacerbates DSS-induced colitis.

(A) Schematic of experimental design. Sex-matched and age-matched littermates were treated at 8-12 weeks of age with tamoxifen for five consecutive days before tissues were analysed 14 days after the last tamoxifen administration (Steady State).

(B) Representative quantification of knock-out efficiency measured on Atg7 transcript level by qRT-PCR in purified primary visceral adipocyte at two weeks post-tamoxifen treatment (n = 4-11/group).

(C) Representative immunoblot for LC3-I and LC3-II protein expression and quantification of LC3 conversion ratio (LC3-II/LC3-I) (n = 3/group).

(D) Schematic of experimental design. Sex-matched and age-matched littermates were treated at 8-12 weeks of age with tamoxifen for five consecutive days and DSS-induced colitis was induced after a two-week washout phase (DSS Day 7).

(E) Body weight development upon DSS treatment (n = 25/group).

(F) Colon length after two weeks post-deletion (steady state; n = 14/group) and after DSS at day 7 (n = 18-22/group).

(G) Representative H&E staining images (10x magnification) of colon sections and quantification of histological score at steady state (n = 9/group) and DSS colitis (n = 18-22/group).

(H) Expression of pro-inflammatory cytokines in colon tissues at 7 days post-DSS induction; n = 18-22/group pooled from three independent experiments. Dotted line represents uninflamed controls.

(I) Absolute number CD45+ immune cells from colons at steady state (n = 13-14/group) or at 7 days post-DSS induction (n = 18-22/group).

(J) Frequency of myeloid cell population in colon at day 7 post-DSS induction (n = 18-25/group).

(K) Absolute number of Ly6C+ monocytes discriminated by the absence or presence of MHCII for infiltrating and inflammatory monocytes respectively (n = 18-25/group).

Data are represented as mean ± s.e.m. (B, C, F, G, H, I) Unpaired Student's t-test. (E) Two-Way repeated measures ANOVA. (J, K) Two-Way ANOVA.
Adipocyte autophagy limits gut inflammation by controlling oxylipin-IL10 (2023)
Figure 3

Figure 3: Autophagy loss reduces secretion of fatty acids from adipocytes.

(A) Ex vivo lipolysis measured by released free fatty acid (left, n = 4-5/group) and glycerol (right, n = 7-8/group) in culture supernatant of adipose tissue explants simulated with isoproterenol (10µM) for 1-2h.

(B) Ex vivo lipolysis measured by released free fatty acid (left, n = 4/group) and glycerol (right, n = 7/group) adipose tissue explants simulated with TNFα (100ng/mL) for 24h before replacing with fresh medium in the absence of TNFα for 3h.

(C) Representative immunoblot for key lipolytic enzymes HSL, pHSL (Ser660) and quantification (n = 10-11/group).

(D) Serum levels of circulating FFAs measured in wild-type and Atg7-deficient mice (n = 13-14/group).

(E) Concentration of individual FFA species in serum in water-treated and DSS-treated mice as measured by FID-GC (n = 12-14/group).

Data are represented as mean ± s.e.m. (A, B, E) Two-Way ANOVA. (B, D) Unpaired Student's t-test. (C) Mann-Whitney test.
Adipocyte autophagy limits gut inflammation by controlling oxylipin-IL10 (2023)
Figure 4

Figure 4: Adipocyte-specific loss of Atgl was dispensable for regulation of intestinal inflammation.

(A) Schematic of experimental design. DSS-induced colitis was induced in sex-matched and age-matched littermates.

(B) Representative quantification of knock-out efficiency measured on Atgl transcript level by qRT-PCR in purified primary visceral adipocyte (n = 3-8/group).

(C) Ex vivo lipolysis assays on Atg7-deficient adipose tissue explants simulated with isoproterenol (10µM) for 1-2h (n = 5-6/group).

(D) Body weight development upon DSS treatment (n = 8/group).

(E) Tissue weights of mWAT and visWAT at day 7 after start of DSS (n = 3-8/group).

(F) Colon length after DSS at day 7 (n = 3-8).

(G) Quantification of histological score at steady state (n = 3/group) and DSS colitis (n = 6-7/group).

(H) Expression of pro-inflammatory cytokines in colon tissues at 7 days post-DSS induction (n = 8/group). Dotted line represents non-inflamed controls.

(I) Absolute number CD45+ immune cells from colons at 7 days post-DSS induction (n = 3-8/group).

(J) Frequency of myeloid cell population in colon at day 7 post-DSS induction (n = 8/group).

(K) Absolute number of Ly6C+ monocytes discriminated by the absence or presence of MHCII for infiltrating and inflammatory monocytes respectively (n = 8/group).

Data are represented as mean ± s.e.m. (B, E-I, K) Unpaired Student's t-test. (D) Two-Way repeated measures ANOVA. (C, J) Two-Way ANOVA.
Adipocyte autophagy limits gut inflammation by controlling oxylipin-IL10 (2023)
Figure 5

Figure 5: Adipocyte autophagy loss activates NRF2-EPHX1 pathway and alters intra-tissual oxylipin balance.

(A) Differential gene expression in visceral adipocytes from water-treated WT and Atg7Ad animals two weeks after tamoxifen treatment.

(B) Differential gene expression in visceral adipocytes from DSS-treated WT and Atg7Ad animals at day 7 post-DSS treatment.

(C) Venn diagram of commonly regulated genes between Atg7-deficient and Atg7-sufficient adipocytes during water- or DSS-treatment.

(D) GSEA enrichment analysis between Atg7-deficient and Atg7-sufficient adipocytes during DSS-treatment.

(E) Fold change expression of NRF2-target genes in primary visceral adipocytes at day 7 after DSS induction from normalized counts of RNAseq dataset (n = 6/group).

(F) Representative immunoblot for NRF2 protein expression and quantification (n = 16-18/group).

(G) Transcriptional expression of Ephx1 and Ephx2 in visceral adipocytes at day 7 after DSS induction pooled from three cohorts (n = 20-25/group).

(H) Representative immunoblot of EPHX1 and EPHX2 in gonadal adipose tissues at day 7 after DSS induction. Asterix indicating non-specific band (n = 14-18/group).

(I) Schematic overview of cytochrome P450-EPHX oxylipin pathway.

(J) Normalized fold change differences in epoxy fatty acid precursor fatty acids, docosahexaenoic acid (DHA), arachidonic acid (AA) and linoleic acid (LA) in mWAT and gWAT (n = 13-14/group).

(K) Normalized ratios of epoxy fatty acid to their corresponding diol fatty acids pairs in mWAT and gWAT (n = 6-8/group).

Data are represented as mean ± s.e.m. (E-H, L) Unpaired Student's t-test. (J,K) Two-Way ANOVA.
Adipocyte autophagy limits gut inflammation by controlling oxylipin-IL10 (2023)
Figure 6

Figure 6: Reduced adipose-tissue derived IL-10 secretion and IL-10 serum levels in adipocyte autophagy-deficient mice during DSS-induced colitis.

(A) Colitis was induced in mice for 7 days and mesenteric adipose tissue explants were cultured with FBS. Cytokine secretion into the supernatant was measured after 24h of culture (n = 4-12).

(B) Colitis was induced in mice for 7 days and adipose tissues were extracted and cultured for 6 hours in serum-starved medium. Secretion of IL-10 and from mesenteric (left panel) and gonadal adipose tissues (right panel) was measured by ELISA. Shapes identify individual experiments (n = 5-15/group).

(C) Representative FACS plots of CD11b+ F4/80+ ATMs in visceral adipose tissue from WT and Atg7Ad mice upon DSS-induced colitis at day 7.

(D) Quantification of IL-10-producing ATMs in visceral adipose tissue from WT and Atg7Ad upon DSS-induced colitis at day 7 (n = 3-6/group).

(E) Frequencies of IL-10-producing cells in visceral adipose tissues from WT and Atg7Ad upon DSS-induced colitis by flow cytometry. (n = 4-6/group).

(F) Serum cytokines upon DSS-induced colitis at day 7 post-induction (n = 17-23/group).

Data are represented as mean ± s.e.m. (A) Multiple t-test. (D-F) Two-Way ANOVA. (B) Two-Way ANOVA with regression for experiment.
Adipocyte autophagy limits gut inflammation by controlling oxylipin-IL10 (2023)
Figure 7

Figure 7: Cytochrome P450-EPHX pathway regulates IL-10 secretion from autophagy-deficient adipose tissues upon DSS-induced intestinal inflammation.

(A) Quantification of Il10 transcript levels in RAW264.7 upon stimulation to epoxy fatty acids (n = 3/group).

(B) Quantification of IL-10 protein levels in the supernatant of RAW264.7 upon stimulation to epoxy fatty acids (n = 3/group).

(C) Quantification of IL-10 protein levels in the supernatant of ex vivo cultured adipose tissues from water- or DSS-treated wild-type mice in the absence or presence of the cytochrome P450 inhibitor 1-ABT (n = 5-9/group).

(D) Quantification of IL-10 protein levels in the supernatant of ex vivo cultured adipose tissues from DSS-induced Atg7Ad mice in the absence or presence of the EPHX1 inhibitor NTPA and EPHX2 inhibitor TPPU (n = 4/group).

(E) Graphical summary of the anti-inflammatory fat-gut crosstalk during intestinal inflammation. Designed using BioRender.

Data are represented as mean ± s.e.m. (A,B) One-Way ANOVA. (C,D) Paired Student's t-test.
High density of REC8 constrains sister-chromatid axes and prevents illegitimate synaptonemal complex formation (2016)
Figure 2

Figure 2. Inter-sister SC formation takes place within sites of local separation of axial elements.

A) Representative wild-type pachytene bivalents; pachytene-like Rec8−/−, zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP1, SYCP3 and ACA. Filled arrowheads indicate sites of LSAEs with detectable SYCP1. Bars, 1 μm. Bellow: graphs showing the percentages of sites of LSAEs with detectable SC assembly along sister-AEs of Stag3 mutant and Smc1β−/− univalents (n=120 and n=68, respectively).

B) Representative zygotene-like Stag3 mutant univalents. Nuclear spreads were immunostained for SYCP3, ACA and: SYCE1, SYCE2 and TEX12. Filled arrowheads indicate SC assembly between sites of LSAEs. Bars, 1 μm

C) Quantification of signal distribution within sites of LSAEs, in Stag3 mutant spermatocytes. Signal distribution measured for SYCP3 and: SYCP1, SYCE1, SYCE2 and TEX12. Empty arrowheads indicate signal intensity peaks that correspond to each of the two sister-chromatid axes, filled arrowheads indicate SYCP1 peaks.
High density of REC8 constrains sister-chromatid axes and prevents illegitimate synaptonemal complex formation (2016)
Figure 3

Figure 3. Localization of α-kleisins REC8, RAD21L and RAD21 in relation to sites with local separation of axial elements.

A) Representative zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP3, SYCE1 and REC8. Bellow: graph showing the percentages of Stag3 mutant axes with sites of LSAEs with and without flanking REC8 foci. 78 axes with LSAEs were analysed. Bars, 1 μm.

B) Representative pachytene-like Rec8−/−, zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP3, SYCE1 and RAD21L. Bars, 1 μm.

C) Representative zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP3, SYCE1 and RAD21. Bars, 1 μm.

D) Schematic representation of univalents axes (black lines) with sites of LSAEs, and relative distribution of REC8 (green), RAD21L grey) and RAD21 (grey).
High density of REC8 constrains sister-chromatid axes and prevents illegitimate synaptonemal complex formation (2016)
Figure 4

Figure 4. Narrow inter-axis distance prevents illegitimate SC formation along sister-chromatids of wild-type X-chromosomes.

A) Representative wild-type pachytene sex chromosomes. Nuclear spreads of pachytene spermatocytes were immunostained for SYCP3. Filled arrowheads indicate the PAR. Dashed area indicated in centre panel corresponds to the magnified view on the right panel. Bars, 10 μm in spreads and 1 μm on insets.

B) Graph indicating the inter-axis distances measured at sites of local separation of axial elements in zygotene-like Stag3 mutant univalents and along pachytene wild-type X-chromosomes. Each measurement in the X-chromosomes corresponds to the median of 3 distances (according to the scheme in figure) (n=54). Each measurement in Stag3 mutant corresponds to 1 distance measured at sites with local separation of axial elements (n=40). Horizontal lines indicate median. p < 0.0001, obtained with two-tailed Mann-Whitney test. Bellow: schematic representation of the XY pair: sister-AEs are coloured in black and grey for easier visualization. Filled arrowhead indicates PAR. Purple bars in scheme indicate regions where inter-axis distances are measured.

C) Representative pachytene wild-type sex chromosomes. Nuclear spreads of pachytene spermatocytes were immunostained for SYCP3 and SYCE2. Dashed area indicated in centre panel corresponds to the magnified view on the right panel. Empty arrowhead indicates two discernable sister-AEs without SYCE2. Bars, 10 μm in spreads and 1 μm on insets.
High density of REC8 constrains sister-chromatid axes and prevents illegitimate synaptonemal complex formation (2016)
Figure 5

Figure 5. Analysis of REC8 distribution along the sister-chromatid axes of wild-type X-chromosomes.

A) Analysis of REC8 distribution in representative pachytene wild-type sex chromosomes. Nuclear spreads of pachytene spermatocytes were immunostained for SYCP3 and REC8. Bars, 10 μm in spreads and 1 μm on insets. Magnified XY pair is indicated by dashed area. Scatterplot showing the number of REC8 foci per X-chromosome. 225 foci identified in 14 X-chromosomes. Horizontal line indicates the mean, error bars indicate s.d. 3.02.

B) Top: representative computationally straightened pachytene wild-type X-chromosomes. Nuclear spreads were immunostained for SYCP3 and REC8. Chromosomes were aligned at the PAR, and subdivided into 25 regular intervals. Bar, 1 μm. Bellow: histogram showing the distribution of the percentages of REC8 foci (n=225) among 25 regular intervals along 14 X-chromosomes. Dotted line indicates mean.

C) Histogram showing the relative frequency of inter-REC8 distances along X-chromosomes.

211 inter-REC8 distances from 14 X-chromosomes. Grey area represents 15% of the chromosome axis length. The respective cumulative distribution function is shown.

D) Histogram showing the best fit of the observed inter-REC8 distances to the gamma distribution. Green curve indicates the best fit of the observed inter-REC8 distances.  indicates the value for which the best fit was obtained, with estimated standard error 0.27.

E) Graph comparing the median distance of REC8 foci with computationally predicted even and random distributions. Median distances between foci subdivided in three categories (11-14, 15-18 and 19-22 REC8 foci per chromosome). Purple asterisk indicates the median distance of all REC8 foci.

F) Scatterplot comparing inter-REC8 distances along on X-chromosomes with the distances between flanking REC8 foci at sites of LSAEs in the Stag3 mutant. 211 inter-REC8 distances measured along X-chromosomes and 10 distances measured between flanking REC8 foci in Stag3 mutant. Grey area represents 15% of the chromosome axis length. Horizontal lines indicate the median. p < 0.0001, obtained with two-tailed Mann-Whitney test.