EMBO/soda-nel huggingface dataset:
Phospholipid oxidation generates potent anti-inflammatory lipid mediators that mimic structurally related pro-resolving eicosanoids by activating Nrf2. (2015)
Figure 4

Epoxycyclopentenone lipids inhibit the inflammatory response of myeloid cells via Nrf2 signaling

A IL-12 production of BMDC from wild-type, Nrf2−/−, Pparg−/−, and Pparg litter mate control mice normalized to medium control (open bars). Cells were treated with the indicated lipids (filled bars) for 60 min prior TLR 7 ligation with R837 (5 μg/ml) for 18 h. Lipids were used at starting concentrations of 40 μM (POVPC, PGPC, and KOdiAPC), 40 μg/ml (OxPAPC), 20 μM (15d-PGJ2), and 1.25 μM (EC), depicted as black bars, and 2-fold serial dilutions thereof (gray bars). Data represent mean ± SEM of triplicates from one of three independent experiments.

B Expression of Nrf2 target genes Hmox1 and Nqo1 in wild-type and Nrf2−/− BMDM stimulated with EC (2 μM) for 60 min. Gene expression levels are presented relative to that of untreated cells after normalization to G6pdx. Data (mean ± SEM) are representative of two independent experiments.

C, D mRNA expression levels of the Nrf2 targets Gclc and Gsta3 (C) and of the pro-inflammatory cytokines IL-6 and IL-12 (D) in wild-type, PPAR-γ-deficient, and Nrf2-deficient BMDM after treatment with EC or 15d-PGJ2 for 60 min followed by LPS treatment for 3 h. Expression levels are normalized to G6pdx. Data represent mean ± SEM of triplicate cultures from one of two independent experiments.

E mRNA expression of the indicated chemokines as determined by qPCR. Wild-type BMDCs were treated with EC (1 μM) or 15d-PGJ2 (20 μM) for 60 min followed by R837 stimulation (5 μg/ml) for 3 h. Expression levels are shown normalized to G6pdx. Data (mean ± SEM, n = 2) are representative of three independent experiments.

Data information: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant; as determined by one-way ANOVA adjusted by Dunnett's multiple comparisons test.
Phospholipid oxidation generates potent anti-inflammatory lipid mediators that mimic structurally related pro-resolving eicosanoids by activating Nrf2. (2015)
Figure 5

EC mitigates sepsis-associated inflammation in vivo

A C57BL/6 mice were treated (i.v.) with 500 μg EC or DPPC control 2 h prior to i.p injection of 150 ng/g LPS together with 800 μg/g D-galactosamine. 4 h after LPS application, lungs were perfused with PBS and embedded in paraffin. Tissue sections were hematoxylin-stained to visualize adherent cells. Bars represent 100 ?m.

B, C Leukocyte adhesion to lung microvascular endothelium as determined by morphometric image analysis of lung tissue sections is presented for individual vessels in (B) and as averages of single mice in (C). Pooled data of two independent experiments are shown (n = 10 for EC, n = 14 for DPPC). Unpaired two-tailed t-test.

D, E C57BL/6 mice were treated with EC or DPPC by intra-tracheal instillation at 18 h (50 μg) and 1.5 h (100 μg) prior to i.p. injection of 150 ng/g LPS and 800 μg/g D-galactosamine. Bar graphs represent absolute numbers of total infiltrating cells and of neutrophils (D). Unpaired two-tailed t-test. Data represent mean ± SEM from one of two independent experiments with at least 6 mice per group. (E) Dot plots depict exemplary gating of lung neutrophils on pregated CD45+ CD11c− SiglecF− BAL cells of EC-/LPS-treated and DPPC-/LPS-treated mice.

F, G The concentrations of IL-6 (F) and IL-12 (G) in the BAL of mice treated as in (D) were quantified by ELISA. Data (mean ± SEM) are representative of two independent experiments with 3 mice per group. ****P < 0.0001; unpaired two-tailed t-test.
Phospholipid oxidation generates potent anti-inflammatory lipid mediators that mimic structurally related pro-resolving eicosanoids by activating Nrf2. (2015)
Figure 6

Structure–function studies identify critical molecular determinants of the anti-inflammatory bioactivity

Dose–response curves showing the modulation of R837-induced (5 μg/ml; 18 h) IL-12 secretion by prior treatment of BMDCs with the indicated synthetic lipids for 1 h.

Chemical structures and ΔEC50 values of the synthetic OxPL variant lipids analyzed in (A), presented relative to EC.

Data information: Mean ± SEM of triplicate determinations are shown.
Phospholipid oxidation generates potent anti-inflammatory lipid mediators that mimic structurally related pro-resolving eicosanoids by activating Nrf2. (2015)
Figure 7

Cyclo-EC is an EC variant with superior anti-inflammatory bioactivity in vitro and in vivo

Chemical structure of the cyclic EC analog cyclo-EC.

Quantification of IL-6 and IL-12 secretion by BMDC treated with 250 nM cEC or EC for 60 min before stimulation with R837 (5 μg/ml; 18 h). Bars represent mean ± SEM from one of three independent experiments. **P < 0.01; ***P < 0.001; one-way ANOVA adjusted by Dunnett's multiple comparisons test.

mRNA expression of the Nrf2 targets Hmox1 and Nqo1 normalized to G6pdx expression. BMDCs were treated for 60 min with 500 nM cEC or DPPC followed by R837 stimulation (5 μg/ml) for 3 h. Data (mean ± SD) are representative of three independent experiments. *P < 0.05; **P < 0.01; unpaired two-tailed t-test.

ΔEC50 values of the indicated lipids and OxPL shown relative to that of EC as determined in (E).

Dose–response curves showing the modulation of R837-induced (5 μg/ml; 18 h) IL-12 secretion in BMDCs by prior treatment with the indicated OxPL derivatives for 1 h.

mRNA quantification of the indicated chemokines relative to G6pdx expression. BMDC were pretreated for 60 min with 500 nM cEC or the variant BisRed prior to R837 stimulation (5 μg/ml) for 3 h. Data (mean ± SD) are representative of three independent experiments. *P < 0.05; **P < 0.01; unpaired two-tailed t-test.

Quantification and characterization of cellular infiltrates in BAL. Groups of six C57BL/6 mice were pretreated i.t. with 50 μg cEC, EC, or BisRed 24 and 2 h before challenge with 150 ng/g LPS in the presence of 800 μg/g D-galactosamine. BAL was harvested 4 h after LPS injection, and inflammatory cells were characterized by FACS analysis. **P < 0.01; ****P < 0.0001; one-way ANOVA with Sidak's multiple comparisons test.

Comparison of the capacity of cEC, EC, and PECPC (1 μM) to license splenic dendritic cells to polarize naïve CD4 T cells into IFN-γ-producing (Th1) and IL-4-producing (Th2) effector cells. Data (mean ± SD) are representative of two independent experiments. One-way ANOVA adjusted by Dunnett's multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
IκBα deficiency imposes a fetal phenotype to intestinal stem cells (2020)
Figure 1

Figure 1. Nuclear localization and chromatin binding of P-IκBα in intestinal crypts.

(A-B) Immunofluorescence (IF) analysis of IκBα in sections from murine small intestine of 2 month-old WT (A) and Lgr5-GFP reporter mice (B). Scale bars, 50μm.

(C-D) IF analysis of P-IκBα in the intestine of 2 month-old WT (C) and Lgr5-GFP reporter mice (D), and quantification of P-IκBα positivity inside the Lgr5-GFP population. A minimum of 30 crypts was counted in 3 Lgr5-GFP mice. Scale bars in C, 25 μm and in D, 50 μm.

(E) Distribution of P-IκBα positive cells according to their position in the intestinal crypt from 200 crypts counted.

(F) Double IF analysis of 2 month-old murine small intestine sections with the indicated antibodies and quantification of P-IκBα positivity inside the EphB2+ population. 70 crypts were counted. Bars represent mean values ± standard error of the mean (s.e.m). Scale bars, 50 μm.

(G) Double IF analysis of murine small intestine sections with the indicated antibodies. Scale bars, 50 μm.

(H) Western blot analysis of cytoplasmic (CYT) and nuclear (NUC) extracts from isolated intestinal crypt cells.

(I) Western blot analysis of crypt nuclear extracts immunoprecipitated with anti-SUMO2/3 antibody.

(J) IF analysis of developing intestines from E12.5 to E17.5 with the indicated antibodies. Scale bars, 25 μm.

(K) Representation of IκBα distribution in the indicated genomic regions obtained from ChIP-sequencing analysis (n=4).
IκBα deficiency imposes a fetal phenotype to intestinal stem cells (2020)
Figure 2

Figure 2. Altered intestinal homeostasis in IκBα-deficient mice.

(A) Representative images of P4 and P6 mice of the indicated IκBα genotypes.

(B-C) Quantification of the animal weight (B) and intestinal length (C) from 50 WT/HET and 15 KO mice analyzed.

D-G (D) Immunohistochemical (IHC), IF ­and Alcian Blue staining with quantification of P6 IκBα WT and KO intestines (D), 2 month-old IκBβ;IκBε KO (E) and 2 month-old IκBαNES mice (F and G). A minimum of 50 crypts/villus was counted per genotype (3 mice).

Data information: In B, C, D, E and G, bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, * p-value<0.05, n.s. no significant. Scale bars in D, E, F and G, 50 μm.
IκBα deficiency imposes a fetal phenotype to intestinal stem cells (2020)
Figure 3

Figure 3. IκBα-deficient intestinal stem cells display a fetal-like phenotype. (A) Representative image of the intestinal populations purified in the cell sorting experiments.

(B) IF analysis of EphB2 in P6 WT and IκBα KO intestine.

(C) P-IκBα IF of EphB2-high or negative sorted cells.

(D) Clustering analysis of the indicated cell populations based on their transcriptional profiles obtained in RNA-seq. 3 mice per genotype were initially processed although 1 EphB2neg KO replicate was excluded from the analysis due to insufficient number of reads in the RNA seq.

(E) Correlation plot of the indicated differentially expressed gene (DEG) sets. Values for DEG in fetal and adult ISC was obtained from Nusse et al, 2012.

(F) Table indicating the top 15 up-regulated genes in the IκBα KO EphB2-high signature including 11 genes in the fetal ISC signature (in orange).

(G) GSEA of EphB2-high cells indicating the enrichment of relevant ISC-related pathways.

(H) Representative image of the Lgr5+ population purified in the cell sorting experiments.

(I) qPCR analysis of the indicated genes in purified Lgr5+ cells from the different IκBα backgrounds. 4 WT and 1 KO mice were analyzed with at least three technical replicates. Bars represent mean values ± standard error of the mean (s.e.m). p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, **p-value<0.005, *p-value<0.05, n.s. no significant.

Data information: Scale bars in B and C, 25 μm.
IκBα deficiency imposes a fetal phenotype to intestinal stem cells (2020)
Figure 4

Figure 4. Normal NF-κB but altered PRC2 function in IκBα-deficient intestine.

(A) IF analysis of c-Rel and RelA/p65 in P6 WT and IκBα KO intestines.

(B) Number of peaks from p65 ChIP-sequencing analysis associated with the different genomic localizations was obtained merging two biological replicates per condition (n=2 P6 WT and n=2 P6 IκBα KO intestinal crypt cells). Data from individual experiments have been deposited at GSE131187.

(C-D) IF analysis of active Notch1 (ICN1) (C) and β-catenin (D) in the indicated IκBα backgrounds at P6 intestines.

(E) IF analysis of the PRC2 protein EZH2 and quantification of the EZH2 positive cells per crypt in n=3 P6 animals per genotype were analyzed (a minimum of 15 crypts was counted per genotype). Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001.

(F) Table indicating the methylation-related genes (PRC2 members and KDM6 demethylase) differentially express between IκBα WT and KO EphB2-high sorted cells (RNA-seq).

(G) Western blot analysis of IκBα IP from crypt nuclear extracts.

(H) Venn diagram representing the distribution of genes detected as differentially methylated in the H3K27me3 ChIP-sequencing analysis across P6 IκBα genotypes (n=2 WT and n=2 KO mice).

(I) Representation of H3K27me3 distribution at embryonic E14.5 (n=2 WT and n=2 KO mice) and postnatal P6 (n=2 WT and n=2 KO mice) (from ChIP-sequencing) and expression levels (from RNA-sequencing) in the genomic region of the indicated fetal ISC genes.

(J) Representation of IκBα (n=4) and H3K27me3 (n=2) distribution (from ChIP-sequencing) and expression levels (from RNA-sequencing) in the genomic region of Litaf.

Scale bars in A, C, D and E, 25 μm.
IκBα deficiency imposes a fetal phenotype to intestinal stem cells (2020)
Figure 5

Figure 5. IκBα deficient-derived organoids display a fetal ISC phenotype. (A) Representative stereoscopic image of P6 WT and IκBα KO-derived intestinal organoids.

(B) IHC analysis with the indicated antibodies and Alcian Blue staining of WT and IκBα KO-derived organoids.

(C) qPCR analysis of the indicated ISC genes in WT and IκBα KO-derived organoids.

(D) Correlation plots of the indicated differentially expressed gene (DEG) sets obtained in expression microarray (5 technical replicates of a minimum of two organoids per genotype were analyzed). Values for DEG in fetal and adult organoids were obtained from Mustata et al, 2013.

(E) qPCR analysis of the indicated fetal genes in WT and IκBα KO-derived organoids.

(F) Western blot analysis of control and IκBα-deleted organoids using CRISPR-Cas9 technology.

(G) Representative stereoscopic image of WT and IκBα-deleted organoids.

Data information: In C and E, 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean (s.e.m). p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, **p-value<0.005, * p-value<0.05. Scale bars in A, 100 μm, and in B and G, 50 μm.
IκBα deficiency imposes a fetal phenotype to intestinal stem cells (2020)
Figure 6

Figure 6. Fetal ISC phenotype of IκBα deficient organoids is PRC2, Notch and IFN dependent.

A) Representative stereoscopic images and Western blot analysis of IκBα KO organoids infected with the indicated sh-RNA for NF-κB elements.

(B) qPCR analysis of the indicated ISC and differentiation genes in the different sh-RNA treated organoids. The canonical NF-κB target gene Cxcl10 is shown as positive control of the experiment.

(C) Representative stereoscopic images of IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ-6438.

(D-E) qPCR analysis of the indicated adult and fetal ISC genes (D) and elements of the Notch and IFN pathway (E) (differentially expressed in IκBα KO organoids) in IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ-6438.

(F) Representative stereoscopic images of IκBα KO organoids treated with dexamethasone as IFN inhibitor and DAPT as Notch inhibitor.

(G) qPCR analysis of IκBα KO organoids untreated or treated with dexamethasone and DAPT.

Data information: In B, D, E and G, 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant. Scale bars in A, 100 μm, and in C and F, 50 μm.
IκBα deficiency imposes a fetal phenotype to intestinal stem cells (2020)
Figure 7

Figure 7. Nuclear accumulation and functional involvement of IκBα in the regenerating intestinal epithelium.

(A) IF analysis of colonic tissue from control and DSS-treated animals including acute damaged (AD) areas and recovery (R) areas.

(B-C) IF analysis of P-IκBα and the indicated markers in the areas of acute damage and recovery (B) and colocalization of P-IκBα with the ISC marker Olfm4 in acute damaged areas (C). Asterisks indicate the presence of rare cells with nuclear P-IκBα.

(D) qPCR analysis of the indicated genes in WT and IκBα KO organoids treated with TNFα and collected at the indicated time-points. 3 technical replicates of organoids from each genotype were analyzed.

(E-F) Representative images of the colonic tissue from 2 months-old DSS-treated WT and IκBα KO mice are shown. Alcian Blue staining was used to identify the mucus-secreting goblet cells in the colonic glands and Ki67 as proliferation marker. Nuclear counterstain is Fast Red. The table shows number of ulcerations present in the intestines of mice analysed (3 WT, 2 IκBα KO).

Data information: Scale bars in A, E and F, 100 μm, and in B and C, 50 μm.
m6A methylation promotes white to beige fat transition by facilitating Hif1a translation (2021)
Figure 1

Figure 1. Ablation of Fto in Adipose Tissue Resists HFD-Induced Obesity.

(A) Representative photographs of 16-week-old male Ftoflox/flox and FtoAKO (Ftoflox/flox; Adipoq-Cre) mice fed a HFD. Scale bar, 2 cm.

(B) The growth curve of Ftoflox/flox and FtoAKO mice fed a HFD (n = 8).

(C) Food intake of Ftoflox/flox and FtoAKO mice fed a HFD.

(D) The ratio of weight of iWAT, eWAT and BAT to body weight in 16-week-old Ftoflox/flox and FtoAKO mice fed a HFD.

(E) Representative photos of iWAT, eWAT and BAT from Ftoflox/flox and FtoAKO mice fed a HFD. Scale bar, 1 cm.

(F) Representative H&E staining of iWAT, eWAT and BAT from Ftoflox/flox and FtoAKO mice fed a HFD. Scale bar, 200 μm.

(G) Adipocyte sizes of iWAT from Ftoflox/flox and FtoAKO mice fed a HFD were estimated from the H&E staining results in (F) using ImageJ. The percentage of cells with the indicated sizes is shown.

(H) A representative image (upper) and H&E staining (lower) of liver from Ftoflox/flox and FtoAKO mice fed a HFD. Upper image, scale bar, 0.5 cm. Lower image, scale bar, 50 μm.

(I-K) Serum levels of glucose (I), triglyceride (J) and free fatty acid (FFA) (K) in Ftoflox/flox and FtoAKO mice fed a HFD followed by fasting.

(L, M) The blood glucose level of Ftoflox/flox and FtoAKO mice fed a HFD after intraperitoneal injection of glucose or insulin for glucose (GTT) (L) and insulin tolerance tests (ITT) (M), respectively.

Data information: The data were presented as the mean ± SD (n = 6). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
m6A methylation promotes white to beige fat transition by facilitating Hif1a translation (2021)
Figure 2

Figure 2. Adipose-Specific Fto Depletion Enhances Energy Expenditure and Induces Browning of White Adipocytes.

(A) O2 consumption of 16-week-old Ftoflox/flox and FtoAKO mice fed a HFD. White and gray areas in the graphs indicate day and night, respectively.

(B) CO2 generation of 16-week-old Ftoflox/flox and FtoAKO mice fed a HFD. White and gray areas in the graphs indicate day and night, respectively.

(C) Energy expenditure of 16-week-old Ftoflox/flox and FtoAKO mice fed a HFD. White and gray areas in the graphs indicate day and night, respectively.

(D) The rectal temperature of Ftoflox/flox and FtoAKO mice fed a HFD at room temperature (n = 6).

(E) The rectal temperature of Ftoflox/flox and FtoAKO mice fed a HFD during acute cold exposure (4°C) and fasting for 6 h.

(F) qPCR analysis of genes involved in thermogenesis, mitochondrial biogenesis and adipogenesis in iWAT of Ftoflox/flox and FtoAKO mice.

(G) Western blot analysis of the protein levels of UCP1, PPARGC1A and PPARG in iWAT of Ftoflox/flox and FtoAKO mice.

(H) Immunohistochemical detection of UCP1 in iWAT from Ftoflox/flox and FtoAKO mice following 6 days cold exposure (4°C). Scale bar, 200 μm.

(I) Immunofluorescence detection of UCP1 in iWAT from Ftoflox/flox and FtoAKO mice following 6 days cold exposure (4°C). Scale bar, 20 μm.

(J) Western blot analysis of FTO protein level in differentiated beige adipocytes from Ftoflox/flox mice infected with retroviruses MSCVhygro expressing vector (Vec) or Cre.

(K) The O2 consumption rate (OCR) in differentiated beige adipocytes described in (J). The average basal and maximal respiration rates are shown in lower panel.

(L) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in (J).

(M) Western blot analysis of UCP1, PPARGC1A and PPARG protein levels in differentiated beige adipocytes described in (J).

(N) Immunofluorescent staining of UCP1 in differentiated beige adipocytes described in (J). Scale bar, 20 μm.

Data information: The data were presented as the mean ± SD of n = 6 mice per group or triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗∗p < 0.01, ∗∗∗p < 0.001.
m6A methylation promotes white to beige fat transition by facilitating Hif1a translation (2021)
Figure 3

Figure 3. FTO Regulates Thermogenic Gene Expression via Targeting HIF1A.

(A) Gene Ontology analysis of up-regulated genes of siCtrl and siFto 3T3-L1 cells. P value was calculated by Fisher's Exact test in DAVID (The Database for Annotation, Visualization and Integrated Discovery).

(B) Western blot analysis of the protein level of HIF1A in iWAT of Ftoflox/flox and FtoAKO mice (n = 6).

(C) qPCR analysis of mRNA level of Hif1a in iWAT of Ftoflox/flox and FtoAKO mice (n = 6).

(D) qPCR analysis of mRNA level of Hif1a in differentiated beige adipocytes from Ftoflox/flox mice infected with retroviruses MSCVhygro expressing Vec or Cre.

(E) Western blot analysis of the protein level of HIF1A in differentiated beige adipocytes described in (D).

(F) qPCR analysis of mRNA levels of Ucp1, Ppargc1a, Prdm16 and Pparg in differentiated beige adipocytes from Ftoflox/flox mice infected with retroviruses MSCVhygro expressing Vec, Cre or transfected with Hif1a siRNA.

(G) Western blot analysis of the protein levels of FTO, HIF1A, UCP1, PPARGC1A and PPARG in differentiated beige adipocytes described in (F).

Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test or ANOVA. ∗∗p < 0.01, ∗∗∗p < 0.001.
m6A methylation promotes white to beige fat transition by facilitating Hif1a translation (2021)
Figure 4

Figure 4. HIF1A Facilitates Browning of White Adipocytes by Activating Transcription of Thermogenic Genes.

(A) Western blot analysis of HIF1A and FTO protein levels in 3T3-L1 cells overexpressing Vec or HIF1A plasmid.

(B) Western blot analysis of HIF1A and FTO protein levels in siCtrl or siHif1a 3T3-L1 cells.

(C) The O2 consumption rate (OCR) in differentiated beige adipocytes from 3T3-L1 cells overexpressing Vec or HIF1A plasmid. The average basal and maximal respiration rates are shown in right panel.

(D) The O2 consumption rate (OCR) in differentiated beige adipocytes from siCtrl or siHif1a 3T3-L1 cells. The average basal and maximal respiration rates are shown in right panel.

(E) Western blot analysis of UCP1, PPARGC1A and PPARG in differentiated beige adipocytes described in (C).

(F) Western blot analysis of UCP1, PPARGC1A and PPARG in differentiated beige adipocytes described in (D).

(G) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in (C).

(H) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in (D).

(I) Immunofluorescent staining of UCP1 in differentiated beige adipocytes described in (D). Scale bar, 20 μm.

(J) ChIP-qPCR analysis of the association of HIF1A with the promoter of thermogenic genes in 3T3-L1 cells differentiated into beige adipocytes. IgG was used as control. Consensus HIF1A binding sites were obtained from JASPAR database (a database of transcription factor binding profiles).

Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.