EMBO/soda-nel huggingface dataset:
Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model (2022)
Figure 6

Figure 6. Quantification of human NOTCH3 ECD protein present in whole blood serum of sham, immunized and non-vaccinated TgN3R182C150 mice (at 3 and 7 months old).

A) NOTCH3 ECD was detected in the whole blood serum of the non-treated TgN3R182C150 mice at three months (n=9) of age and further increased at seven months (n=6) of age. Serum from Notch3 -/- and C57Bl6/J WT mice were included as negative controls. Statistical analysis was performed using unpaired student t test with Welch's correction. (3 m.o. vs 7 m.o. ***P=0.0005). Error bars indicate standard error of the mean (SEM).

B) NOTCH3 ECD in the TgN3R182C150 mice was significantly reduced in the vaccinated (n=10) TgN3R182C150 mice vs sham (n=9). Statistical analysis was performed using unpaired student t test with Welch's correction. (Sham vs Vaccinated *P=0.0196). Error bars indicate standard error of the mean (SEM).
Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model (2022)
Figure 7

Figure 7. Microglia activation and NOTCH3 ECD expression in sham, immunized and non-vaccinated TgN3R182C150 mice at 7 months.

A) Representative images show microglia stained with anti-CD68 antibody (red) and Iba1 antibody (green). Scale bar=20µm.

B) Quantification of CD68-stained area revealed a significant increase in the % of microglia and microglia area between N3 EGF1-5-immunized (n=6), sham (n=4) and non-vaccinated TgN3R182C150 (n=5) mice. Statistical significance was assessed using an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. (% microglia with CD68 staining: 7 m.o. vs. Sham ns P=0.7393; Sham vs. Vaccinated **P=0.0087. CD68 staining (% of microglia area): 7 m.o. vs. Sham ns P=0.9131; Sham vs. Vaccinated *P=0.0157, ns=non-significant). Error bars indicate standard error of the mean (SEM).

C) Representative images of TgN3R182C150, sham- and NOTCH3 EGF1-5-immunized mice at 7 months of age stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an antibody against microglia (Iba1, green). Scale bar=20µm.

D) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3 ECD stained area and average size per microglia revealed no alterations between the NOTCH3 EGF1-5-immunized (n=11), sham (n=8) and non-vaccinated TgN3R182C150 (n=6) mice at 7 months of age. Statistical significance was assessed using an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. (% microglia with NOTCH3 ECD deposits: 7 m.o. vs. Sham ns P=0.7997; Sham vs. Vaccinated ns P=0.0526. NOTCH3 ECD deposits (% of microglia area): 7 m.o. vs. Sham ns P=0.5362; Sham vs. Vaccinated ns P=0.8059. NOTCH3 ECD deposits (number/1000 um2): 7 m.o. vs. Sham ns P=0.8392; Sham vs. Vaccinated ns P=0.5777. NOTCH3 ECD deposits size: 7 m.o. vs. Sham ns P=0.6664; Sham vs. Vaccinated ns P=0.8787, ns=non-significant). Error bars indicate standard error of the mean (SEM).
Redox signals at the ER-mitochondria interface control melanoma progression (2019)
Figure 1

Figure 1: TMX1 and NFAT1 expression is elevated in human melanoma cell lines and patient samples

A, B mRNA levels of TMX1 (A) and NFAT1 (B) in primary melanocytes, keratinocytes and melanoma cell lines.

C Representative immunoblot (from 3) depicting protein abundance of TMX1 and NFAT1 in melanocytes and melanoma cell lines; actin was used as a loading control. Indicated band intensities were normalized to actin and background signal was subtracted.

D Melan-A (brown), TMX1 (red-brown) and NFAT1 (deep red) staining (IHC) in a primary stage 4 high risk nodular melanoma patient I (out of four), the enlarged regions show healthy tissue (panels 2, 5 and 8) and tumor tissue (panels 3, 6 and 9). Arrows indicate Melan-A positive melanocytes.

E TMX1 (red-brown) and NFAT1 (deep red) staining of healthy human skin and increasing melanoma stages; P1-P13 refer to the donor patient number.

Data information: In (D-E), Scale bar 50 µm. In (A-B), data are are normalized to the expression of TBP and are presented as mean ± SEM (n≥3). Statistical significance was addressed using unpaired, two-tailed Student t-test, *P < 0.05; **P < 0.01; ***P < 0.005.
Redox signals at the ER-mitochondria interface control melanoma progression (2019)
Figure 2

Figure 2: TMX1 or TMX3 silencing impair NFAT1 nuclear translocation in melanoma cells

A, B Insulin (50 µg/mL) induced the translocation of NFAT1-GFP to the nucleus of WM3734 cells. (A) Ratio images (F/F0), (B) corresponding endpoint quantification (n=5).

C-H Reduced nuclear translocation of NFAT1-GFP in TMX1 or TMX3 silenced (siRNA) melanoma cells. (C) Images show NFAT1-GFP fluorescence intensity before and after stimulation with thapsigargin (Tg; 1 µM) in WM3734 cells. (D) Corresponding time-dependent nuclear import of NFAT1 as a change of F/F0. (E) Normalized endpoint quantification. Same analysis was performed with Mel Juso cells with (F) images, (G) time-dependent nuclear import and (H) normalized endpoint quantification.

Data information: In (A), (C) and (F) scale bars: 10 µm. In (A) color code: blue=0, red=3. In (E) and (H), data are presented as mean ± SEM (n values: WM3734, control=142, TMX1 kd=116, TMX3 kd=148; Mel Juso, control=75, TMX1 kd=47, TMX3 kd=67). Statistical significance was addressed using unpaired, two-tailed Student t-test, *P < 0.05; **P < 0.01.
Redox signals at the ER-mitochondria interface control melanoma progression (2019)
Figure 3

Figure 3: Silencing of TMX1 or TMX3 causes ROS production

A-C (A) Thapsigargin (Tg) induced Fura-2 based cytosolic Ca2+ imaging in Ringer's buffer containing 0.25 mM Ca2+. (B) Quantification of basal cytosolic calcium levels and (C) SOCE quantification (plateau-basal) for WM3734 after stable silencing of TMX1 (two clones).

D, E (D) Cytosolic Ca2+ imaging (Fura-2) and (E) SOCE quantification (plateau-basal) for WM3734 after transient silencing of TMX1 or TMX3.

F-H Cellular H2O2 (HyPer) was evaluated in two melanoma cell lines upon TMX1 kd and TMX3 kd. (F) Exemplary ratiometric images (F505 nm / F420 nm) are shown for WM3734 and Mel Juso. Quantification of basal cellular H2O2 in WM3734 (G) and Mel Juso (H) cells.

Data information: In (B-C), data are presented as mean ± SEM (n values: WM3734, control=939, TMX1 kds 1=988, TMX1 kds 2=508). In (E), data are presented as mean ± SEM (n values: WM3734, control=30, TMX1 kd=49, TMX3 kd=52). In (G-H), data are presented as mean ± SEM (n values: WM3734: control=168, TMX1 kd=209, TMX3 kd=192; Mel Juso: control=297, TMX1 kd=343, TMX3 kd=440). Statistical significance was addressed using unpaired, two-tailed Student t-test, **P < 0.01. In (F) scale bars: 10 µm; color code: WM3734: color code: blue=0, red=3; Mel Juso: color code: blue=0, red=1.5.
Redox signals at the ER-mitochondria interface control melanoma progression (2019)
Figure 4

Figure 4: ROS inhibit NFAT1 translocation via oxidation of calcineurin

A WM3734 cells were pre-incubated for 5 min with extracellular H2O2 (0 - 500 µM). Tg-stimulated NFAT1-GFP nuclear translocation was measured as F/F0.

B The IC50 of H2O2 inhibition of NFAT1 nuclear import was determined to be 13.2 µM (red mark).

C Tg-induced NFAT1-GFP nuclear import in transient TMX1 silenced WM3734 cells and after pre-incubation with 100 µM N-acetylcysteine (NAC) for 48 h.

D Endpoint quantification of the data from (C) and upon treatment with NAC, PEG-catalase (50 U/mL), or DTT (1 mM).

E Cytosolic calcineurin activity (CaNAR2) in WM3734 cells with transient knockdown of TMX1 or TMX3 measured upon Tg stimulation.

F, G Quantification of the basal calcineurin activity and the maximal calcineurin activity (plateau-basal).

H Normalized Tg-induced cytosolic calcineurin activity in Mel Juso cells after 5 min pre-incubation with extracellular H2O2 (0 - 200 µM).

I Quantification of the maximum cytosolic calcineurin activity (plateau-basal).

J, K Quantification of the basal calcineurin activity and the maximal calcineurin activity (plateau-basal) 8 h after treatment with the antioxidant NAC (100 µM) in WM3734 with transient knockdown of TMX1.

Data information: In (A-B), data show an average of at least 4 single cells from one experiment. In (D), data are presented as mean ± SEM (n values: control=73, control+NAC= 19, TMX1 kd=57, TMX1 kd+NAC=63, TMX1 kd+catalase=58, TMX1 kd+DTT=22). In (F-G), data are presented as mean ± SEM (n values: control=49, TMX1 kd=48, TMX3 kd=63). In (I), data are presented as boxplots (center line: median; box: 25 % and 75 % percentile; whiskers: 1.5 times interquartile range; outliers are shown as dots.) (n≥20). In (J-K) data are presented as mean ± SEM (n values: control=15, TMX1 kd=24, TMX1 kd+NAC=19). Statistical significance was addressed using unpaired, two-tailed Student t-test, *P < 0.05; **P < 0.01; ***P < 0.005.
Redox signals at the ER-mitochondria interface control melanoma progression (2019)
Figure 5

Figure 5: TMX1 silencing causes elevated ROS via mitochondria and NADPH oxidase 4

A-C Mitochondrial H2O2 (mito-HyPer) was measured in two cell lines upon transient TMX knockdown. Exemplary ratiometric images (F505 nm / F420 nm) are shown (A); corresponding quantification for (B) WM3734 and (C) Mel Juso.

D-F Mitochondrial Ca2+ uptake in WM3734 with stable TMX1 knockdown was measured using a mitochondria-targeted calcium sensor (4mt-D3cpV) (D). Cells were exposed to 0.25 mM Ca2+ containing Ringer's buffer and Tg. (E) Quantification of basal mitochondrial Ca2+ levels and (F) mitochondrial Ca2+ uptake (basal-plateau).

G-H Mitochondrial volume was determined using MitoTracker DeepRed in WM3734 cells with stable knockdown of TMX1. The mean mitochondrial volume was modeled based on microscopy, the quantification of mean volume (µm3) and mean surface (µm2) is depicted in (G) and (H) respectively.

I-J Peripheral mitochondria were quantified in WM3734 cells with stable knockdown of TMX1. A peripheral mask was applied based on the membrane staining (CellMask Green) and mitochondria covered area (MitoTracker DeepRed) was evaluated (see Fig. 9). Representative images (I) and quantification (J).

K-N Representative electron micrographs of control and 1205Lu cells with stable TMX1 knockdown (K). Corresponding quantification of the distance between (L) mitochondria and plasma membrane, (M) mitochondria and ER, and (N) the length of the MAM (mitochondria-ER contact site).

O-P Cellular H2O2 concentration (HyPer) 48 h after transient silencing of TMX1 and / or NOX4 in WM3734 cells or after inhibiting NOX4 with GKT137831 (140 nM) (O) Representative ratiometric images (F505 nm / F420 nm) and (P) quantification.

Data information: In (B-C), data are presented as mean ± SEM (n values: WM3734: control=546, TMX1 kd=510, TMX3 kd=621; Mel Juso: control=416, TMX1 kd=418, TMX3 kd=442). In (E-F), data are presented as mean ± SEM (n values: control=62, TMX1 kds=45). In (G-H), data are presented as boxplots (n values: control=101, TMX1 kds=90; center line: median; box: 25 % and 75 % percentile; whiskers: 1.5 times interquartile range; outliers are shown as dots). In (J), data are presented as mean ± SEM (n values: control=163, TMX1 kds=116). In (L), data are presented as mean ± SEM (n values: control=100, TMX1 kds= 100). In (M-N), data are presented as mean ± SEM (n values: control=60, TMX1 kds= 60). In (P), data are presented as mean ± SEM (n values: control=837, TMX1 kd=888, TMX1 kd + NOX4 kd=793, TMX1 kd + GKT=844). Statistical significance was addressed using unpaired, two-tailed Student t-test, *P < 0.05; **P < 0.01; ***P < 0.005. Scale bars in (A, I and O): 10 µm, (K): 1 µm. Color code in (A) WM3734: blue=0, red=6; Mel Juso: blue=0, red=12; (O): blue=0, red=4.
Redox signals at the ER-mitochondria interface control melanoma progression (2019)
Figure 6

Figure 6: TMX1, TMX3 and NFAT1 control melanoma proliferation and invasion

A Proliferation (48 h) of melanoma cell lines measured after transient knockdown of TMX1 or TMX3.

B Proliferation (48 h) of melanoma cell lines with stable TMX1 knockdown (two clones).

C Proliferation (24 h) of melanoma cell lines after transient knockdown of NFAT1.

D Proliferation of WM3734 48 h after transient knockdown of TMX1 and treatment with NAC (100 µM) or mTEMPO (100 nM).

E Transwell migration (48 h) of WM3734 after transient knockdown of TMX1 or TMX3.

F Transwell migration (48 h) of WM3734 after transient knockdown of TMX1 and treatment with NAC (100 µM) or mTEMPO (1 µM).

G Transwell migration (48 h) of melanoma cell lines after preventing NFAT-calcineurin interaction with dipyridamole (40 µM) or inhibition of calcineurin with cyclosporine A (CsA, 2 µM).

H Invasion of WM3734 with stable TMX1 knockdown (two clones) through Matrigel for 96 h.

I-K Mouse xenograft model to assess tumor growth of WM3734 with stable TMX1 knockdown (TMX1 kds; two clones) in immunodeficient mice. (I) Tumor growth in the first 19 days after injection; (J) the corresponding tumor volume quantified after 19 days and (K) after 45 days.

Data information: In (A-G), data are presented as mean ± SEM (n=3). In (H), data are presented as mean ± SEM (n=2). In (I-K), data are presented as mean ± SEM from 7 animals for each group. Statistical significance was addressed using unpaired, two-tailed Student t-test, *P < 0.05; **P < 0.01; ***P < 0.005.
Redox signals at the ER-mitochondria interface control melanoma progression (2019)
Figure 7

Figure 7: NFAT1 regulates tumor associated genes and with TMX and BRAF, they affect disease outcome

A Kaplan-Meier survival plots and log rank for the correlation between mRNA expression levels and survival probability of melanoma patients, separated in groups with high or low expression of any of the three genes of interest (GOI), TMX1, TMX3 and NFAT1. For survival probabilities for the individual genes, please refer to Figure EV5.

B mRNA expression levels of NFAT1, TMX1 and TMX3 in BRAF WT and BRAF V600E melanoma patients.

C-D Kaplan-Meier survival plots and log rank for the correlation between mRNA expression levels and survival probability of melanoma patients, separated in groups with (C) BRAF WT and (D) BRAF V600E and with high or low expression of NFAT1.

E Venn diagram showing the respective numbers of differentially expressed genes after NFAT1 knockdown.

F From the differentially expressed genes in (E), mitochondrion-localized and redox-related proteins were more than by chance downregulated after NFAT1 knockdown.

G Differentially expressed genes, categorized by Gene Ontology terms annotations covering established hallmarks of cancer, after NFAT1 knockdown, presented as fold-enrichment.

Data information: In (A, C, D), shaded areas depict the 95 %-confidence intervals of the survival probabilities and significance was assessed using log-rank tests. In (B), expression levels are based on the FPKM values of the TCGA samples (center line: median; box: 25 % and 75 % percentile; whiskers: 1.5 times interquartile range). In (F-G), data are presented as fold-enrichment of the genes affected by knockdown of NFAT1. The significance of the fold change in (F) and (G) was determined with a hypergeometric test, adjustment by the Benjamini-Hochberg procedure, FDR=0.05, *adjusted P < 0.05; ***adjusted P < 0.005.
Tissue resident macrophages actively suppress Il-1β release via a reactive prostanoid/IL-10 pathway (2020)
Figure 1

Fig. 1 Aberrant cytokine release from LPS-stimulated Gata6-deficient resident peritoneal macrophages.

(A and B) Gata6-WT and Gata6-KOmye peritoneal macrophages (pMɸ) were unstimulated (-) or stimulated with TLR2L Pam3CSK4 (500 ng.ml-1), TLR3L Poly I:C (1 µg.ml-1), TLR4L purified LPS (100 ng.ml-1), TLR5L Flagellin (100 ng.ml-1), TLR7 and 8L R848 (1 µg.ml-1) or TLR9L CpG ODN1826 (5 µM). Culture supernatants were collected 24 h after the start of stimulation and IL-1β and TNF ELISA were performed. n=5, two-way ANOVA analysis with Tukeys multiple comparison post-test.

(C, D) IL-1β (C) and TNF (D) ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 24 h with the indicated LPS concentrations. n=3, two-way ANOVA analysis with Sidak's multiple comparison post-test.

(E, F) IL-1β (E) and TNF (F) ELISA from Gata6-WT and Gata6-KOmye pMɸ stimulated with LPS (100 ng.ml-1) for the indicated times. n=3, two-way ANOVA analysis with Sidak's multiple comparison post-test.

(G) IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 18 h with 100 ng.ml-1 LPS or recombinant IL-1 receptor antagonist (rIL-1ra), n=4 to 5, two-way ANOVA analysis with Tukey's multiple comparison post-test.

(H) IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 18 h with 100 ng.ml-1 LPS or 100 ng.ml-1 recombinant TNF (recTNF), n=4 to 5, two-way ANOVA analysis with Tukey's multiple comparison post-test.

Data information: Data are expressed as mean +/- SEM. *p<0.05, **p<0.01, ***p<0.001
Tissue resident macrophages actively suppress Il-1β release via a reactive prostanoid/IL-10 pathway (2020)
Figure 2

Fig. 2 The aberrant release of IL-1β by Gata6-KO-deficient pMɸ follows classical inflammasome activation.

(A) Normalised expression of Tlr and Cd14 genes following microarray analysis performed on unstimulated cell-sorted Gata6-WT and Gata6-KOmye pMɸ. Data are shown as mean +/- SEM from three biological replicates. Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by the Benjamin-Hochberg procedure. n.d = non detectable.

(B) Mean fluorescence intensity (MFI) of extracellular TLR2, TLR4 and CD14 expression on naïve Gata6-WT and Gata6-KOmye pMɸ. n = 4 to 7 individual mice per group.

(C, D) Il1b and Tnf mRNA relative expression (C) and IL-1β Western blot protein analysis (D) of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are representative of at least 3 independent experiments. Western blot was performed on whole cell lysates.

(E) Representative dot plot, percentage and mean fluorescence intensity (MFI) analysis of pro-IL-1β+ Gata6-WT and Gata6-KOmye pMФ flow cytometry analysis 3 h after stimulation with 100 ng.ml-1 LPS. n = at least 3 independent experiments.

(F) Nlrp3 mRNA relative expression of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h. Data shown are pooled from 3 independent experiments.

(G) Western blot protein analysis of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 6 h. Data shown are representative of at least 3 independent experiments. Western blot was performed on whole cell lysates.

(H, I) IL-1β ELISA (H) and Western blot protein analysis (I) of supernatants collected from Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS and either vehicle control (Vh, DMSO) or 10 µM MCC950 for 24 h (n=5). Data shown in (H) are pooled from 5 independent replicates.

(J, K) Caspase1 (Casp1) mRNA relative expression (J) and Western blot protein analysis (K) of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are pooled from 3 independent experiments.

(L) IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS and either vehicle control (Vh, DMSO) or Ac-YVAD-cmk for 24 h. Data shown are pooled of 5 independent replicates.

(M) IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h, followed by a 30 min pulse with either vehicle control (Vh), 5 mM ATP or 20 µM Nigericin. Data shown are pooled of 5 independent replicates.

(N) Representative picture of confocal immunofluorescence analysis of Gata6-WT and Gata6-KOmye pMФ stimulated with 100 ng.ml-1 LPS for 3 h, followed by a 30 min pulse with 5 mM ATP. The white arrows show ASC specks. Scale bar = 10 µm.

Data information: Data are expressed as mean +/- SEM and analysis were performed using two-way ANOVA analysis Tukey's multiple comparison post-test unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001
Tissue resident macrophages actively suppress Il-1β release via a reactive prostanoid/IL-10 pathway (2020)
Figure 3

Fig. 3 Prostanoid production is imbalanced in Gata6-KOmye pMФ.

(A) IL-1β ELISA analysis of supernatants of Gata6-WT and Gata6-KOmye pMɸ in monoculture or co-cultured (ratio 1:1) and stimulated for 24 h with 100 ng.ml-1 LPS. Data shown are pooled from 3 independent experiments. One-way ANOVA statistical analysis with Tukey's multiple comparison test was performed.

(B) IL-1β ELISA analysis of supernatants of Gata6-WT and Gata6-KOmye pMɸ co-cultured in the same well (co-culture) or using Transwell system (ratio 1:1) and stimulated for 24 h with 100 ng.ml-1 LPS. Data shown are pooled from 3 independent experiments. One-way ANOVA statistical analysis with Tukey's multiple comparison test was performed.

(C) Microarray analysis of Ptgis, Tbxas1, Ptges2 and Ptgs1 expression from Gata6-WT and Gata6-KOmye pMɸ isolated from naïve mice. Data is shown as mean +/- SEM from three biological replicates. Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by the Benjamin-Hochberg procedure.

(D) Western blot analysis of Ptgis protein level of unstimulated pMФ from Gata6-WT and Gata6-KOmye mice.

(E) Mass spectrometry analysis of 6-keto-PGF1α, TXB2 and PGE2 content of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated for 3 h with 100 ng.ml-1 LPS. n = 6. Two-way ANOVA statistical analysis with Tukey's multiple comparison post-test was performed.

(F) Representation of the variation of the prostanoid synthesis pathway in Gata6-KOmye pMФ created using Cytoscape software. Circle shape represent lipids, diamond shape enzymes, yellow downregulation and purple upregulation of the expression/production in Gata6-KOmye cells. The size of the circles represents relative levels observed in Gata6-KOmye cells.

Data information: Data is shown as mean +/- SEM. *p<0.05, **p<0.01, ***p<0.001
Tissue resident macrophages actively suppress Il-1β release via a reactive prostanoid/IL-10 pathway (2020)
Figure 4

Fig. 4 The Gata6-PGI2-IL-10 pathway controls IL-1β production in Gata6-WT pMФ.

(A) IL-10 ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS. Data shown are representative of at least 5 independent experiments.

(B, C) IL-10 (B) and IL-1β (C) ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, beraprost (1 and 10 µM), cicaprost (1 and 10 µM), Iloprost (1 or 10 nM) or vehicle control (Vh, DMSO). Data shown are representative of at least 3 independent experiments.

(D, E) IL-10 (D) and IL-1β (E) ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM beraprost, 10 µM PGE2, 10 µM U46619, 100 µM picotamide or vehicle control (Vh, methyl acetate). Data shown are representative of at least 3 independent experiments.

(F) IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 ng.ml-1 IL-10, 10 µM beraprost or vehicle control (Vh). Data shown are representative of at least 3 independent experiments.

(G) IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM beraprost, 5 µg.ml-1 αIL-10R or isotype antibody. Data shown are representative of at least 3 independent experiments.

(H, I) IL-1β (H) and IL-10 (I) ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM MCC950, 5 µg.ml-1 αIL-10R or isotype antibody All stimulations were performed for 16 h. Data shown are representative of at least 3 independent experiments.

(J) IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 ng.ml-1 IL-10 and 5 mM ATP. Data shown are a pool of 3 independent experiments.

Data information: Data are expressed as mean +/- SEM and two-way ANOVA statistical analysis with Tukey's multiple comparison post-test were performed. *p<0.05, **p<0.01, ***p<0.001