EMBO/soda-nel huggingface dataset:
Genome-wide cooperation of EMT-transcription factor ZEB1 with YAP and AP-1 in breast cancer (2020)
Figure 4

Figure 4. ZEB1, YAP and AP-1 factors together activate target gene transcription.

(A) qRT-PCR in TGFß treated MCF10A cells after siRNA mediated knockdown of ZEB1, FOSL1 or YAP compared to control knockdown cells. Knockdown efficiency of all three factors is shown on the left. The right panel shows their effects on 11 ZEB1/YAP/AP-1 activated target genes. n = 3; shown is mean ± s.e.m.; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.; unpaired two-tailed Student's t-test.

(B) Overlap of ZEB1, YAP and JUN peaks at the promoter of CYR61, CTGF and ANKRD1 and in a known enhancer region of the DOCK9 gene.

(C) ZEB1-only peak at the promoter of LLGL2, a well-known ZEB1 repressed target.

(D) Luciferase reporter assay in MDA-MB-231 cells comparing control and transient siRNA-mediated knockdown of ZEB1. Reporter constructs of regulatory regions of genes shown in (B) and (C). n = 3-4; shown is mean ± s.e.m.; *P ≤ 0.05; ***P ≤ 0.001., unpaired two-tailed Student's t-test.

(E) Luciferase reporter assays with the ANKRD1 promoter and DOCK9 enhancer constructs in MCF7 cells upon overexpression of ZEB1, YAP and JUN/FOSL1 (AP-1) or different combinations of the factors. n = 5 (ANKRD1) and n = 3 (DOCK9); shown is mean ± s.e.m.; *P ≤ 0.05; ratio paired t-test. For reporter assays, firefly luciferase activity was normalized to co-transfected renilla luciferase.
Genome-wide cooperation of EMT-transcription factor ZEB1 with YAP and AP-1 in breast cancer (2020)
Figure 5

Figure 5. The canonical high affinity ZEB1 motif is dispensable for the formation of a ZEB1 activator complex.

(A) Enrichment of the ZEB1, AP-1 and TEAD4 DNA-binding motifs is shown, as identified by HOMER known motif analysis on 200 bp regions centred on different subsets of ZEB1 peak summits. An extended list of the identified motifs can be found in Appendix Table S3 and S4.

(B) Hypergeometric testing showed that while there is a significant association between the AP-1 and TEAD4 motif within ZEB1 peaks, both motifs are associated with the absence of the ZEB1 consensus motif (Z-box). Odds ratio (OR): measure of association between categorical variables with OR>1 indicating a positive association. P-value from Fisher's exact test.

(C) The upper panel shows a schematic representation of the ANKRD1 luciferase reporter constructs. The lower panel shows luciferase reporter assays with these different ANKRD1 promoter constructs in MCF7 cells upon overexpression of ZEB1, YAP and JUN/FOSL1 or different combinations of the factors. n = 4-5; shown is mean ± s.e.m.; *P ≤ 0.05; **P ≤ 0.01; ratio paired t-test. Firefly luciferase activity was normalized to co-transfected renilla luciferase. The wild type assay corresponds to the assay shown in Figure 4E.

(D) Left panel: EMSA using nuclear extracts from MDA-MB-231 and labelled TEAD binding side as probe (black arrow). Competition of specific binding complex (read arrow) with cold probe (c), anti-JUN, -panTEAD, and -ZEB1 antisera. Right panel: recombinant GST-ZEB1, but not GST-Ctrl, only binds to Z-box.
Genome-wide cooperation of EMT-transcription factor ZEB1 with YAP and AP-1 in breast cancer (2020)
Figure 6

Figure 6. Differential association of ZEB1-only and ZEB1/YAP/JUN peaks with active and repressed genomic elements.

(A) Enrichment of the ZEB1, AP-1 and TEAD4 DNA-binding motifs identified by HOMER known motif analysis on 200 bp regions centred on ZEB1 peak summits associated with repressed or activated genomic regions identified by ATAC-seq. An extended list of the identified motifs can be found in Appendix Table S6 and S7.

(B) Comparison of ZEB1 ChIP-seq peaks with ZEB1-dependent changes in chromatin activation status identified by ATAC-seq on control and ZEB1 knock-down MDA-MB-231 cells. Percentage of ZEB1-only (n=5,963) and ZEB1/YAP/JUN peaks (n=1,993) falling into ZEB1 repressed or activated genomic regions are shown.

(C) Genome browser tracks of ZEB1, YAP and JUN ChIP-seq signal intensity in wild type cells overlaid to ATAC-seq signal intensity in wild type, control (shCtrl) and ZEB1 (shZEB1) knock-down MDA-MB-231 cells. One known ZEB1 repressed target (CDH1) and one activated target (ANKRD1) are shown.
Genome-wide cooperation of EMT-transcription factor ZEB1 with YAP and AP-1 in breast cancer (2020)
Figure 7

Figure 7. ZEB1, FOSL1 and their common target genes are specifically enriched in breast cancer cell lines of the claudin-low subtype.

(A) Correlation of ZEB1 mRNA expression with the expression of YAP, FOSL1 and JUN across all breast cancer cell lines included in the cancer cell line encyclopedia (CCLE). rs: Spearman's rank correlation coefficient.

(B) Heatmap of RNA-seq expression data of all CCLE breast cancer cell lines.
Genome-wide cooperation of EMT-transcription factor ZEB1 with YAP and AP-1 in breast cancer (2020)
Figure 8

Figure 8. Clinical relevance of ZEB1, FOSL1 and their common activated target genes in breast cancer.

(A) Kaplan Meier plots from survival analyses of human ER-/PR- breast cancers showing distant metastasis free survival (DMFS) based on the expression of the indicated genes or gene combinations. HR: hazard ratio. p-value from log-rank test.

(B) Hypergeometric testing showed a significant association of nuclear ZEB1 and FOSL1 expression on the protein level as determined by immunohistochemistry on 81 human breast cancer samples. The upper panel shows staining of a serial section of a negative and a positive case. Note that stromal cells (asterisks) are negative for FOSL1, and partially positive for ZEB1 and YAP, and that expression of ZEB1 in tumour cells (arrows) of positive cases is weaker than in stromal cells. Odds ratio (OR): measure of association between categorical variables with OR > 1 indicating a positive association. P-value from Fisher's exact test. Scale bar = 20 µm.

(C) Heatmap of mRNA expression data of ZEB1, YAP, JUN and 9 selected activated ZEB1/YAP/AP-1 target genes in breast cancer patient samples (GSE18229). Genes were selected for known roles in tumour promoting properties like cell migration. Tumours in which ≥ 6 out of 9 genes of the selected ZEB1/YAP/AP-1 target gene set were expressed higher than the 60th percentile of expression were defined as "tumour promoting gene set high"; when expression of ≥ 6 out of the 9 genes was below the 40th percentile, tumours were defined as "tumour promoting gene set low". Tumours were annotated according to their molecular subtype.

(D) Correlation of ZEB1 mRNA expression with selected activated ZEB1/YAP/AP-1 target genes which have known functions in cell migration in breast cancer patient samples (GSE18229).

(E) Kaplan Meier plots from survival analyses of human breast cancers patient samples (GSE18229) showing relapse free survival (RFS) based on the combined expression of the 9 selected ZEB1/YAP/AP-1 activated targets shown in (C). HR: hazard ratio. p-value from log-rank test.

(F) Kaplan Meier plots from survival analyses of human ER-/PR- breast cancers showing distant metastasis free survival (DMFS) based on the combined expression of the 9 selected ZEB1/YAP/AP-1 activated targets shown in (C). HR: hazard ratio. p-value from log-rank test.
Lipid and protein content profiling of isolated native autophagic vesicles (2022)
Figure 1

Figure 1. FACS-mediated isolation of autophagic vesicles.

A. Schematic representation of the antibody-based FACS-mediated isolation method. TL total lysate; P1-2 pellet fractions; S1-2 supernatants.

B. Western blot analysis of purified autophagic vesicles. Isolations were performed using antibodies directed against LC3B or all GABARAP isoforms, respectively, and are represented with total lysate (TL). Depicted are representative blots of 14 independent approaches.

C. Quantification of fluorophore-labelled events in WT and ATG5 KO HeLa cells. Shown percentages represent the relative number of detected events of 3 independent experiments.

D. Quantification of fluorophore-labelled events in WT and FIP200 KO MEFs. Shown percentages represent the relative number of detected events of 3 independent experiments.

E. Co-localization of fluorescence signals linked to antibodies directed against LC3B and all GABARAP isoforms. Shown percentages represent the average distribution of 3 independent experiments, excluding double negative events.

Data information: C-E. Statistics are depicted as mean ± SD; t-test (C+D) or One-Way ANOVA (E); *p ≤ 0.05; ***p ≤ 0.001.
Lipid and protein content profiling of isolated native autophagic vesicles (2022)
Figure 2

Figure 2. Isolated autophagic vesicles are sealed.

A. Differential interference contrast microscopy images of purified autophagic vesicles at high (I) or low (II) dilution. Images are representative of 3 independent approaches. Scale bar = 10 µm.

B. Negative stain electron microscopy images of isolated vesicles. Scale bar = 500 nm.

C. Size evaluation of isolated vesicles. The diameters of approx. 60 individual vesicles were determined using EM images. Statistics are depicted as mean ± SD.

D. Western blot analysis of isolated autophagic vesicles upon proteinase K digestion. Mechanically opened vesicles served as positive control. For negative control, isolates were incubated with BSA instead of proteinase K. Depicted are two different blots that are representative for 5 independent experiments
DOCK5 Regulates Energy Balance and Hepatic Insulin Sensitivity by Targeting mTORC1 Signaling (2019)
Figure 1

Figure 1 Changes in body weight and energy expenditure in 5-month-old mice.

Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months.

A Body weight curve.

B Cumulative body weight.

C Average daily food intake.

D Fasting and fed blood glucose.

E Rectal temperature.

F Locomotor tolerance.

G 24-h oxygen consumption.

H Respiratory exchange ratio (RER: VCO2/VO2).

I Energy expenditure.

Data information: Data are expressed as the mean ± SEM (n = 6 mice for each group). SD, standard chow diet; HFD, high-fat diet. P-values were determined with two-way ANOVA, # p < 0.05, ## p < 0.01 vs. SD-WT; *p < 0.05, **p < 0.01 vs. HFD-WT.
DOCK5 Regulates Energy Balance and Hepatic Insulin Sensitivity by Targeting mTORC1 Signaling (2019)
Figure 2

Figure 2 Deletion of DOCK5 increases susceptibility to HFD- and lipid-induced insulin resistance.

Eight-week-old male WT and DOCK5-/- mice were fed a SD or HFD for 3 months.

A Blood glucose levels and area under curve (AUC) during the glucose tolerance test.

B Blood glucose levels and AUC during the insulin tolerance test.

C Experimental procedure and hyperinsulinemic-euglycemic clamp (HEC) protocol.

D Glucose infusion rates (GIR).

E Glucose disposal rate (GDR).

F Hepatic glucose production (HGP).

G Percentage of suppression of hepatic glucose production (HGP).

H Experimental procedure for lipid infusion and the hyperinsulinemic-euglycemic clamp (HEC).

I Glucose infusion rates (GIR) during lipid infusion.

Data information: SD, standard chow diet; HFD, high-fat diet; AUC, the area under the curve for glucose. Data are means ± SEM (n = 5 or 6 mice for each group). P-values were determined with two-way ANOVA, # p < 0.05, ## p < 0.01 vs. SD-WT; **p < 0.01 vs. HFD-WT.
DOCK5 Regulates Energy Balance and Hepatic Insulin Sensitivity by Targeting mTORC1 Signaling (2019)
Figure 3

Figure 3 Deletion of DOCK5 increases the expression of gluconeogenic genes, inhibits insulin signaling and activates the mTOR pathway in the liver.

Eight-week-old male WT and DOCK5-/-mice were fed a SD or a HFD for 3 months. Mice were treated with insulin (1 U/kg) or control (PBS) by intraperitoneal injection for 10 minutes; animals were killed and livers were collected.

A The expression of expression of phosphoenolpyruvate carboxykinase(PEPCK) and glucose-6- phosphatase(G6Pase) mRNA and protein.

B Total and phosphorylated insulin receptor (InsR), IRS-1ser1101 and Akt.

C Total and phosphorylated mTOR, S6K1, and the levels of Raptor and Rictor protein.

Data information: SD, standard chow diet; HFD, high-fat diet. Data are means ± SEM (n = 2 independent cell cultures). P-values were determined with t-test, # p < 0.05, ## p < 0.01 vs. SD-WT; **p < 0.01 vs. HFD-WT.
DOCK5 Regulates Energy Balance and Hepatic Insulin Sensitivity by Targeting mTORC1 Signaling (2019)
Figure 4

Figure 4 DOCK5 regulates gluconeogenesis, insulin, and mTOR/mTORC1 (Raptor) signaling in vitro.

Hep1-6 cells were infected with pCDNA3.1-DOCK5 (DOCK5 +) or pCDNA3.1 (DOCK5-) for 48 h. Hep1-6 cells and MPHs isolated from WT and DOCK5-/- mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min.

A The mRNA and protein expression of PEPCK and G6Pase in Hep1-6 cells.

B Total and phosphorylated InsR, IRS-1Ser1101 and Akt in Hep1-6 cells.

C Total and phosphorylated mTOR and S6K1, and the levels of Raptor and Rictor protein in Hep1-6 cells.

D The mRNA and protein expression of PEPCK and G6Pase in MPHs.

E Total and phosphorylated InsR, IRS-1Ser1101 and Akt in MPHs.

F Total and phosphorylated mTOR and S6K1, and the levels of Raptor and Rictor protein in MPHs.

Data information: GlcN, glucosamine. Data are means ± SEM of at least two independent experiments. P-values were determined with t-test ,**p < 0.01 vs. lane 6.
DOCK5 Regulates Energy Balance and Hepatic Insulin Sensitivity by Targeting mTORC1 Signaling (2019)
Figure 5

Figure 5 Adult onset, liver-specific deletion of Raptor attenuates insulin resistance (IR) and glucose intolerance induced by DOCK5 knockdown in HFD-fed mice.

Eight-week-old male Raptor flox/flox mice (Raptorflox/flox Cre+) were fed a HFD for 12 weeks and injected with AAV8-shDOCK5 ± AAV8-Cre or AAV8-GFP (at a dose of 3 × 1011 vg / 200 μL/ mouse) via the tail-vein 14 days prior to the in vivo study.

A Schematic representation of the strategy used to produce liver-specific Raptor knockout (L-Raptor-KO) mice.

B Fasting and fed blood glucose 14 days post-infection.

C Blood glucose levels and AUC during glucose tolerance tests.

D Blood glucose levels and AUC during insulin tolerance tests.

E Glucose infusion rates (GIR).

F Glucose disposal rate (GDR).

G Hepatic glucose production (HGP).

H Percentage of suppression of hepatic glucose production (HGP).

Data information: AUC, the area under the curve for glucose. Data are means ± SEM (n = 5-6 mice for each group). P-values were determined with t-test ,**p < 0.01 vs. GFP group; # p <0.05, ## p <0.01 vs. shDOCK5 group.
DOCK5 Regulates Energy Balance and Hepatic Insulin Sensitivity by Targeting mTORC1 Signaling (2019)
Figure 6

Figure 6 Deletion of liver Raptor attenuates the role of the DOCK5 deficiency on gluconeogenesis and insulin signaling in vivo and vitro.

Male Raptor flox/flox mice (8 weeks) were fed a HFD for 12 weeks and injected with AAV8- GFP or AAV8-shDOCK5 or AAV8-shDOCK5 + AAV8-Cre via the tail vein.

A The mRNA and protein expression of PEPCK and G6Pase in the liver.

B Total and phosphorylated InsR, IRS-1Ser1101 and Akt in the liver.

C The levels of DOCK5 and Raptor protein, and total and phosphorylated mTOR and S6K1 in the liver.

D-F MPHs from Raptor flox/flox mice were infected with Ad-GFP or Ad-shDOCK5 or Ad-shDOCK5 + Ad-Cre. The lysates were immunoblotted with indicated antibodies or β-actin.

D The expression of PEPCK and G6Pase mRNA and protein.

E Total and phosphorylated InsR, IRS-1Ser1101 and Akt.

F The levels of DOCK5 and Raptor protein, and total and phosphorylated mTOR and S6K1.

Data information: Data are means ± SEM (n = 2 mice for each group or 1 cell cultures). P-values were determined with Student's t-test, **p < 0.01 vs. GFP group; # p <0.05, ## p <0.01 vs. shDOCK5 group.
DOCK5 Regulates Energy Balance and Hepatic Insulin Sensitivity by Targeting mTORC1 Signaling (2019)
Figure 7

Figure 7 DOCK5 regulates mTOR signaling via its Rac guanine nucleotide exchange factors (GEF) activity, depending on the molecular interaction between DOCK5 and Raptor.

A Rac1 GEF activation in MPHs from WT and DOCK5-/- mice.

B SEW2871 treatment promotes Rac1 GEF activation and eliminates the influence of a DOCK5 deficiency on Raptor / S6K1 signaling in the mouse primary hepatocytes (MPHs) of DOCK5-/- mice.

C Total and phosphorylated mTOR and S6K1 and the level of Raptor protein expression in plasmids expressing a WT DOCK5 or mutant DOCK5 treated-Hep1-6 cells.

D Coimmunoprecipitation (Co-IP) analysis of DOCK5 and Raptor expression in Hepa1-6 cells. After transfected with indicated plasmids, lysates from cells were immunoprecipitated (IP) with anti-DOCK5 antibody or normal IgG, and then immunoblotted (IB) with the indicated antibody.

E DOCK5 binding to Raptor fragments (445 - 887 aa) during transient expression in Hepa1-6 cells.

F The DOCK5 and Raptor binding sites were identified using mutant DOCK5 (DHR-2V1559A) and Raptor fragments (445 - 887 aa) based on a Co-IP assay. VA, mutant DOCK5 DHR-2; His, His-DHR-2.

Data information: Data are means ± SEM (n = 1), P-values were determined with t-test, *p < 0.05, **p < 0.01 vs. DOCK5-/- MPHs without SEW2871 treatment group; *p < 0.05, **p < 0.01 vs. plasmids expressing a WT DOCK5 treated-Hep1-6 cells group
Exosome-like nanoparticles from Mulberry bark prevent DSS-induced colitis via the AhR/COPS8 (2021)
Figure 1

Fig 1. MBELNs activate AhR signalling.

(A) Mice were orally administered with Mulberry bark derived exosome-like nanoparticles (MBELNs) (10 x 109 particles/100 µl/mouse) or phosphate buffered saline (PBS) for seven days. Heat map showing influence of MBELNs on colonic gene expression from three biological replicates.

(B) In vitro assessment of MBELN-dependent induction of aryl hydrocarbon receptor (AhR) promoter using HEPA1.1 cells (contain AhR responsive luciferase reporter construct). Data are mean ± SEM from three biological replicates. **P<0.01, ***P<0.001 using one-way ANOVA.

(C) Representative images showing expression of pAhR in MC38 cells and Caco2 cells treated with MBELNs from three biological replicates. Scale bar 20 μm.

(D) Western blot (top) and graphical representation of fold changes (bottom) for AhR in MC38 cells, Caco2 cells and colon epithelial cells after treatment with MBELNs. Data are mean ± SEM of three biological replicates. *P<0.05 using Student's t-test.

(E) Fecal microbiota were analysed following administration of MBELNs (10 x 109 particle/100 µl/dose/day/mouse) for 7 days to C57BL/6 mice. Alpha diversity (Shannon index) was calculated at the family level and is displayed as a bar-and-whiskers plot for each individual combination of control and MBELN treatment. The centre line represents the median and the box encloses the 1st and 3rd quartiles ("hinges"). The upper and lower whiskers represent the furthermost points from the respective hinges, which are no more than 1.5 interquartile ranges from the hinge. The individual points are overlaid. Data are presented from five mice or biological replicates.

(F) Beta diversity analysis, biplots- samples (points) and bacterial taxa at family level (axes) are simultaneously projected on the two-dimensional canonical axes (CA1 and CA2). Data are presented from five mice or biological replicates.

(G) Pictorial representation of Dextran sulfate sodium (DSS; 2%) induced colitis development in mice.

(H) Graph showing loss of weight following DSS-induced colitis with and without MBELN treatment. Data are mean ± SEM of seven biological replicates, **P<0.01 using Mann-Whitney test.

(I) Representative image showing changes in colon morphology and length following DSS-induced colitis with concurrent treatment with MBELNs.

(J) Column graph showing changes in colon length. Data are mean ± SEM from seven biological replicates, **P<0.01 using one-way ANOVA.

(K-L) Hematoxylin and eosin (HE) staining to show histological changes in colon tissue (left) and a graph showing severity score (right) based on histological data. Scale bar 200 μm. Data are mean ± SEM of seven biological replicates, **P<0.01 using Student's t-test.

(M) Enzyme-linked immunosorbent assay (ELISA) for interleukin (IL)-6 and IL-1β from colon tissue. Data are mean ± SEM from seven biological replicates, **P<0.01 using one-way ANOVA.
Exosome-like nanoparticles from Mulberry bark prevent DSS-induced colitis via the AhR/COPS8 (2021)
Figure 2

Fig 2. MBELN treatment leads to inducing the expression of COPS8 via the AhR signalling pathway.

(A) Mass spectrometry (MS) analysis of aryl hydrocarbon receptor (AhR) pulldown protein from Mulberry bark derived exosome-like nanoparticles (MBELNs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated MC38 cell lysate. Data from three technical replicates are shown.

(B) Co-immunoprecipitation of COP9/COP9 Constitutive Photomorphogenic Homolog Subunit 8 (COPS8) bound AhR from MC38 cell lysate treated with MBELNs. Data are mean ± SEM from three biological replicates, **P<0.01 using Student's t-test.

(C) Western blot analysis showing expression of COPS8 in mice ileum crypts. Data are mean ± SEM of three biological replicates, **P<0.01 using Student's t-test.

(D) Representative images showing effect of MBELNs with and without TCDD (1 nm)/ carbobenzoxy-Leu-Leu-leucinal (MG132; 20 µM) on co-expression of AhR and COPS8 using immunofluorescence. Scale bar 10 μm, images are from three biological replicates.

(E) Western blot showing MBELNs, TCDD and MG132 effect on co-expression of AhR and COPS8. The data represent as mean ± SEM from three biological replicates **P<0.01, ***P<0.001 using one-way ANOVA.

(F) Heat map showing MS analysis of AhR pulldown protein from MC38 cells treated with MBELNs in combination with proteasome inhibitor, MG132. Data are represented from three technical replicates.

(G) Expression of Cullin 1 (CUL1) and neddylated CUL1 (N-CUL1) following MBELN treatment using Western blot. Data are mean ± SEM from three biological replicates. **P<0.01 using Student's t-test.

(H-I) Western blot showing expression of COPS8 in wildtype (Wild) and AhR-KO MC38 cells and expression of AhR in wild and COPS8-KO MC38 cells. MC38 cells were KO using CRISPR/ CAS9 KO plasmid transfection. Data represents mean ± SEM from three biological replicates, **P<0.01, NS- nonsignificant, using one-way ANOVA.