CiteAb

Fig.1.C showing Western Blotting from the publication: Proximity interactome of lymphatic VE-cadherin reveals mechanisms of junctional remodeling and reelin secretion.

Fig.2.B showing Western Blotting in a Canis lupus familiaris (Domestic dog) sample from the publication: HIF2α-dependent Dock4/Rac1-signaling regulates formation of adherens junctions and cell polarity in normoxia.

Fig.3.A showing Immunohistochemistry-immunofluorescence from the publication: ZO-1 interacts with YB-1 in endothelial cells to regulate stress granule formation during angiogenesis.

Fig.5.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: Targeting the Endothelin-1 pathway to reduce invasion and chemoresistance in gallbladder cancer cells.

Fig.2.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Targeting the Endothelin-1 pathway to reduce invasion and chemoresistance in gallbladder cancer cells.

Fig.2.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: Targeting the Endothelin-1 pathway to reduce invasion and chemoresistance in gallbladder cancer cells.

Fig.2.E showing Western Blotting in a Homo sapiens (Human) sample from the publication: Targeting the Endothelin-1 pathway to reduce invasion and chemoresistance in gallbladder cancer cells.

Fig.6.A showing Immunohistochemistry from the publication: Wnt activation disturbs cell competition and causes diffuse invasion of transformed cells through NF-κB-MMP21 pathway.

Fig.3.A showing Western Blotting from the publication: Wnt activation disturbs cell competition and causes diffuse invasion of transformed cells through NF-κB-MMP21 pathway.

Fig.7.E showing Western Blotting from the publication: Gain-of-function mutant p53 together with ERG proto-oncogene drive prostate cancer by beta-catenin activation and pyrimidine synthesis.

Fig.2.D showing Western Blotting from the publication: Quercetin's Dual Mode of Action to Counteract the Sp1-miR-27a Axis in Colorectal Cancer Cells.

Fig.4.A showing Western Blotting from the publication: Sema3C signaling is an alternative activator of the canonical WNT pathway in glioblastoma.

Fig.2.D showing Western Blotting from the publication: Characterization of Inflammatory Signals in BV-2 Microglia in Response to Wnt3a.

Fig.2.B showing Western Blotting from the publication: Characterization of Inflammatory Signals in BV-2 Microglia in Response to Wnt3a.

Fig.1.E showing Immunocytochemistry-immunofluorescence from the publication: Characterization of Inflammatory Signals in BV-2 Microglia in Response to Wnt3a.

Fig.1.D showing Western Blotting from the publication: Characterization of Inflammatory Signals in BV-2 Microglia in Response to Wnt3a.

Fig.2.A showing Western Blotting from the publication: Uncoupled nitric oxide synthase activity promotes colorectal cancer progression.

Fig.5.A showing Western Blotting from the publication: HOXB3 drives WNT-activation associated progression in castration-resistant prostate cancer.

Fig.3.A showing Immunocytochemistry-immunofluorescence from the publication: Endothelial dysfunction in Marfan syndrome mice is restored by resveratrol.

Fig.5.D showing Western Blotting from the publication: Chemotherapy induces feedback up-regulation of CD44v6 in colorectal cancer initiating cells through β-catenin/MDR1 signaling to sustain chemoresistance.

Fig.4.A showing Immunocytochemistry-immunofluorescence from the publication: Small-molecule Wnt inhibitors are a potential novel therapy for intestinal fibrosis in Crohns disease.

Fig.1.A showing Immunohistochemistry-immunofluorescence from the publication: Small-molecule Wnt inhibitors are a potential novel therapy for intestinal fibrosis in Crohns disease.

Fig.8.A showing Western Blotting from the publication: Selectively Targeting Breast Cancer Stem Cells by 8-Quinolinol and Niclosamide.

Fig.7.A showing Western Blotting from the publication: Thyroid Hormone Induces Oral Cancer Growth via the PD-L1-Dependent Signaling Pathway.

Fig.3.D showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Lack of extracellular matrix switches TGF-β induced apoptosis of endometrial cells to epithelial to mesenchymal transition.

174 images found

The E3 ubiquitin ligase MARCH2 controls TNF-α mediated inflammation by autoubiquitination.

In Cell Communication and Signaling : CCS on 31 May 2025 by Chathuranga, K., Rathnapala, P., et al.

Regulation of the nuclear factor-kappa B (NF-kB) signaling pathway is a major host homeostatic mechanism for controlling hyper-inflammation or chronic inflammation. Despite extensive research, the regulatory factors of NF-kB signaling required to preserve homeostasis and control inflammatory disorders are not fully understood. Moreover, the role of MARCH2 in chronic inflammation models and the regulation of MARCH2 activation remain to be elucidated.
We monitored disease severity and mortality in MARCH2-/- or MARCH2+/+ mice induced experimental colitis. Susceptibility to DSS-induced experimental colitis was determined by various methods, including Swiss roll assay and fluorescein isothiocyanate (FITC)-dextran treatment, respectively. RNA-sequencing was conducted to recognize the inflammatory response-related genes in the distal colon of colitis-induced mice. Enzyme-linked immunosorbent assay (ELISA) was used to measure the cytokines and chemokines with in vitro and in vivo samples. Affinity purification and LC-MS/MS analysis were used to identify the MARCH2 interacting proteins and posttranslational modifications. The underlying mechanism was elucidated using immunoblotting, co-immunoprecipitation, ubiquitination assay, and confocal microscopy.
Here, we report that MARCH2-/- mice were more susceptible to experimental inflammatory bowel disease (IBD) due to the massive production of cytokines. Stimulation by inflammatory cytokines such as TNF induces dimerization of MARCH2 at a later stage and dimerized MARCH2 undergoes K63-linked autoubiquitination at lysine 127 and 238, which promotes NEMO recognition, ubiquitination and proteasomal degradation. We also show an interaction between MARCH2 and MARCH8 in resting cells that inhibits MARCH2 activation. Taken together, these findings provide new insights into the molecular mechanism of MARCH2 and suggest a crucial role of MARCH2 in the modulation of inflammation and cellular homeostasis.
Our results indicate that MARCH2 plays a critical role in regulating NEMO/IKKγ under the inflammatory and resting conditions, thereby suppressing excessive or unexpected inflammatory responses. Our findings here not only demonstrate a biological role of MARCH2 in inflammatory signaling pathways but also provide a novel insight in the underlying mechanism.
© 2025. The Author(s).

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Integration of focal adhesion morphogenesis and polarity by DOCK5 promotes YAP/TAZ-driven drug resistance in TNBC.

In Molecular Omics on 12 May 2025 by Pascual-Vargas, P., Arias-Garcia, M., et al.

YAP and TAZ are transcriptional co-activators that are inhibited by sequestration in the cytoplasm. Cellular signalling pathways integrate soluble, mechanical (cytoskeleton, adhesion), and geometric (cell size, morphology) cues to regulate the translocation of YAP/TAZ to the nucleus. In triple-negative breast cancer (TNBC) cells, both signalling and morphogenesis are frequently rewired, leading to increased YAP/TAZ translocation, which drives proliferation, invasion, and drug resistance. However, whether this increased YAP/TAZ translocation is due to alterations in upstream signalling events or changes in cell morphology remains unclear. To gain insight into YAP/TAZ regulation in TNBC cells, we performed multiplexed quantitative genetic screens for YAP/TAZ localisation and cell shape, enabling us to determine whether changes in YAP/TAZ localisation following gene knockdown could be explained by alterations in cell morphology. These screens revealed that the focal adhesion (FA)-associated RhoGEF DOCK5 is essential for YAP/TAZ nuclear localisation in TNBC cells. DOCK5-defective cells exhibit defects in FA morphogenesis and fail to generate a stable, polarised leading edge, which we propose contributes to impaired YAP/TAZ translocation. Mechanistically, we implicate DOCK5's ability to act as a RacGEF and as a scaffold for NCK/AKT as key to its role in FA morphogenesis. Importantly, DOCK5 is essential for promoting the resistance of LM2 cells to the clinically used MEK inhibitor Binimetinib. Taken together, our findings suggest that DOCK5's role in TNBC cell shape determination drives YAP/TAZ upregulation and drug resistance.

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CDX2 downregulation regulates intrinsic WNT pathway activation, dictating metastasis in APC and CTNNB1 wildtype colorectal cancer.

In Oncogene on 6 April 2025 by Wouters, V. M., Helderman, R. F. C. P. A., et al.

Colorectal cancer (CRC) can be divided into 4 subtypes of which consensus molecular subtype 4 (CMS4) is associated with metastasis and poor survival. Previously, we reported that the KPN mouse model resembles human CMS4. Strikingly, although tumor formation in this model is slow and limited, effective metastasis is observed. To understand this aggressive behavior, we compared two distinct in vitro KPN models, organoids and tumoroids. The organoid model only carries the original mutations, while the tumoroids are derived from in vivo grown tumors that underwent selection during development. Here, we reveal that tumoroids harbor endogenous WNT pathway activity, which can be driven by tankyrase activity and Cdx2 downregulation. Importantly, WNT pathway activation was heterogeneous in nature, subject to regulation and allowed for a mixture of WNT-driven and YAP-driven cells within tumoroids. This unique type of WNT pathway activation is not crucial for colonic tumor growth, but results in metastatic spreading. Intriguingly, these findings reflect a specific subset of aggressive human CMS4 cancers that display low CDX2 expression and lack of classical WNT pathway mutations, while having a higher tendency to metastasize. Together, these data propose a novel mechanism for WNT pathway activation that drives metastasis formation in aggressive CRC.
© 2025. The Author(s).

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Clonal memory of colitis accumulates and promotes tumor growth

Preprint on Research Square on 27 March 2025 by Buenrostro, J., Nagaraja, S., et al.

Abstract Chronic inflammation is a well-established risk factor for cancer, but the underlying molecular mechanisms remain unclear. Using a mouse model of colitis, we demonstrate that colonic stem cells retain an epigenetic memory of inflammation following disease resolution, characterized by a cumulative gain of activator protein 1 (AP-1) transcription factor activity. Further, we develop SHARE-TRACE, a method that enables simultaneous profiling of gene expression, chromatin accessibility and clonal history in single cells, enabling high resolution tracking of epigenomic memory. This reveals that inflammatory memory is propagated cell-intrinsically and inherited through stem cell lineages, with certain clones demonstrating dramatically stronger memory than others. Finally, we show that colitis primes stem cells for amplified expression of regenerative gene programs following oncogenic mutation that accelerate tumor growth. This includes a subpopulation of tumors that have exceptionally high AP-1 activity and the additional upregulation of pro-oncogenic programs. Together, our findings provide a mechanistic link between chronic inflammation and malignancy, revealing how long-lived epigenetic alterations in regenerative tissues may contribute to disease susceptibility and suggesting potential therapeutic strategies to mitigate cancer risk in patients with chronic inflammatory conditions.

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Ultimate paths of least resistance: Intrinsically disordered links as developmental resets in regulatory protein networks

Preprint on BioRxiv : the Preprint Server for Biology on 18 March 2025 by Badyaev, A. V., Moreno, C. S., et al.

ABSTRACT Development and evolution require both stability and adaptability, yet how these opposite properties are reconciled is unclear. Here, we show that intrinsically disordered proteins (IDPs) act as reset mechanisms in conserved regulatory networks facilitating developmental transitions by integrating physical processes with genetic regulation. By tracing the ontogeny of mesenchymal cells in avian beak primordia, we demonstrate that mechanosensitive IDPs mediate shifts between physical cell states via dosage-dependent binding plasticity, converting stochastic protein variation into discreet regulatory networks. The disorder-enabled connectivity in these proteins resets their regulatory specialization and promotes population divergence. Comparative analyses across avian proteomes confirm that binding plasticity in transcriptional IDPs drives their diverse regulatory associations and accelerates their evolution. By resetting specialized states in conserved regulatory networks, IDPs flexibly regulate developmental pathways and reconcile precision with evolvability.

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Proximity interactome of lymphatic VE-cadherin reveals mechanisms of junctional remodeling and reelin secretion.

In Nat Commun on 4 September 2024 by Serafin, D. S., Harris, N. R., et al.

Fig.1.C

Fig.1.C showing Western Blotting from the publication: Proximity interactome of lymphatic VE-cadherin reveals mechanisms of junctional remodeling and reelin secretion.

Validation of a functional VE-cadherin-V5-miniTurbo that recapitulates native VE-cadherin AM-induced junctional rearrangement in LECs.A Schematic of adrenomedullin (AM)-mediated rearrangement of VE-cadherin; a central component on inter-endothelial adherens junctions. AM induces the rapid rearran... more

Validation of a functional VE-cadherin-V5-miniTurbo that recapitulates native VE-cadherin AM-induced junctional rearrangement in LECs.A Schematic of adrenomedullin (AM)-mediated rearrangement of VE-cadherin; a central component on inter-endothelial adherens junctions. AM induces the rapid rearrangement of discontinuous junctions to continuous VE-cadherin junctions. B Experimental workflow used to obtain the VE-cadherin interactome during junctional rearrangements (predominantly discontinuous with 0 nM AM; predominantly continuous with 100 nM AM). C LECs were transduced with VE-cadherin-V5-miniTurbo (VE-cad-mT) lentivirus and treated with vehicle or 100 nM AM and 50 µM biotin for a 2 h labeling period to evaluate fusion protein expression and biotinylation by western blot after streptavidin affinity-purification. Anti-V5 evaluated fusion protein expression, streptavidin probed biotinylated proteins, ß-catenin probed a known VE-cadherin-interactor, and actin served as a load control. Shown is a representative experiment. A minimum of three independent experiments were performed. D Confocal microscopy to evaluate the cellular localization of VE-cadherin-mT fusion protein in transduced primary LECs treated with vehicle or 100 nM AM and 50 µM biotin for a 2 h labeling period. Cells were stained for VE-cadherin (magenta) to detect VE-cadherin-mT fusion proteins as well as native VE-cadherin, streptavidin (green) to stain for biotinylated proteins, and DAPI (cyan) to mark cell nuclei. Scale bar = 20 µm. A minimum of three independent experiments were performed. Source data are provided with this paper. less

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ZO-1 interacts with YB-1 in endothelial cells to regulate stress granule formation during angiogenesis.

In Nat Commun on 23 May 2024 by El Bakkouri, Y., Chidiac, R., et al.

Fig.3.A

Fig.3.A showing Immunohistochemistry-immunofluorescence from the publication: ZO-1 interacts with YB-1 in endothelial cells to regulate stress granule formation during angiogenesis.

Stress-inducing treatments decrease ZO-1 expression levels in ECs.a Arsenite treatment decreases ZO-1 localization at EC junctions without affecting the localization of VE-cadherin and β-catenin. The fluorescence intensity of ZO-1 (red), VE-cadherin (green) and β-catenin (blue) was measured in co... more

Stress-inducing treatments decrease ZO-1 expression levels in ECs.a Arsenite treatment decreases ZO-1 localization at EC junctions without affecting the localization of VE-cadherin and β-catenin. The fluorescence intensity of ZO-1 (red), VE-cadherin (green) and β-catenin (blue) was measured in confluent cells in the absence (NT) or presence of sodium arsenite (500 µM; 30 min). The fluorescence intensity of each condition is normalized to the average intensity of the control (n = 20 cell junctions per condition). Unpaired two-tailed Student’s t test. b, c, d Effects of increasing concentrations of arsenite (30 min; n = 3) (b), thapsigargin (2 h; n = 3) (c) or H2O2 (30 min; n = 3) (d) on expression levels of ZO-1, VE-cadherin and β-catenin in HUVECs (b and d) or BAECs (c). Protein expression levels were determined by immunoblotting and β-actin serves as a loading control. Comparisons of basal expression levels versus treated conditions were performed for each. e Kinetics of the effects of arsenite treatment (500 µM) on expression levels of ZO-1, VE-cadherin and β-catenin in HUVECs. Protein expression levels were determined by immunoblotting and β-actin serves as a loading control (n = 4 independent experiments). f Effect of the proteasome inhibitor MG132 (40 µM; 60 min) on arsenite-induced (500 µM; 30 min) downregulation of ZO-1 in HUVECs. ZO-1 expression levels were determined by immunoblotting and β-actin serves as a loading control (n = 6 independent experiments). Effects of a recovery period post-arsenite treatment (1 h; 500 µM) on ZO-1 levels (n = 4 independent experiments) (g) and percentage of HUVECs with SGs (n = 10 fields of view per condition and at least 15 cells/field) (h). b–f Each point of the line graphs refers to the quantification of protein levels relative to β-actin. b–h Groups were compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests; ns not significant. Data are presented as mean values ± SEM. Source data are provided in the Source Data file. less

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Targeting the Endothelin-1 pathway to reduce invasion and chemoresistance in gallbladder cancer cells.

In Cancer Cell Int on 10 December 2023 by Rodas, F., Vidal-Vidal, J. A., et al.

Fig.2.B

Fig.2.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Targeting the Endothelin-1 pathway to reduce invasion and chemoresistance in gallbladder cancer cells.

Dual ETRs antagonism with macitentan (MAC) promotes ET-1 signaling downregulation in GBC cells. a Transcript levels of ET-1 target genes were measured by RT-qPCR in two GBC cell lines, 1TKB (blue) and 2TKB (red) treated with 1 µM MAC for 24 h. b Total β-catenin and NF-kB (p65) levels were measure... more

Dual ETRs antagonism with macitentan (MAC) promotes ET-1 signaling downregulation in GBC cells. a Transcript levels of ET-1 target genes were measured by RT-qPCR in two GBC cell lines, 1TKB (blue) and 2TKB (red) treated with 1 µM MAC for 24 h. b Total β-catenin and NF-kB (p65) levels were measured by western blot in NOZ GBC cells treated with 100 nM ET-1 and/or 1 µM MAC for 24 h. Representative images from three independent experiments are shown. c Cytosolic and nuclear β-catenin and NF-kB (p65) were measured by western blot in 1TKB and 2TKB GBC cells treated with 1 µM MAC for 24 h. Lamin B1 was used as a nuclear marker. d Indirect immunofluorescence of NOZ cells treated with 1 µM MAC and/or 100 nM ET1 for 24 h. β-catenin was visualized using a specific antibody and the nucleus was stained with DAPI. e Cycloheximide stability assay of β-catenin in NOZ cells treated with 100 nM ET-1 and/or 1 µM MAC between 0 and 4 h. f Reporter activity of β-catenin was measured by luciferase reporter assay using the TOP/FOB Flash system. Values reported for luciferase activity were used to calculate the TOP/FOP ratios (mean ± SD). Representative images from three independent experiments are shown. Data represent averages ± SDs (n = 3). ANOVA and Student’s tests were used. *p < 0.05 less

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Targeting the Endothelin-1 pathway to reduce invasion and chemoresistance in gallbladder cancer cells.

In Cancer Cell Int on 10 December 2023 by Rodas, F., Vidal-Vidal, J. A., et al.

Fig.2.C

Fig.2.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: Targeting the Endothelin-1 pathway to reduce invasion and chemoresistance in gallbladder cancer cells.

Dual ETRs antagonism with macitentan (MAC) promotes ET-1 signaling downregulation in GBC cells. a Transcript levels of ET-1 target genes were measured by RT-qPCR in two GBC cell lines, 1TKB (blue) and 2TKB (red) treated with 1 µM MAC for 24 h. b Total β-catenin and NF-kB (p65) levels were measure... more

Dual ETRs antagonism with macitentan (MAC) promotes ET-1 signaling downregulation in GBC cells. a Transcript levels of ET-1 target genes were measured by RT-qPCR in two GBC cell lines, 1TKB (blue) and 2TKB (red) treated with 1 µM MAC for 24 h. b Total β-catenin and NF-kB (p65) levels were measured by western blot in NOZ GBC cells treated with 100 nM ET-1 and/or 1 µM MAC for 24 h. Representative images from three independent experiments are shown. c Cytosolic and nuclear β-catenin and NF-kB (p65) were measured by western blot in 1TKB and 2TKB GBC cells treated with 1 µM MAC for 24 h. Lamin B1 was used as a nuclear marker. d Indirect immunofluorescence of NOZ cells treated with 1 µM MAC and/or 100 nM ET1 for 24 h. β-catenin was visualized using a specific antibody and the nucleus was stained with DAPI. e Cycloheximide stability assay of β-catenin in NOZ cells treated with 100 nM ET-1 and/or 1 µM MAC between 0 and 4 h. f Reporter activity of β-catenin was measured by luciferase reporter assay using the TOP/FOB Flash system. Values reported for luciferase activity were used to calculate the TOP/FOP ratios (mean ± SD). Representative images from three independent experiments are shown. Data represent averages ± SDs (n = 3). ANOVA and Student’s tests were used. *p < 0.05 less

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Targeting the Endothelin-1 pathway to reduce invasion and chemoresistance in gallbladder cancer cells.

In Cancer Cell Int on 10 December 2023 by Rodas, F., Vidal-Vidal, J. A., et al.

Fig.2.E

Fig.2.E showing Western Blotting in a Homo sapiens (Human) sample from the publication: Targeting the Endothelin-1 pathway to reduce invasion and chemoresistance in gallbladder cancer cells.

Dual ETRs antagonism with macitentan (MAC) promotes ET-1 signaling downregulation in GBC cells. a Transcript levels of ET-1 target genes were measured by RT-qPCR in two GBC cell lines, 1TKB (blue) and 2TKB (red) treated with 1 µM MAC for 24 h. b Total β-catenin and NF-kB (p65) levels were measure... more

Dual ETRs antagonism with macitentan (MAC) promotes ET-1 signaling downregulation in GBC cells. a Transcript levels of ET-1 target genes were measured by RT-qPCR in two GBC cell lines, 1TKB (blue) and 2TKB (red) treated with 1 µM MAC for 24 h. b Total β-catenin and NF-kB (p65) levels were measured by western blot in NOZ GBC cells treated with 100 nM ET-1 and/or 1 µM MAC for 24 h. Representative images from three independent experiments are shown. c Cytosolic and nuclear β-catenin and NF-kB (p65) were measured by western blot in 1TKB and 2TKB GBC cells treated with 1 µM MAC for 24 h. Lamin B1 was used as a nuclear marker. d Indirect immunofluorescence of NOZ cells treated with 1 µM MAC and/or 100 nM ET1 for 24 h. β-catenin was visualized using a specific antibody and the nucleus was stained with DAPI. e Cycloheximide stability assay of β-catenin in NOZ cells treated with 100 nM ET-1 and/or 1 µM MAC between 0 and 4 h. f Reporter activity of β-catenin was measured by luciferase reporter assay using the TOP/FOB Flash system. Values reported for luciferase activity were used to calculate the TOP/FOP ratios (mean ± SD). Representative images from three independent experiments are shown. Data represent averages ± SDs (n = 3). ANOVA and Student’s tests were used. *p < 0.05 less

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Wnt activation disturbs cell competition and causes diffuse invasion of transformed cells through NF-κB-MMP21 pathway.

In Nat Commun on 3 November 2023 by Nakai, K., Lin, H., et al.

Fig.3.A

Fig.3.A showing Western Blotting from the publication: Wnt activation disturbs cell competition and causes diffuse invasion of transformed cells through NF-κB-MMP21 pathway.

MMP21 plays a crucial role in basal extrusion of β-cat ΔN/RasV12 cells surrounded by β-cat ΔN cells.a,b Establishment of MDCK mCherry-β-cateninΔ131 and MDCK-pTR GFP-RasV12 mCherry-β-cateninΔ131 cells. a Constitutive expression of mCherry-β-cateninΔ131 and tetracycline-induced expression of RasV12... more

MMP21 plays a crucial role in basal extrusion of β-cat ΔN/RasV12 cells surrounded by β-cat ΔN cells.a,b Establishment of MDCK mCherry-β-cateninΔ131 and MDCK-pTR GFP-RasV12 mCherry-β-cateninΔ131 cells. a Constitutive expression of mCherry-β-cateninΔ131 and tetracycline-induced expression of RasV12. b TOP FLASH reporter assay. Indicated cell lines were co-transfected with TOP FLASH or FOP FLASH and Renilla expressing vector, and subjected to measurement of luciferase activity. Data are mean ± s.e.m. *P = 0.0015; **P = 0.0032, unpaired two-tailed t-test; n = three independent experiments. c Immunofluorescence images of β-cat ΔN/RasV12 cells cultured alone or surrounded by β-cat ΔN cells. d Quantification of cell fates of β-cat ΔN/RasV12 cells cultured alone or surrounded by β-cat ΔN cells. Data are mean ± s.e.m. n = three independent experiments. *P = 0.0190 for basal extrusion between two conditions, unpaired two-tailed t-test. e Experimental design and heatmap for differentially expressed genes between β-cat ΔN/RasV12 cells cultured alone and β-cat ΔN/RasV12 cells co-cultured with β-cat ΔN cells. f Volcano plot showing the distribution of fold changes and corrected P-values calculated by unpaired two-tailed t-test in β-cat ΔN/RasV12 cells mixed with β-cat ΔN cells compared with a single culture. The red or blue dots represent upregulated or downregulated genes with statistical significance, respectively. The upregulated and downregulated genes are shown in Supplementary Data 1. g q-PCR analysis of MMP-related metalloproteases. β-cat ΔN/RasV12 cells were co-cultured with β-cat ΔN cells or cultured alone, and subjected to q-PCR analysis. Values are shown as fold change relative to β-cat ΔN/RasV12 cells cultured alone. Data are mean ± s.e.m. n = three independent experiments. *P = 0.0221, unpaired two-tailed t-test. h Immunofluorescence images of MMP21. β-cat ΔN/RasV12 cells were cultured alone or co-cultured with β-cat ΔN cells in the presence of 2.5 μg ml−1 BFA. Cells were stained with Hoechst 33342 (blue) and anti-MMP21 antibody (white). i Quantification of fluorescence intensity of MMP21. Values are expressed as a ratio relative to β-cat ΔN/RasV12 cells cultured alone. Data are mean ± s.e.m. *P < 0.001, unpaired two-tailed t-test; n = 286 and 284 cells pooled from three independent experiments. j Establishment of MDCK-pTR GFP-RasV12 mCherry-β-cateninΔ131 cells stably expressing MMP21 shRNA1 or MMP21 shRNA2. Knockdown of MMP21 was confirmed by western blotting. k Immunofluorescence images of β-cat ΔN/RasV12 cells, β-cat ΔN/RasV12 cells expressing MMP21 shRNA1 or MMP21 shRNA2 surrounded by β-cat ΔN cells. Scale bars, 10 μm (c, h, k). l Quantification of basal extrusion. Data are mean ± s.e.m. n = three independent experiments. *P = 0.0186; **P = 0.0001, unpaired two-tailed t-test. less

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Gain-of-function mutant p53 together with ERG proto-oncogene drive prostate cancer by beta-catenin activation and pyrimidine synthesis.

In Nat Commun on 3 August 2023 by Ding, D., Blee, A. M., et al.

Fig.7.E

Fig.7.E showing Western Blotting from the publication: Gain-of-function mutant p53 together with ERG proto-oncogene drive prostate cancer by beta-catenin activation and pyrimidine synthesis.

LEF1/TCF O’PROTAC inhibits PSG expression and growth of TMPRSS2-ERG and mutant p53-positive PCa PDX tumors.a Sequence alignment between the DNA oligonucleotide used in LEF1/TCF O’PROTAC (OP) and the consensus DNA binding elements of each member of the LEF/TCF family. b Western blot analysis of in... more

LEF1/TCF O’PROTAC inhibits PSG expression and growth of TMPRSS2-ERG and mutant p53-positive PCa PDX tumors.a Sequence alignment between the DNA oligonucleotide used in LEF1/TCF O’PROTAC (OP) and the consensus DNA binding elements of each member of the LEF/TCF family. b Western blot analysis of indicated proteins in VCaP cells treated with control or LEF1/TCF OP (100 nM) for 48 h. c MTS assay in VCaP cells treated with control or LEF1/TCF OP (100 nM). d Sanger sequencing confirmation of C238Y mutation in LuCaP 23.1 PDX tumor samples. e Western blot analysis of indicated proteins in organoids derived from LuCaP 23.1 PDXs (PDXO). f–h LuCaP 23.1 PDXOs were treated with indicated OP (100 nM) and/or deoxynucleotides and harvested for Western blot analysis at 48 h after treatment (f) or cultured for 3 days followed by photographing (g) and quantification of the diameters of organoids (h). n.s., not significant. i–k Representative images of LuCaP 23.1 PDX tumors at 21 days after OP treatment (10 mg/kg every other day) (i), growth curve of tumors over the 21-day treatment period (j) and weight of tumors at day 21 of OP treatment (k). n.s., not significant. l Body weight of mice at 21 days after OP treatment as in (j). n.s., not significant. m, n Representative IHC images of the indicated proteins from tumors shown in (i) and quantification of IHC staining of the indicated proteins. See details of staining scoring and index in Methods. The western blot assays in (b, e) and (f) were repeated two independent times with similar results. Data in (c) was shown as mean ± s.d. from five replicates. Data in (h) was shown as mean ± s.d. from a total of 45 organoids randomly obtained from three independent experiments. Data in (j) and (k) were shown as mean ± s.d. from six PDXs. Data in l was shown as mean ± s.d. from six mice. Data in (n) was shown as mean ± s.d. from three independent experiments. For each experiment, five independent fields were enrolled for the calculation. Two-way ANOVA was performed in (c,j) and (l). Two-tailed Student’s t test was performed in (h,k) and (n). less

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HOXB3 drives WNT-activation associated progression in castration-resistant prostate cancer.

In Cell Death Dis on 27 March 2023 by Zhu, S., Yang, Z., et al.

Fig.5.A

Fig.5.A showing Western Blotting from the publication: HOXB3 drives WNT-activation associated progression in castration-resistant prostate cancer.

HOXB3 can be degraded by β-TrCP in destruction complex depend on the kinase activity of CKIα, see also Fig. S3.A Western blot of indicated proteins CO-IP with HOXB3 in LNCaP-AI cells cultured in medium with or without WNT3A. B Graph showing HOXB3 structure with distinguishable functional domains ... more

HOXB3 can be degraded by β-TrCP in destruction complex depend on the kinase activity of CKIα, see also Fig. S3.A Western blot of indicated proteins CO-IP with HOXB3 in LNCaP-AI cells cultured in medium with or without WNT3A. B Graph showing HOXB3 structure with distinguishable functional domains distinguished [24]: HTLV-II domain (N-terminal), Homeobox domain, and DUF4074 domain (C-terminal). C Autoradiography of 35S-AXIN1 pulled by GST-fused full-length or truncated HOXB3 as indicated. Input: western blot of GST fused truncated HOXB3 the same as samples used in pull-down experiments. D Western blot of β-TrCP and HOXB3 in LNCaP-AI cells treated with siRNAs targeting control (siCtrl) or β-TrCP (siβ-TrCP). E Western blot of AXIN1 and β-TrCP CO-IP with HOXB3 in LNCaP-AI cells after deletion of HOXB3, IKKβ, CKIα, or GSK3. Inputs: Western blot of HOXB3, AXIN1 and β-TrCP in the same lysates. F Western blot of AXIN1, β-TrCP and HOXB3 CO-IP with CKIα in WNT3A-treated LNCaP-AI cells that harvested at different time points as indicated. Inputs: Western blot of CKIα, HOXB3, AXIN1 and β-TrCP in the same lysates as above. G Graph showing phospho-degrons in HOXB3 and other proteins that may be recognized by CKIα, IKKβ, or GSK3. Diagrammatic sketch in frame showing three induced mutants of HOXB3 (M1, M2, and M3 for short) and mutation sites. H Top: western blot of HOXB3, CKIα and β-actin in LNCaP-AI cells transfected with siCtrl, siHOXB3 or siCKIα, followed by reconstituting with vector or siRNA-insensitive HOXB3, either wild-type (wt) or its mutants. Cells with S181/185 A mutation became insensitive to CKIα. Bottom: qRT-PCR analyses of CDCA3 expressions in the same cells as above. Error bars indicate mean ± SD. *p < 0.05, **p < 0.01, #p < 0.05 relative to line 4, ##p < 0.01 relative to line 4. less

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Endothelial dysfunction in Marfan syndrome mice is restored by resveratrol.

In Sci Rep on 28 December 2022 by Mieremet, A., van der Stoel, M., et al.

Fig.3.A

Fig.3.A showing Immunocytochemistry-immunofluorescence from the publication: Endothelial dysfunction in Marfan syndrome mice is restored by resveratrol.

Normalization of EC morphology after resveratrol treatment in the ascending aorta in MFS mice. (a) Representative en face IF confocal images showing β-catenin (green), and DAPI (blue) of ascending aorta from 36 weeks old Fbn1C1041G/+ MFS mice treated for 3 weeks with either vehicle control or RES... more

Normalization of EC morphology after resveratrol treatment in the ascending aorta in MFS mice. (a) Representative en face IF confocal images showing β-catenin (green), and DAPI (blue) of ascending aorta from 36 weeks old Fbn1C1041G/+ MFS mice treated for 3 weeks with either vehicle control or RESV. Region of interest (ROI) highlights EC junctional linearity. Arrow indicates the direction of blood flow. Scale bar: 25 μm. (b) Violin plot showing quantification of EC alignment normalized to the direction of flow. Data distribution was analyzed by Kolmogorov–Smirnov test, * indicates p < 0.05. (c–e) Bar plots of EC morphology parameters in the ascending aorta from 36 weeks old Fbn1C1041G/+ MFS mice treated for 3 weeks with either vehicle control or RESV. All data are represented as mean ± SD with indicated datapoints for vehicle treatment (N = 6; M/F 0/6) n = 24 images (total 360 ECs) and RESV treatment (N = 6; M/F 0/6) n = 24 images (total 360 ECs). (f) Representative en face IF confocal images showing p-eNOS (Ser1177) (red) and DAPI (blue) of ascending aorta from 36 weeks old WT, Fbn1C1041G/+ MFS mice treated for 3 weeks with either vehicle control or RESV. Line of quantification indicated as white line. (g) Line plot of p-eNOS quantification over a length of 4 µm perpendicular over the EC membrane in WT, MFS-CTRL, and MFS-RESV. (h, i) Bar plots comparing peak intensity and area under the curve (AUC) of p-eNOS. Data represents mean mean ± SD with indicated datapoints for WT (N = 4; M/F 0/4) n = 16 images (total 80 ECs), vehicle treatment (N = 4; M/F 0/4) n = 16 images (total 80 ECs), and RESV treatment ((N = 4; M/F 0/4) n = 16 images (total 80 ECs). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 analyzed by students t-test or one-way ANOVA with Holm-Sidak post-test. less

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Chemotherapy induces feedback up-regulation of CD44v6 in colorectal cancer initiating cells through β-catenin/MDR1 signaling to sustain chemoresistance.

In Front Oncol on 5 November 2022 by Ghatak, S., Hascall, V. C., et al.

Fig.5.D

Fig.5.D showing Western Blotting from the publication: Chemotherapy induces feedback up-regulation of CD44v6 in colorectal cancer initiating cells through β-catenin/MDR1 signaling to sustain chemoresistance.

CICs isolated from resistant cells demonstrate resistance to FOLFOX treatment through WNT3A/β-catenin signaling. (A), QPCR (Upper panel) and western blot (Lower Panel) data are shown for CD44v6 mRNA (Upper panel) and protein expression (Lower panel) in CICs isolated from SQ tumors of SW480-FR, SW... more

CICs isolated from resistant cells demonstrate resistance to FOLFOX treatment through WNT3A/β-catenin signaling. (A), QPCR (Upper panel) and western blot (Lower Panel) data are shown for CD44v6 mRNA (Upper panel) and protein expression (Lower panel) in CICs isolated from SQ tumors of SW480-FR, SW480-OXA, SW-5-FU, and SW480-S cells that were treated with or without either 1 x FOLFOX for 12 hours, Inset: CICs isolated from SQ tumors of SW480-FR, SW480-OXA, SW-5-FU, and SW480-S cells that were treated with either 1 x FOLFOX, or 1.2 ng/ml of WNT inhibitor LGK974 (IC50, for LGK974 in SW480-S and SW480-FR cells are 0.8 ng/ml and 1.15 ng/ml [data not shown]), or 1 x FOLFOX + LGK974 for 12 hours. QPCR analysis was done with total RNA extracted from these treated cells and data are shown for CD44v6 mRNA. (B), Secretion of WNT3A was measured by ELISA in sensitive and FR cells of SW480 after treatment with DMSO. Or 1 x FOLFOX, or 1.2ng/ml of LGK974, or 1 x FOLFOX + LGK974 for the indicated times. (C), Sensitive and FR cells of SW480 were transfected with 50 ng TOPFlash and 50 ng TK-Renilla vectors, or with 50 ng FOPFlash and 50 ng TK-Renilla vectors. The TOPFlash/FOPFlash promoter was activated by treatment with FOLFOX (1x) for 12 hours. Cells were lysed and subjected to luciferase measurements. (D-E), Validations of β-catenin shRNAs (β-catenin sh1 and β-catenin sh2) (D) and of constitutively active β-actin (E) used in the following experiments (H-K) were examined. In “D”, the indicated shRNA mediated knockdown and the corresponding knock-in (KI) gene transfections were dune as described in Methods. Target proteins were analyzed by WB analysis (β-tubulin, internal control). (F), CD44v6 negative PD-FR/NON-CICs were transfected with either TOPFlash and control TK-Renilla vectors, or with FOPFlash and TK-Renilla vectors together with increasing time of incubation with CD44v6 cDNAs. After 48 hours, the cells were stimulated with or without 20 ng/ml WNT3A for the indicated times. Then the cells were lysed and subjected to luciferase measurements (upper panel) or, in parallel, to WB analysis for CD44v6 and Flag. (G), PD-FR CICs were transfected with NT sh1, or with CD44v6 sh1. After 48 hours, cells were analyzed for WNT3A stimulated β-catenin/TCF4 promoter luciferase activity as shown in upper panel or, in parallel, to WB analysis with the indicated proteins (lower panel). (H, J), SW480-FR CICs were transfected with NT sh1, or CD44v6 sh1 (v6 sh1), or β-catenin sh1, or treated with DMSO, or 1.2 ng/ml of LGK974. 48 hours after the transfections, and 12 hours after the LGK974 treatment, cell growth was assessed by counting colonies in a clonogenic growth assay (H), and apoptosis was assessed by the Annexin V positive stain assay (J). (I, K), SW480-FR Non-CICs were transfected with vector control, v6 cDNA, or CA β-catenin cDNA. 48 hours after the transfections, cell growth was assessed by clonogenic growth assay (I), and apoptosis was assessed by the Annexin V positive stain assay (K). Data are presented as Mean ± SD from n = 3-4 replicates in three independent experiments. All WB data are representative of 4 independent experiments. (A) *P < 0.05 was considered significant for red asterisks, CD44v6 mRNA levels of FOLFOX treated cells were compared with the DMSO treated cells; *P < 0.05 considered significant for the green and blue asterisks, CD44v6 mRNA levels of 1.2 ng/ml, LGK974 and FOLFOX + LGK974 treated cells were compared with DMSO, or FOLFOX treated controls. (B), *P < 0.05, was considered significant, secreted WNT3A in LGK974 treated cells of SW480-S and SW480-FR were compared with their respective DMSO treated controls. (C), *P < 0.05, was considered significant, FOLFOX treated cells of SW480-S and SW480-FR were compared with their respective DMSO treated controls. (D-E), *P < 0.05, was considered significant, WNT3A treated PD-FR NON-CICs (D) and PD-FR CICs (E) at various time points were compared with their respective untreated controls. (F, H), *P < 0.05, was considered significant, v6 shRNA1, β-catenin shRNA1, and LGK974 treated clonogenic growth (F), and Annexin V positive (H) CICs were compared with their appropriate vector + NTshRNA, and DMSO controls. (G, I), *P < 0.05, was considered significant, v6 cDNA, CA-β-catenin CDNA overexpressed clonogenic growth (G), and Annexin V positive (I) NON-CICs were compared with their appropriate vector controls. less

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Small-molecule Wnt inhibitors are a potential novel therapy for intestinal fibrosis in Crohns disease.

In Clin Sci (Lond) on 14 October 2022 by Lewis, A., Sanchez, S., et al.

Fig.4.A

Fig.4.A showing Immunocytochemistry-immunofluorescence from the publication: Small-molecule Wnt inhibitors are a potential novel therapy for intestinal fibrosis in Crohns disease.

Small-molecule Wnt inhibitors of FZD8 and Wnt5B inhibit Collagen-I expression in intestinal fibroblasts(A) β-catenin and Collagen-I protein levels were also determined by IF in treated CCD-18Co cells with Wnt-C59 (C59), 3235-0367 (C1), or the vehicle control (DMSO) in combination with TGFβ. Repre... more

Small-molecule Wnt inhibitors of FZD8 and Wnt5B inhibit Collagen-I expression in intestinal fibroblasts(A) β-catenin and Collagen-I protein levels were also determined by IF in treated CCD-18Co cells with Wnt-C59 (C59), 3235-0367 (C1), or the vehicle control (DMSO) in combination with TGFβ. Representative images are provided. (B) β-catenin levels are presented as a ratio of the nuclear:cytoplasmic levels of the protein (n=2). (C,D) Cytosolic Collagen-I levels are also provided (n=3), along with cell counts for each treatment (n=3). (E) Pro-Collagen-Iα1 levels in the media were determined by ELISA (n=3). (F) Representative western blot showing the effects of 48 h of treatments on Fibronectin, dephosphorylated ABC levels, and total β-catenin protein levels. (G) β-catenin protein levels are presented as a ratio of ABC:β-catenin as determined by quantification of the IF images (n=3). (H) Fibronectin levels are expressed normalised to the loading control (β-Actin); DL = density levels. Differences between treatments were determined by a paired t-test to account for different cell passages. In general, data are presented as fold-changes with panels B–E, G, and H as box plots showing 25th to 75th percentiles, median (horizontal bar), and the smallest and largest value (whiskers). Significant results relative to control are indicated by * symbol (*<0.05, **<0.01, ***<0.001). A bar indicates specific statistical comparisons. less

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Small-molecule Wnt inhibitors are a potential novel therapy for intestinal fibrosis in Crohns disease.

In Clin Sci (Lond) on 14 October 2022 by Lewis, A., Sanchez, S., et al.

Fig.1.A

Fig.1.A showing Immunohistochemistry-immunofluorescence from the publication: Small-molecule Wnt inhibitors are a potential novel therapy for intestinal fibrosis in Crohns disease.

Altered β-catenin expression associated with intestinal fibrosis in SCD patients(A) FFPE sections from a colorectal adenocarcinoma block were also used for a positive to control IHC and IF protein analysis, given the link between APC mutations in patients, altered Wnt signalling, and changes in β... more

Altered β-catenin expression associated with intestinal fibrosis in SCD patients(A) FFPE sections from a colorectal adenocarcinoma block were also used for a positive to control IHC and IF protein analysis, given the link between APC mutations in patients, altered Wnt signalling, and changes in β-catenin staining. Nuclear accumulation of β-catenin in epithelial cells (EC) within the tumour are shown by the yellow arrow. The IF further shows clear colocalisation (yellow) of β-catenin (488) and Vimentin (555) in the cytoplasm of fibroblastic cells (pink arrow); DAPI staining demarcates the cell nuclei. (B) Representative IHC and IF images of β-catenin in the mucosa (M) overlying SCD and patient-matched NSCD areas (n=6) and MP of SCD and NSCD intestine (n=5). In the images, positively stained EC, endothelial cells surrounding blood vessels (BV) and stromal cells, including cells that morphologically resembled fibroblasts (indicated by arrows) are observed. (C) Enlarged (Zoom) IHC sections from SCD and NSCD mucosa highlighting negatively (yellow arrow) and positively (green arrow) stained fibroblastic cells are highlighted. (D–G) Quantification of the total number and percentage of stromal β-catenin-positive cells in the mucosa and the percentage of β-catenin-positive cells in the MP, excluding endothelial cells associated with BV, demonstrates transmural increases in β-catenin throughout SCD tissues. Differences between SCD and NSCD samples were determined by a paired t-test. Significant results are indicated by * symbol (*<0.05, **<0.01, ***<0.001). less

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