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).
Product Citations: 1,016
In Oncogene on 6 April 2025 by Wouters, V. M., Helderman, R. F. C. P. A., et al.
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Cancer Research
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In Aging and Disease on 11 March 2025 by Sharma, G., Lee, Y. H., et al.
Age-related alterations in the skeletal system are linked to decreased bone mass, a reduction in bone strength and density, and an increased risk of fractures and osteoporosis. Therapeutics are desired to stimulate bone regeneration and restore imbalance in the bone remodeling process. Quercetin (Qu), a naturally occurring flavonoid, induces osteogenesis; however, its solubility, stability, and bioavailability limit its therapeutic use. Nanoformulation can improve the physical properties of Qu and enhance its bioactivity and bioavailability. Further, localized delivery of Qu nanoformulations at the site of bone defects could ensure high local concentration, augmenting its osteogenic properties. Thus, this study aims to synthesize selenium nanoparticles-based Qu nanoformulation (Qu-SeNPs) and evaluate their osteogenic stimulation ability along with localized bone regeneration ability. Here, the spontaneously synthesized Qu-SeNPs showed uniform size distribution and rough flower-shaped morphology. The confocal images indicate improved cellular uptake and even cellular distribution of Qu-SeNPs in osteoblasts, resulting in increased osteogenic activity as indicated by enhanced expression of early and late osteoprogenitor differentiation markers. Qu-SeNPs also decreased osteoblasts' RANKL/OPG ratio and inhibited osteoclast formation. Mechanistically, Qu-SeNPs activate critical signaling pathways, including WNT and BMP, and utilize the miR-206/Connexin43 pathway to enhance osteogenesis. In vivo, experiments utilizing a drill-hole bone defect model in mice indicate that hydrogel-mediated localized delivery of Qu-SeNPs significantly accelerates bone defect healing. Thus, well-characterized and mechanistic, detailed synthesized Qu-SeNPs can restore bone remodeling, and Qu-SeNPs embedded in hydrogels may improve Qu cellular uptake and bioavailability in clinical settings, enabling innovative orthopedic and regenerative therapies for bone loss/defects.
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Clonal memory of colitis accumulates and promotes tumor growth
Preprint on BioRxiv : the Preprint Server for Biology on 17 February 2025 by Nagaraja, S., Ojeda-Miron, L., et al.
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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Cancer Research
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A clinical drug candidate that triggers non-apoptotic cancer cell death
Preprint on Research Square on 11 February 2025 by Dixon, S., Leak, L., et al.
Abstract Small molecules that induce non-apoptotic cell death are of fundamental mechanistic interest and may be useful to treat certain cancers. Here, we report that tegavivint, a drug candidate undergoing human clinical trials, can activate a unique mechanism of non-apoptotic cell death in sarcomas and other cancer cells. This lethal mechanism is distinct from ferroptosis, necroptosis and pyroptosis and requires the lipid metabolic enzyme trans-2,3-enoyl-CoA reductase (TECR). TECR is canonically involved in the synthesis of very long chain fatty acids but appears to promote non-apoptotic cell death in response to CIL56 and tegavivint via the synthesis of the saturated long-chain fatty acid palmitate. These findings outline a lipid-dependent non-apoptotic cell death mechanism that can be induced by a drug candidate currently being tested in humans.
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Cancer Research
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In Biology on 3 February 2025 by Porreca, V., Corbella, E., et al.
Pigment epithelium-derived factor (PEDF) is a multifunctional soluble glycoprotein, primarily known for its potent anti-angiogenic properties. In recent years, its ability to counteract cell proliferation and motility has generated interest in PEDF as a potential tumor suppressor. In the intrahepatic Cholangiocarcinoma (iCCA), PEDF, Thrombospondin 1 (THBS1), and Thrombospondin 2 (THBS2) are expressed and released into the tumor microenvironment (TME), where they promote lymphangiogenesis at the expense of the neoangiogenic program, aiding the dissemination of cancer cells via lymphatic vessels. Recently, we demonstrated that THBS1 and THBS2 directly affect iCCA cells, exacerbating their malignant behavior, while the direct role of PEDF remains to be elucidated. In this study, through a cell-based assay and molecular analysis, we investigate the direct function of PEDF on two well-established iCCA cell lines. Our results show that PEDF affects cancer cell motility in a paracrine manner, reducing their migratory and invasive capabilities. Notably, our data suggest that the PEDF-induced inhibition of motility in iCCA cells occurs through the MAPK/ERK signaling pathway, as indicated by the reduced phosphorylation of ERK1/2. Overall, this study provides the first evidence of PEDF acting as a tumor suppressor in iCCA.
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Cancer Research
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In Nat Commun on 23 May 2024 by El Bakkouri, Y., Chidiac, R., et al.
Fig.3.A
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IHC-IF
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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...
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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.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Cancer Cell Int on 10 December 2023 by Rodas, F., Vidal-Vidal, J. A., et al.
Fig.5.D
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Homo sapiens (Human)
ET-1 signaling blockage with macitentan sensitizes GBC cells to gemcitabine. a ECE1, ETAR and ETBR protein levels were detected by western blot in NOZ cells treated with 14 µM GEM and/or 1 µM MAC for 24 h. b Cells were seeded in 96-well plates for 24 h and treated with increased concentrations of...
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ET-1 signaling blockage with macitentan sensitizes GBC cells to gemcitabine. a ECE1, ETAR and ETBR protein levels were detected by western blot in NOZ cells treated with 14 µM GEM and/or 1 µM MAC for 24 h. b Cells were seeded in 96-well plates for 24 h and treated with increased concentrations of gemcitabine (GEM) alone or in combination with 1 μM MAC for 72 h. Cells were incubated with MTS reagent for 2 h and absorbance was measured at 490 nm and plotted as percentage. c Cell viability of NOZ cells treated with 14 µM GEM and/or 1 µM MAC for 72 h measured by crystal violet assay. d ZEB1, β-catenin, NF-kB and cleaved PARP (cl-PARP) detection by western blot in NOZ cells treated with 14 µM GEM and/or 1 µM MAC for 24 h. Data represent averages ± SDs (n = 3). ANOVA test was used. *p < 0.05
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Collected and cropped from Cancer Cell Int by CiteAb, provided under a CC-BY license
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In Nat Commun on 3 November 2023 by Nakai, K., Lin, H., et al.
Fig.6.A
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IHC
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Expression of MMP21 and nuclear β-catenin, p-ERK, or nuclear p65 positively correlate in early colon cancer.a Representative immunostaining patterns of MMP21, β-catenin, p-ERK, p65, p-Akt, or Cyclin E1. Paraffin-embedded sections of clinical samples were stained with MMP21, β-catenin, p-ERK, p65,...
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Expression of MMP21 and nuclear β-catenin, p-ERK, or nuclear p65 positively correlate in early colon cancer.a Representative immunostaining patterns of MMP21, β-catenin, p-ERK, p65, p-Akt, or Cyclin E1. Paraffin-embedded sections of clinical samples were stained with MMP21, β-catenin, p-ERK, p65, p-Akt, or Cyclin E1 antibody and were counterstained with hematoxylin or methyl green. Staining intensities were classified as weak, moderate, or strong. Scale bars, 20 μm. b Quantified H-scores of MMP21, nuclear β-catenin, p-ERK, nuclear p65, p-Akt, and Cyclin E1 in normal and tumor tissues. n = 9 independent patient-derived samples. c Correlation analysis between the H-score of MMP21 and that of nuclear β-catenin, p-ERK, nuclear p65, p-Akt, or Cyclin E1 in tumor tissues. Scatter plots of MMP21 H-score and nuclear β-catenin, p-ERK, nuclear p65, p-Akt, or Cyclin E1 H-score are shown. Correlation coefficient r and P-values calculated based on a t-distribution with 7 degrees of freedom are also shown.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Nat Commun on 3 November 2023 by Nakai, K., Lin, H., et al.
Fig.3.A
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WB
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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...
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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.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Nat Commun on 3 August 2023 by Ding, D., Blee, A. M., et al.
Fig.7.E
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WB
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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...
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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).
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Antioxidants (Basel) on 2 August 2023 by Fosso, E., Leo, M., et al.
Fig.2.D
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WB
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Quercetin impairs the main proliferation pathways in HCT116 cells. Total protein extracts from HCT116 cells treated with 50 µM Qc for 24 h or with the vehicle alone were analyzed by a Western blot for the phosphorylated form of ERK (A), AKT (B), and mTOR (C) or for β-catenin levels (D). β-Actin i...
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Quercetin impairs the main proliferation pathways in HCT116 cells. Total protein extracts from HCT116 cells treated with 50 µM Qc for 24 h or with the vehicle alone were analyzed by a Western blot for the phosphorylated form of ERK (A), AKT (B), and mTOR (C) or for β-catenin levels (D). β-Actin is reported as a protein loading control in (A–C), α-Tubulin is used for normalization in (D). Statistical significance is considered when * p < 0.05, and *** p < 0.001 (t-test).
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Collected and cropped from Antioxidants (Basel) by CiteAb, provided under a CC-BY license
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In Nat Commun on 20 April 2023 by Hao, J., Han, X., et al.
Fig.4.A
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WB
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Sema3C promotes β-catenin nuclear localization.a Western blots of β-catenin in cytosolic and nuclear fractions (left) of GSCs after Sema3C knockdown (right). Western blots were repeated at least twice. b Quantification of nuclear β-catenin positive cells of GSCs expressing FLAG vector or FLAG-Sem...
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Sema3C promotes β-catenin nuclear localization.a Western blots of β-catenin in cytosolic and nuclear fractions (left) of GSCs after Sema3C knockdown (right). Western blots were repeated at least twice. b Quantification of nuclear β-catenin positive cells of GSCs expressing FLAG vector or FLAG-Sema3C (n = 3 independent experiments in 387 GSCs Two-tailed T-test p = 0.0066, error bars: S.D.; n = 4 independent experiments in 4121 GSCs, Two-tailed T-test p = 0.0088, error bars: S.D.). c, d Left panels: representative immunofluorescence images of β-catenin (green) in 387 (c) and 4121 (d) GSCs after control shNT and shSema3C knockdown. Nuclei were counterstained with DAPI (blue). Right panels: quantification of nuclear β-catenin positive cell fraction (n = 3 independent experiments, Two-tailed T-test, p = 0.0012 (387 GSCs); p < 0.0001 (4121 GSCs) error bars: S.D. Scale bar 50 μM). Source data are provided as a Source data file.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Biomedicines on 7 April 2023 by Li, C., Wu, Y., et al.
Fig.1.D
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WB
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BV-2 cell activation led by LPS and IFN-γ inhibited the expression of β-catenin. A Griess reaction kit detected NO production in the supernatant (a–c). (a) NO production in the supernatant after incubating BV-2 cells for 24 h with LPS (100 ng/mL), IFN-γ (20 ng/mL), or both. One-way ANOVA, F (3, 8...
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BV-2 cell activation led by LPS and IFN-γ inhibited the expression of β-catenin. A Griess reaction kit detected NO production in the supernatant (a–c). (a) NO production in the supernatant after incubating BV-2 cells for 24 h with LPS (100 ng/mL), IFN-γ (20 ng/mL), or both. One-way ANOVA, F (3, 8) = 52,697, **** p < 0.0001, n = 3. (b) The dose-dependent reaction of IFN-γ combined with LPS (100 ng/mL) induced NO production in BV-2 cells. One-way ANOVA, F (5, 30) = 1410, **** p < 0.0001,* p = 0.0191, n = 3. (c) The time course of NO production in LPS/IFN-γ-induce BV-2 cells. One-way ANOVA, F (8,18) = 3232, **** p < 0.0001, n = 3. (d) Western blot showing expression of β-catenin in BV-2 cells. Top representative bands. Bottom, data summary. Two-tailed unpaired t-test, F, DFn, Dfd (4.798, 2, 2), ** p = 0.0050, n = 3 (e) Left, immunofluorescence showing expression of β-catenin (red). Right, data summary. Two-tailed unpaired t-test, F, DFn, Dfd (1.375, 2, 2), ** p = 0.0089, n = 3.
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Collected and cropped from Biomedicines by CiteAb, provided under a CC-BY license
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In Biomedicines on 7 April 2023 by Li, C., Wu, Y., et al.
Fig.1.E
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ICC-IF
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BV-2 cell activation led by LPS and IFN-γ inhibited the expression of β-catenin. A Griess reaction kit detected NO production in the supernatant (a–c). (a) NO production in the supernatant after incubating BV-2 cells for 24 h with LPS (100 ng/mL), IFN-γ (20 ng/mL), or both. One-way ANOVA, F (3, 8...
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BV-2 cell activation led by LPS and IFN-γ inhibited the expression of β-catenin. A Griess reaction kit detected NO production in the supernatant (a–c). (a) NO production in the supernatant after incubating BV-2 cells for 24 h with LPS (100 ng/mL), IFN-γ (20 ng/mL), or both. One-way ANOVA, F (3, 8) = 52,697, **** p < 0.0001, n = 3. (b) The dose-dependent reaction of IFN-γ combined with LPS (100 ng/mL) induced NO production in BV-2 cells. One-way ANOVA, F (5, 30) = 1410, **** p < 0.0001,* p = 0.0191, n = 3. (c) The time course of NO production in LPS/IFN-γ-induce BV-2 cells. One-way ANOVA, F (8,18) = 3232, **** p < 0.0001, n = 3. (d) Western blot showing expression of β-catenin in BV-2 cells. Top representative bands. Bottom, data summary. Two-tailed unpaired t-test, F, DFn, Dfd (4.798, 2, 2), ** p = 0.0050, n = 3 (e) Left, immunofluorescence showing expression of β-catenin (red). Right, data summary. Two-tailed unpaired t-test, F, DFn, Dfd (1.375, 2, 2), ** p = 0.0089, n = 3.
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Collected and cropped from Biomedicines by CiteAb, provided under a CC-BY license
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In Biomedicines on 7 April 2023 by Li, C., Wu, Y., et al.
Fig.2.B
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WB
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Overexpression of Wnt3a did not alter the morphology of BV-2 cells. (a) Treatment of BV-2 cells with RM Wnt3a at different concentrations. The cell viability was evaluated by CCK-8 assay. One-way ANOVA, F (7, 16) = 1.705, p > 0.05, n = 3. (b) Western blot analysis of expression of β-catenin in BV...
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Overexpression of Wnt3a did not alter the morphology of BV-2 cells. (a) Treatment of BV-2 cells with RM Wnt3a at different concentrations. The cell viability was evaluated by CCK-8 assay. One-way ANOVA, F (7, 16) = 1.705, p > 0.05, n = 3. (b) Western blot analysis of expression of β-catenin in BV-2 cells after RM Wnt3a. Top, representative bands. Bottom, data summary. One-way ANOVA, F (4, 9) = 38.54, ** p < 0.005, n = 3. (c) Western blot analysis of expression of Wnt3a in pcDNA3.1-Wnt3aV-2 cells. Top, representative bands. Bottom, data summary. Two-tailed unpaired t-test, F, DFn, Dfd (43.85, 2, 2), ** p = 0.0037, n = 3. (d) Western blot analyzed the expression of β-catenin in pcDNA3.1-Wnt3aV-2 cells. Top, representative bands. Bottom, data summary. Two-tailed unpaired t-test, F, DFn, Dfd (51.34, 2, 2), * p = 0.0264, n = 3. (e) Immunofluorescence showing the morphological changes of BV2 cells following treatment of LPS plus IFN-γ, but not Wnt3a. Scale bar = 20 μm. Left, representative images. Right, data summary. One-way ANOVA, F(2, 15) = 104.1, **** p < 0.0001, n = 3.
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Collected and cropped from Biomedicines by CiteAb, provided under a CC-BY license
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In Biomedicines on 7 April 2023 by Li, C., Wu, Y., et al.
Fig.2.D
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WB
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Overexpression of Wnt3a did not alter the morphology of BV-2 cells. (a) Treatment of BV-2 cells with RM Wnt3a at different concentrations. The cell viability was evaluated by CCK-8 assay. One-way ANOVA, F (7, 16) = 1.705, p > 0.05, n = 3. (b) Western blot analysis of expression of β-catenin in BV...
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Overexpression of Wnt3a did not alter the morphology of BV-2 cells. (a) Treatment of BV-2 cells with RM Wnt3a at different concentrations. The cell viability was evaluated by CCK-8 assay. One-way ANOVA, F (7, 16) = 1.705, p > 0.05, n = 3. (b) Western blot analysis of expression of β-catenin in BV-2 cells after RM Wnt3a. Top, representative bands. Bottom, data summary. One-way ANOVA, F (4, 9) = 38.54, ** p < 0.005, n = 3. (c) Western blot analysis of expression of Wnt3a in pcDNA3.1-Wnt3aV-2 cells. Top, representative bands. Bottom, data summary. Two-tailed unpaired t-test, F, DFn, Dfd (43.85, 2, 2), ** p = 0.0037, n = 3. (d) Western blot analyzed the expression of β-catenin in pcDNA3.1-Wnt3aV-2 cells. Top, representative bands. Bottom, data summary. Two-tailed unpaired t-test, F, DFn, Dfd (51.34, 2, 2), * p = 0.0264, n = 3. (e) Immunofluorescence showing the morphological changes of BV2 cells following treatment of LPS plus IFN-γ, but not Wnt3a. Scale bar = 20 μm. Left, representative images. Right, data summary. One-way ANOVA, F(2, 15) = 104.1, **** p < 0.0001, n = 3.
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Collected and cropped from Biomedicines by CiteAb, provided under a CC-BY license
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In Front Oncol on 1 April 2023 by Alam, A., Smith, S. C., et al.
Fig.2.A
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SP decreases β-catenin expression in vivo and ex vivo.(A) Western blot analysis for active (non-phosphorylated) β-catenin expression in HTC-116 and HT-29 cells treated with 20 µM SP. (B) LET/TCF luciferase promotor activity assay in HCT-116 cells incubated with 20 µM SP for 3 and 5 days. Results ...
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SP decreases β-catenin expression in vivo and ex vivo.(A) Western blot analysis for active (non-phosphorylated) β-catenin expression in HTC-116 and HT-29 cells treated with 20 µM SP. (B) LET/TCF luciferase promotor activity assay in HCT-116 cells incubated with 20 µM SP for 3 and 5 days. Results are normalized to untreated cells and reported as ± SD. Student’s t-test was used to assess statistical significance (*p< 0.05). (C)Ex vivo immunofluorescence analysis of β-catenin in CRC tumors. At 2 weeks after the final DSS treatment, animals were administered SP in their drinking water for 3 weeks as described in Materials and Methods. Tumors were excised from colon, and histological slides were made and stained for β-catenin expression. Representative sections are shown with β-catenin staining in green and DAPI nuclear staining in blue. (D) Quantification of β-catenin fluorescence was normalized to DAPI for control (N = 3) and SP treated (N = 3). Values are shown as ± SD; *p< 0.05 by t-test.
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Collected and cropped from Front Oncol by CiteAb, provided under a CC-BY license
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In Cell Death Dis on 27 March 2023 by Zhu, S., Yang, Z., et al.
Fig.5.A
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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 ...
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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.
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
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In Sci Rep on 28 December 2022 by Mieremet, A., van der Stoel, M., et al.
Fig.3.A
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ICC-IF
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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...
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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.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In Front Oncol on 5 November 2022 by Ghatak, S., Hascall, V. C., et al.
Fig.5.D
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WB
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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...
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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.
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Collected and cropped from Front Oncol by CiteAb, provided under a CC-BY license
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In Clin Sci (Lond) on 14 October 2022 by Lewis, A., Sanchez, S., et al.
Fig.4.A
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ICC-IF
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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...
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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.
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Collected and cropped from Clin Sci (Lond) by CiteAb, provided under a CC-BY license
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In Clin Sci (Lond) on 14 October 2022 by Lewis, A., Sanchez, S., et al.
Fig.1.A
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IHC-IF
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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...
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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).
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Collected and cropped from Clin Sci (Lond) by CiteAb, provided under a CC-BY license
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In Int J Mol Sci on 4 October 2022 by Cámara-Sánchez, P., Díaz-Riascos, Z. V., et al.
Fig.8.A
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WB
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Effect of NCS and PTX treatments in the Wnt/β-Catenin signaling pathway in MDA-MB-231 cells. β-Catenin, total- and phosphorylated-GSK3-β protein levels expression levels after PTX and NCS treatment, both alone and in combination (1:2 ratio), were determined. (A–C) Representative β-Catenin and GSK...
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Effect of NCS and PTX treatments in the Wnt/β-Catenin signaling pathway in MDA-MB-231 cells. β-Catenin, total- and phosphorylated-GSK3-β protein levels expression levels after PTX and NCS treatment, both alone and in combination (1:2 ratio), were determined. (A–C) Representative β-Catenin and GSK3-β protein levels after drug treatments. The β-actin protein expression level was used as a loading control. (B–D) Graphs represent the quantification band intensity signal referred to β-actin expression, represented as mean ± SEM of three independent experiments. Statistic t-test analysis of NCS and combined therapy was performed in comparison with control non-treated cells (black asterisks, ** p < 0.01, *** p < 0.001 and **** p < 0.0001) as well as with PTX treatment (blue asterisks, * p < 0.05 and ** p < 0.01).
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Collected and cropped from Int J Mol Sci by CiteAb, provided under a CC-BY license
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In Cells on 29 September 2022 by Su, K. W., Lin, H. Y., et al.
Fig.7.A
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WB
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Thyroid hormone (T4)-induced PD-L1-dependent protein expression and cell proliferation in oral cancer cells. (A). Oral cancer OEC-M1 cells were seeded in six-well trays and were transfected with either PD-L1 shRNA or a scrambled plasmid (0.5 μg/well). Prior to treatment, cells were refed with hor...
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Thyroid hormone (T4)-induced PD-L1-dependent protein expression and cell proliferation in oral cancer cells. (A). Oral cancer OEC-M1 cells were seeded in six-well trays and were transfected with either PD-L1 shRNA or a scrambled plasmid (0.5 μg/well). Prior to treatment, cells were refed with hormone-stripped FBS-containing medium and treated with T4 (10−7 M) for 24 h. Cells were harvested, and Western blot analyses of total β-catenin, pβ-catenin, PD-L1, and pERK1/2 were conducted. Data are presented as the mean ± SD. Results are from three independent studies (n = 4) ** p < 0.01, compared to the control. I.O.D., intensity of the optical density. (B). Oral cancer OEC-M1 cells were seeded in 24-well trays and were transfected with either PD-L1 shRNA or a scrambled plasmid (0.1 μg/well). Prior to treatment, cells were refed with hormone-stripped FBS-containing medium and treated with T4 (10−7 M) for 72 h. Cells were harvested for cell viability assay. Data are presented as the mean ± SD. Results are from three independent studies (n = 6) * p < 0.05, ** p < 0.01, *** p < 0.001, compared to the control; # p < 0.05, ### p < 0.001, compared to T4, and $ p < 0.05, $$$ p < 0.001, compared to PD-L1 shRNA group.
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Collected and cropped from Cells by CiteAb, provided under a CC-BY license
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In Sci Rep on 1 September 2022 by Ruiz-Mitjana, A., Navaridas, R., et al.
Fig.1.A
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ICC-IF
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Mus musculus (House mouse)
(A) Cytokeratin, E-cadherin, β-catenin and vimentin immunofluorescence on epithelial endometrial cells grown as 2D monolayers or 3D organoids show no differences in epithelial markers expression. Cells were counterstained with Hoechst to visualize nuclei. Scale bars: 25 µm. (B) Confocal images an...
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(A) Cytokeratin, E-cadherin, β-catenin and vimentin immunofluorescence on epithelial endometrial cells grown as 2D monolayers or 3D organoids show no differences in epithelial markers expression. Cells were counterstained with Hoechst to visualize nuclei. Scale bars: 25 µm. (B) Confocal images and quantification of cleaved caspase-3 immunofluorescence on endometrial cells grown as 2D monolayers or 3D organoids untreated (UN) or treated with 10 or 20 ng/ml TGF-β for 72 h. Cells were counterstained with phalloidin to visualize actin cytoskeleton and morphology and Hoechst to visualize nuclei. Scale bars: 25 µm. Values are mean and error bars represent mean ± S.E.M. **P < 0.01; ***P < 0.001 by one-way ANOVA, followed by the Tukey’s multiple comparison test.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In Sci Rep on 1 September 2022 by Ruiz-Mitjana, A., Navaridas, R., et al.
Fig.3.B
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ICC-IF
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Mus musculus (House mouse)
(A) Phase contrast images of 2D endometrial epithelial monolayers untreated (UN) or treated for 72 h with TGF-β 20 ng/ml. (B) Representative confocal images of β-catenin and E-cadherin immunofluorescences corresponding to 2D endometrial epithelial monolayers untreated (UN) or treated for 72 h wit...
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(A) Phase contrast images of 2D endometrial epithelial monolayers untreated (UN) or treated for 72 h with TGF-β 20 ng/ml. (B) Representative confocal images of β-catenin and E-cadherin immunofluorescences corresponding to 2D endometrial epithelial monolayers untreated (UN) or treated for 72 h with TGF-β 20 ng/ml. Cells were counterstained with Hoechst to visualize nuclei. Scale bars: 25 µm. (C) Representative confocal images of double cytokeratin and vimentin immunofluorescence of 2D endometrial epithelial monolayers untreated (UN) or treated for 72 h with TGF-β 20 ng/ml. Cells were counterstained with Hoechst to visualize nuclei. Scale bars: 25 µm. (D) Western blot analysis of E-cadherin, β-catenin and vimentin expression on lysates obtained from 2D monolayer stimulated for 72 h with TGF-β 20 ng/ml. Membrane was re-blotted with tubulin to check for equal protein loading.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In Sci Rep on 1 September 2022 by Ruiz-Mitjana, A., Navaridas, R., et al.
Fig.3.D
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WB
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Mus musculus (House mouse)
(A) Phase contrast images of 2D endometrial epithelial monolayers untreated (UN) or treated for 72 h with TGF-β 20 ng/ml. (B) Representative confocal images of β-catenin and E-cadherin immunofluorescences corresponding to 2D endometrial epithelial monolayers untreated (UN) or treated for 72 h wit...
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(A) Phase contrast images of 2D endometrial epithelial monolayers untreated (UN) or treated for 72 h with TGF-β 20 ng/ml. (B) Representative confocal images of β-catenin and E-cadherin immunofluorescences corresponding to 2D endometrial epithelial monolayers untreated (UN) or treated for 72 h with TGF-β 20 ng/ml. Cells were counterstained with Hoechst to visualize nuclei. Scale bars: 25 µm. (C) Representative confocal images of double cytokeratin and vimentin immunofluorescence of 2D endometrial epithelial monolayers untreated (UN) or treated for 72 h with TGF-β 20 ng/ml. Cells were counterstained with Hoechst to visualize nuclei. Scale bars: 25 µm. (D) Western blot analysis of E-cadherin, β-catenin and vimentin expression on lysates obtained from 2D monolayer stimulated for 72 h with TGF-β 20 ng/ml. Membrane was re-blotted with tubulin to check for equal protein loading.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In Sci Rep on 1 September 2022 by Ruiz-Mitjana, A., Navaridas, R., et al.
Fig.6.B
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ICC-IF
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Mus musculus (House mouse)
Representative confocal images of double cytokeratin and vimentin (A), β-catenin (B) or E-cadherin (C) immunofluorescence corresponding to 2D endometrial epithelial monolayers isolated from Cre:ER+/−; SMAD2f/f; SMAD3f/f, plated and treated with tamoxifen (TAM) to induce Cre:ER-mediated deletion o...
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Representative confocal images of double cytokeratin and vimentin (A), β-catenin (B) or E-cadherin (C) immunofluorescence corresponding to 2D endometrial epithelial monolayers isolated from Cre:ER+/−; SMAD2f/f; SMAD3f/f, plated and treated with tamoxifen (TAM) to induce Cre:ER-mediated deletion of SMAD2 and SMAD3 alleles or left untreated (NO TAM). Images were captured after 3 days of tamoxifen-induced deletion. Cells were counterstained with Hoechst to visualize nuclei. Scale bars: 25 µm. D. Western blot analysis of vimentin expression on 2D endometrial epithelial monolayers isolated from Cre:ER+/−; SMAD2f/f; SMAD3f/f. Cells were treated (TAM) or not (NO TAM) with tamoxifen to induce Cre:ER-mediated deletion of SMAD2 and SMAD3 alleles and then stimulated or not with TGF-β for 72 h. Membranes were also blotted with β-actin antibody to ensure deletion of these proteins. E. RT-qPCR relative quantification of Cdh2 and Twist1 mRNA expression of 2D endometrial epithelial monolayers isolated from Cre:ER+/−; SMAD2f/f; SMAD3f/f. Cells were plated and treated (TAM) or not (NO TAM) with tamoxifen to induce Cre:ER-mediated deletion of SMAD2 and SMAD3 alleles. Values are mean and error bars represent mean ± S.E.M. *P < 0.001 by one-way ANOVA.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
Image 1 of 119
In Sci Rep on 1 September 2022 by Ruiz-Mitjana, A., Navaridas, R., et al.
Fig.7.B
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ICC-IF
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Mus musculus (House mouse)
Representative confocal images of double cytokeratin and vimentin (A) β-catenin (B) or E-cadherin (C) immunofluorescence corresponding to 2D endometrial epithelial monolayers isolated from Cre:ER+/−; SMAD2f/f; SMAD3f/f. Cells were plated and treated with tamoxifen (TAM) to induce Cre:ER-mediated ...
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Representative confocal images of double cytokeratin and vimentin (A) β-catenin (B) or E-cadherin (C) immunofluorescence corresponding to 2D endometrial epithelial monolayers isolated from Cre:ER+/−; SMAD2f/f; SMAD3f/f. Cells were plated and treated with tamoxifen (TAM) to induce Cre:ER-mediated deletion of SMAD2 and SMAD3 alleles or left untreated (NO TAM) and then stimulated for 72 h with 20 ng/ml of TGF-β. Images were captured after 3 days of tamoxifen-induced deletion. Cells were counterstained with Hoechst to visualize nuclei. Scale bars: 25 µm.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In Sci Rep on 1 September 2022 by Ruiz-Mitjana, A., Navaridas, R., et al.
Fig.9.A
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ICC-IF
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Mus musculus (House mouse)
(A) Representative confocal images cytokeratin, E-cadherin, β-catenin and vimentin, on 3D organoid cultures from Cre:ER+/−; SMAD2f/f; SMAD3f/f endometrial epithelial cells treated with tamoxifen to induce SMAD2/3 deletion. Scale bars: 25 µm. (B) Immunofluorescence corresponding to 2D endometrial ...
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(A) Representative confocal images cytokeratin, E-cadherin, β-catenin and vimentin, on 3D organoid cultures from Cre:ER+/−; SMAD2f/f; SMAD3f/f endometrial epithelial cells treated with tamoxifen to induce SMAD2/3 deletion. Scale bars: 25 µm. (B) Immunofluorescence corresponding to 2D endometrial epithelial monolayers isolated from Cre:ER+/−; SMAD2f/f; SMAD3f/f, plated and treated with tamoxifen (TAM) to induce Cre:ER-mediated deletion of SMAD2 and SMAD3 alleles then treated for 72 h with 5% Matrigel diluted in basal culture medium or left untreated (NO ECM). Images were captured after 3 days of tamoxifen-induced deletion. Cells were counterstained with Hoechst to visualize nuclei. Scale bars: 25 µm. (C) RT-qPCR relative quantification of vimentin mRNA expression of 2D endometrial epithelial monolayers isolated from Cre:ER+/−; SMAD2f/f; SMAD3f/f. Cells were plated and treated (TAM) or not (NO TAM) with tamoxifen to induce Cre:ER-mediated deletion of SMAD2 and SMAD3 alleles, then treated for 72 h with 5% Matrigel diluted in basal culture medium or left untreated (NO ECM). Results are expressed as mRNA expression relative to untreated (NO ECM). Values are mean ± S.E.M. *P < 0.01, by t test analysis.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
Image 1 of 119