CiteAb

Fig.6.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

Fig.6.E showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

Fig.5.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

Fig.4.E showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

Fig.4.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

Fig.3.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

Fig.1.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

Fig.1.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

Fig.3.A showing Western Blotting from the publication: JNK-Dependent cJun Phosphorylation Mitigates TGFβ- and EGF-Induced Pre-Malignant Breast Cancer Cell Invasion by Suppressing AP-1-Mediated Transcriptional Responses.

Fig.2.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β family ligands exhibit distinct signalling dynamics that are driven by receptor localisation.

Fig.5.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β family ligands exhibit distinct signalling dynamics that are driven by receptor localisation.

Fig.4.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β family ligands exhibit distinct signalling dynamics that are driven by receptor localisation.

Fig.4.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β family ligands exhibit distinct signalling dynamics that are driven by receptor localisation.

Fig.1.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β family ligands exhibit distinct signalling dynamics that are driven by receptor localisation.

Fig.1.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β family ligands exhibit distinct signalling dynamics that are driven by receptor localisation.

Fig.3.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β family ligands exhibit distinct signalling dynamics that are driven by receptor localisation.

Fig.2.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β family ligands exhibit distinct signalling dynamics that are driven by receptor localisation.

Fig.2.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β family ligands exhibit distinct signalling dynamics that are driven by receptor localisation.

Fig.4.B showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Combinatorial Smad2/3 Activities Downstream of Nodal Signaling Maintain Embryonic/Extra-Embryonic Cell Identities during Lineage Priming.

Fig.11.C showing Immunocytochemistry-immunofluorescence in a Homo sapiens (Human) sample from the publication: Prostaglandin E2 Antagonizes TGF-β Actions During the Differentiation of Monocytes Into Dendritic Cells.

Fig.11.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Prostaglandin E2 Antagonizes TGF-β Actions During the Differentiation of Monocytes Into Dendritic Cells.

Fig.2.C showing Western Blotting from the publication: The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development.

Fig.2.D showing Western Blotting from the publication: The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development.

Fig.2.F showing Western Blotting from the publication: The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development.

Fig.2.B showing Western Blotting from the publication: The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development.

33 images found

Extracellular Vesicles Secreted by Cancer-Associated Fibroblasts Drive Non-Invasive Cancer Cell Progression to Metastasis via TGF-β Signalling Hyperactivation.

In Journal of Extracellular Vesicles on 1 March 2025 by Teixeira, A. F., Wang, Y., et al.

Metastasis is the leading cause of cancer-related deaths. Cancer-associated fibroblasts (CAFs) are abundant components within the tumour microenvironment, playing critical roles in metastasis. Although increasing evidence supports a role for small extracellular vesicles (sEVs) in this process, their precise contribution and molecular mechanisms remain unclear, compromising the development of antimetastatic therapies. Here, we establish that CAF-sEVs drive metastasis by mediating CAF-cancer cell interaction and hyperactivating TGF-β signalling in tumour cells. Metastasis is abolished by genetically targeting CAF-sEV secretion and consequent reduction of TGF-β signalling in cancer cells. Pharmacological treatment with dimethyl amiloride (DMA) decreases CAFs' sEV secretion, reduces TGF-β signalling levels in tumour cells and abrogates metastasis and tumour self-seeding. This work defines a new mechanism required by CAFs to drive cancer progression, supporting the therapeutic targeting of EV trafficking to disable the driving forces of metastasis.
© 2025 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.

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Analysis of the DNA-binding properties of TGF-β-activated Smad complexes unveils a possible molecular basis for cellular context-dependent signaling.

In The FASEB Journal on 15 August 2024 by Itoh, Y., Miyake, K., et al.

Transforming growth factor-β (TGF-β) is a pleiotropic cytokine that modulates a wide variety of cellular responses by regulating target gene expression. It principally transmits signals via receptor-activated transcription factors Smad2 and Smad3, which form trimeric complexes with Smad4 upon activation and regulate gene expression by binding to genomic DNA. Here, we examined the mechanisms by which TGF-β regulates the transcription of target genes in a cell context-dependent manner by screening a double-stranded DNA oligonucleotide library for DNA sequences bound to endogenous activated Smad complexes. Screening was performed by cyclic amplification of selected targets (CASTing) using an anti-Smad2/3 antibody and nuclear extracts isolated from three cell lines (A549, HepG2, and HaCaT) stimulated with TGF-β. The preference of the activated Smad complexes for conventional Smad-binding motifs such as Smad-binding element (SBE) and CAGA motifs was different in HepG2 than in the other two cell lines, which may indicate the distinct composition of the activated Smad complexes. Several transcription factor-binding motifs other than SBE or CAGA, including the Fos/Jun-binding motifs, were detected in the enriched sequences. Reporter assays using sequences containing these transcription factor-binding motifs together with Smad-binding motifs indicated that some of the motifs may be involved in cell type-dependent transcriptional activation by TGF-β. The results suggest that the CASTing method is useful for elucidating the molecular basis of context-dependent Smad signaling.
© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.

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Spatial Single Cell Analysis of Proteins in 2D Human Gastruloids Using Iterative Immunofluorescence.

In Current Protocols on 1 October 2023 by Freeburne, E., Teague, S., et al.

During development, cell signaling instructs tissue patterning, the process by which initially identical cells give rise to spatially organized structures consisting of different cell types. How multiple signals combinatorially instruct fate in space and time remains poorly understood. Simultaneous measurement of signaling activity through multiple signaling pathways and of the cell fates they control is critical to addressing this problem. Here we describe an iterative immunofluorescence protocol and computational pipeline to interrogate pattern formation in a 2D model of human gastrulation with far greater multiplexing than is possible with standard immunofluorescence techniques. This protocol and computational pipeline together enable imaging followed by spatial and co-localization analysis of over 27 proteins in the same gastruloids. We demonstrate this by clustering single cell protein expression, using techniques familiar from scRNA-seq, and linking this to spatial position to calculate spatial distributions and cell signaling activity of different cell types. These methods are not limited to patterning in 2D gastruloids and can be easily extended to other contexts. In addition to the iterative immunofluorescence protocol and analysis pipeline, Support Protocols for 2D gastruloid differentiation and producing micropatterned multi-well slides are included. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Iterative immunofluorescence Basic Protocol 2: Computational analysis pipeline Support Protocol 1: Generating micropatterned multi-well slides Support Protocol 2: Differentiation of 2D gastruloids.
© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

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Theophylline Attenuates BLM-Induced Pulmonary Fibrosis by Inhibiting Th17 Differentiation.

In International Journal of Molecular Sciences on 5 January 2023 by Park, S. J., Hahn, H. J., et al.

Idiopathic pulmonary fibrosis (IPF) is a chronic and refractory interstitial lung disease. Although there are two approved drugs for IPF, they were not able to completely cure the disease. Therefore, the development of new drugs is required for the effective treatment of IPF. In this study, we investigated the effect of theophylline, which has long been used for the treatment of asthma, on pulmonary fibrosis. The administration of theophylline attenuated the fibrotic changes of lung tissues and improved mechanical pulmonary functions in bleomycin (BLM)-induced pulmonary fibrosis. Theophylline treatment suppressed IL-17 production through inhibiting cytokines controlling Th17 differentiation; TGF-β, IL-6, IL-1β, and IL-23. The inhibition of IL-6 and IL-1β by theophylline is mediated by suppressing BLM-induced ROS production and NF-κB activation in epithelial cells. We further demonstrated that theophylline inhibited TGF-β-induced epithelial-to-mesenchymal transition in epithelial cells through suppressing the phosphorylation of Smad2/3 and AKT. The inhibitory effects of theophylline on the phosphorylation of Smad2/3 and AKT were recapitulated in BLM-treated lung tissues. Taken together, these results demonstrated that theophylline prevents pulmonary fibrosis by inhibiting Th17 differentiation and TGF-β signaling.

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Combination therapy with anamorelin and a myostatin inhibitor is advantageous for cancer cachexia in a mouse model.

In Cancer Science on 1 October 2022 by Hanada, K., Fukasawa, K., et al.

Cancer cachexia is a multifactorial disease that causes continuous skeletal muscle wasting. Thereby, it seems to be a key determinant of cancer-related death. Although anamorelin, a ghrelin receptor agonist, has been approved in Japan for the treatment of cachexia, few medical treatments for cancer cachexia are currently available. Myostatin (MSTN)/growth differentiation factor 8, which belongs to the transforming growth factor-β family, is a negative regulator of skeletal muscle mass, and inhibition of MSTN signaling is expected to be a therapeutic target for muscle-wasting diseases. Indeed, we have reported that peptide-2, an MSTN-inhibiting peptide from the MSTN prodomain, alleviates muscle wasting due to cancer cachexia. Herein, we evaluated the therapeutic benefit of myostatin inhibitory D-peptide-35 (MID-35), whose stability and activity were more improved than those of peptide-2 in cancer cachexia model mice. The biologic effects of MID-35 were better than those of peptide-2. Intramuscular administration of MID-35 effectively alleviated skeletal muscle atrophy in cachexia model mice, and the combination therapy of MID-35 with anamorelin increased food intake and maximized grip strength, resulting in longer survival. Our results suggest that this combination might be a novel therapeutic tool to suppress muscle wasting in cancer cachexia.
© 2022 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

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TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

In J Biol Chem on 21 March 2021 by Motizuki, M., Koinuma, D., et al.

Fig.6.B

Fig.6.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

Activation of Rac1 during TGF-β–induced motility. A, time course showing Rac1 activation after TGF-β stimulation. A549 cells were stimulated with 1 ng/ml TGF-β1 for 1–12 h. The amount of active, GTP-loaded Rac1 was determined by using a GST pull-down assay. Rac1 was detected by immunoblotting. B,... more

Activation of Rac1 during TGF-β–induced motility. A, time course showing Rac1 activation after TGF-β stimulation. A549 cells were stimulated with 1 ng/ml TGF-β1 for 1–12 h. The amount of active, GTP-loaded Rac1 was determined by using a GST pull-down assay. Rac1 was detected by immunoblotting. B, Rac1 activation assay using A549, A549-S3KO cells, or those expressing Smad3 or Smad3(RA/KP). Cells were stimulated with 1 ng/ml TGF-β1 for 12 h before harvesting. Cell lysates were subsequently prepared and subjected to a Rac1 activation assay as in A. C, chamber migration assay using A549 cells pretreated with either a TβRI kinase inhibitor SB431542 (5 μM), a PI3K inhibitor LY294002 (10 μM), or 0.1% DMSO (vehicle-only control) for 1 h. Scale bars: 10 μm. Error bars represent SD (n = 5). p values were determined by Student's t-test. ∗p < 0.01. D, Rac1 activation assay using A549 cells pretreated with LY294002, SB431542, or 0.1% DMSO, followed by stimulation with 1 ng/ml TGF-β1 for 12 h. Cell lysates were subsequently prepared and subjected to a Rac1 activation assay as in (A). E, TGF-β-induced phosphorylation of Akt (S473) in A549-S3KO cells expressing Smad3(RA/KP). Cells were pretreated with either 10 μM LY294002 or 0.1% DMSO for 1 h and stimulated with 1 ng/ml TGF-β1 for either 1 h or 4 h. Cell lysates were analyzed by immunoblotting using phospho-Akt or phospho-Smad2 antibodies; α-tubulin was used as a loading control. F, effects of a ROCK inhibitor Y27632 on TGF-β-induced Rac1 activation in A549-S3KO expressing either Smad3 or Smad3(RA/KP). Cells were treated with TGF-β (1 ng/ml) and/or Y27632 (25 μM) for 12 h on collagen-coated plate. Cell lysates were subsequently prepared and subjected to a Rac1 activation assay as in (A). One representative result from two independent experiments is shown (B–F). less

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TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

In J Biol Chem on 21 March 2021 by Motizuki, M., Koinuma, D., et al.

Fig.6.E

Fig.6.E showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

Activation of Rac1 during TGF-β–induced motility. A, time course showing Rac1 activation after TGF-β stimulation. A549 cells were stimulated with 1 ng/ml TGF-β1 for 1–12 h. The amount of active, GTP-loaded Rac1 was determined by using a GST pull-down assay. Rac1 was detected by immunoblotting. B,... more

Activation of Rac1 during TGF-β–induced motility. A, time course showing Rac1 activation after TGF-β stimulation. A549 cells were stimulated with 1 ng/ml TGF-β1 for 1–12 h. The amount of active, GTP-loaded Rac1 was determined by using a GST pull-down assay. Rac1 was detected by immunoblotting. B, Rac1 activation assay using A549, A549-S3KO cells, or those expressing Smad3 or Smad3(RA/KP). Cells were stimulated with 1 ng/ml TGF-β1 for 12 h before harvesting. Cell lysates were subsequently prepared and subjected to a Rac1 activation assay as in A. C, chamber migration assay using A549 cells pretreated with either a TβRI kinase inhibitor SB431542 (5 μM), a PI3K inhibitor LY294002 (10 μM), or 0.1% DMSO (vehicle-only control) for 1 h. Scale bars: 10 μm. Error bars represent SD (n = 5). p values were determined by Student's t-test. ∗p < 0.01. D, Rac1 activation assay using A549 cells pretreated with LY294002, SB431542, or 0.1% DMSO, followed by stimulation with 1 ng/ml TGF-β1 for 12 h. Cell lysates were subsequently prepared and subjected to a Rac1 activation assay as in (A). E, TGF-β-induced phosphorylation of Akt (S473) in A549-S3KO cells expressing Smad3(RA/KP). Cells were pretreated with either 10 μM LY294002 or 0.1% DMSO for 1 h and stimulated with 1 ng/ml TGF-β1 for either 1 h or 4 h. Cell lysates were analyzed by immunoblotting using phospho-Akt or phospho-Smad2 antibodies; α-tubulin was used as a loading control. F, effects of a ROCK inhibitor Y27632 on TGF-β-induced Rac1 activation in A549-S3KO expressing either Smad3 or Smad3(RA/KP). Cells were treated with TGF-β (1 ng/ml) and/or Y27632 (25 μM) for 12 h on collagen-coated plate. Cell lysates were subsequently prepared and subjected to a Rac1 activation assay as in (A). One representative result from two independent experiments is shown (B–F). less

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TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

In J Biol Chem on 21 March 2021 by Motizuki, M., Koinuma, D., et al.

Fig.3.B

Fig.3.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

Signaling of TGF-β-induced cell motility is transmitted through a distinct region of Smad3 from other EMT-associated responses. A, schematic presentation of N1133 and N3311 chimeric proteins. N1133 (Smad3 with Met-1 to Val-65 from Smad1); N3311 (Smad3 with Gln-98 to Ser-144 from Smad1). B, A549-S... more

Signaling of TGF-β-induced cell motility is transmitted through a distinct region of Smad3 from other EMT-associated responses. A, schematic presentation of N1133 and N3311 chimeric proteins. N1133 (Smad3 with Met-1 to Val-65 from Smad1); N3311 (Smad3 with Gln-98 to Ser-144 from Smad1). B, A549-S3KO cells were infected with lentivirus carrying cDNA expressing either the N1133 or N3311 chimera. Expression and TGF-β-induced phosphorylation of N1133 and N3311 were verified by immunoblotting with indicated antibodies; α-tubulin was used as a loading control. C, CAGA-Luc assay of A549-S3KO cells expressing N1133 or N3311. Luciferase assay was carried out using (CAGA)12-MLP-Luc as a reporter plasmid. Error bars represent SD (n = 3). D, light microscopic images, (E) expression of E-cadherin detected by immunoblotting, (F) formation of actin stress fibers visualized by Rhodamine-phalloidin staining, (G) immunofluorescence labeling of E-cadherin, and (H) chamber migration assay in A549-S3KO cells expressing wild-type Smad3, N1133, or N3311 in either the presence or absence of 1 ng/ml TGF-β1. Scale bars: 10 μm. Error bars represent SD (n = 5). p values were determined by Student's t-test. ∗p < 0.01. I, DNA affinity precipitation assay (DNAP) was performed using biotinylated 3xCAGA as a probe. Total cell lysates (input) and proteins in cell lysates bound to the probe were analyzed by immunoblotting with indicated antibodies; α-tubulin was used as a loading control. One representative result from two independent experiments is shown (C, H, I). less

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TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

In J Biol Chem on 21 March 2021 by Motizuki, M., Koinuma, D., et al.

Fig.1.C

Fig.1.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

EMT-associated cellular responses that are dependent on Smad3. A, SMAD3 knockout A549 cells (A549-S3KO) were prepared using CRISPR/Cas9-mediated genome editing. A549-S3KO cells were infected with lentivirus carrying SMAD3 cDNA. Expression and TGF-β-induced phosphorylation of Smad3 were verified b... more

EMT-associated cellular responses that are dependent on Smad3. A, SMAD3 knockout A549 cells (A549-S3KO) were prepared using CRISPR/Cas9-mediated genome editing. A549-S3KO cells were infected with lentivirus carrying SMAD3 cDNA. Expression and TGF-β-induced phosphorylation of Smad3 were verified by immunoblotting with the indicated antibodies; α-tubulin was used as a loading control. B–E, A549 cells, A549-S3KO cells, or those expressing wild-type Smad3 were incubated in either the presence or absence of 1 ng/ml TGF-β1 for 24 h. B, light microscopic photographs. C, expression of E-cadherin was determined by immunoblotting with anti-E-cadherin; α-tubulin was used as a loading control. D, immunofluorescence detection of E-cadherin. E, formation of actin stress fibers in A549-S3KO cells expressing wild-type Smad3. F-actin was stained using Rhodamine-phalloidin. F, chamber migration assay or (G) wound healing assay of A549 cells, A549-S3KO cells, or those expressing wild-type Smad3 in either the presence or absence of 1 ng/ml TGF-β1. Quantification is shown in the right. H, to evaluate growth rates of A549 cells, A549-S3KO cells, or those expressing wild-type Smad3 in either the presence or absence of 1 ng/ml TGF-β1, cell numbers were counted. TGF-β was added at day1. Scale bars: 10 μm (B and D–F) and 200 μm (G). Error bars represent SD (n = 5 for F, G). p values were determined by Student's t-test. ∗p < 0.01. One representative result from two independent experiments is shown (F–H). less

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TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

In J Biol Chem on 21 March 2021 by Motizuki, M., Koinuma, D., et al.

Fig.1.A

Fig.1.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

EMT-associated cellular responses that are dependent on Smad3. A, SMAD3 knockout A549 cells (A549-S3KO) were prepared using CRISPR/Cas9-mediated genome editing. A549-S3KO cells were infected with lentivirus carrying SMAD3 cDNA. Expression and TGF-β-induced phosphorylation of Smad3 were verified b... more

EMT-associated cellular responses that are dependent on Smad3. A, SMAD3 knockout A549 cells (A549-S3KO) were prepared using CRISPR/Cas9-mediated genome editing. A549-S3KO cells were infected with lentivirus carrying SMAD3 cDNA. Expression and TGF-β-induced phosphorylation of Smad3 were verified by immunoblotting with the indicated antibodies; α-tubulin was used as a loading control. B–E, A549 cells, A549-S3KO cells, or those expressing wild-type Smad3 were incubated in either the presence or absence of 1 ng/ml TGF-β1 for 24 h. B, light microscopic photographs. C, expression of E-cadherin was determined by immunoblotting with anti-E-cadherin; α-tubulin was used as a loading control. D, immunofluorescence detection of E-cadherin. E, formation of actin stress fibers in A549-S3KO cells expressing wild-type Smad3. F-actin was stained using Rhodamine-phalloidin. F, chamber migration assay or (G) wound healing assay of A549 cells, A549-S3KO cells, or those expressing wild-type Smad3 in either the presence or absence of 1 ng/ml TGF-β1. Quantification is shown in the right. H, to evaluate growth rates of A549 cells, A549-S3KO cells, or those expressing wild-type Smad3 in either the presence or absence of 1 ng/ml TGF-β1, cell numbers were counted. TGF-β was added at day1. Scale bars: 10 μm (B and D–F) and 200 μm (G). Error bars represent SD (n = 5 for F, G). p values were determined by Student's t-test. ∗p < 0.01. One representative result from two independent experiments is shown (F–H). less

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JNK-Dependent cJun Phosphorylation Mitigates TGFβ- and EGF-Induced Pre-Malignant Breast Cancer Cell Invasion by Suppressing AP-1-Mediated Transcriptional Responses.

In Cells on 21 November 2019 by Sundqvist, A., Voytyuk, O., et al.

Fig.3.A

Fig.3.A showing Western Blotting from the publication: JNK-Dependent cJun Phosphorylation Mitigates TGFβ- and EGF-Induced Pre-Malignant Breast Cancer Cell Invasion by Suppressing AP-1-Mediated Transcriptional Responses.

EGF and TGFβ differentially induced phospho-cJun activity. (a) MCF10A MII cells were incubated for 24 h in serum-starvation medium (insulin, cholera toxin, hydrocortisone, 0.2% FBS) without EGF, subsequently treated with 20 ng/mL EGF and/or 5 ng/mL TGFβ for the indicated time periods, and then an... more

EGF and TGFβ differentially induced phospho-cJun activity. (a) MCF10A MII cells were incubated for 24 h in serum-starvation medium (insulin, cholera toxin, hydrocortisone, 0.2% FBS) without EGF, subsequently treated with 20 ng/mL EGF and/or 5 ng/mL TGFβ for the indicated time periods, and then analyzed by immunoblotting for P-S63-cJun, cJun, P-JNK, cFos, P-ERK1/2, P-Smad3, Smad2/3, and PAI1. Tubulin was included as loading control; a representative experiment is shown. The levels of P-S63-cJun and (total) cJun of three independent biological replicates were quantified by densitometry to determine the effects of EGF and TGFβ on the relative ratio of the levels of P-S63-cJun and cJun in time, depicted in the graph. For proper comparison of the data of the different experiments, the ratio of the levels of P-S63-cJun and cJun at the 30 min EGF time point of each experiment was set at 1.0. (b) MCF10A MII cells were incubated for 24 h in EGF-containing serum-starvation medium (EGF, insulin, cholera toxin, hydrocortisone, 0.2% FBS) or in starvation medium lacking EGF as indicated, treated with 5 ng/mL TGFβ for the indicated time periods, and analyzed by immunoblotting for P-S63-cJun, cJun, Fra1, and P-ERK1/2. Tubulin was included as loading control; a representative experiment is shown. The levels of P-S63-cJun and (total) cJun of four independent biological replicates were quantified to determine the effects of EGF and/or TGFβ on the relative ratio of the levels of P-S63-cJun and cJun in time, depicted in the graph. For proper comparison of the data of the different experiments, the ratio of the levels of P-S63-cJun and cJun at the 0 h TGFβ + EGF time point of each experiment was set at 1.0. *p < 0.05, **p < 0.01, ***p < 0.001 (c) MCF10A MII cells were incubated for 24 h in EGF-containing serum-starvation medium, subsequently stimulated for 1 h with TGFβ in the presence or absence of the indicated TGFβRI, MEK, and JNK inhibitors, and analyzed by immunoblotting for P-S63-cJun, cJun, P-ERK1/2, and P-Smad3. The inhibitors were added 30 min before TGFβ. A background band was included as loading control (L.c.); (d) Schematic model to explain the data in panels (a–c) in view of the current literature. EGF signaling via cell-membrane-localized EGFR triggers phosphorylation and activation of ERK and JNK, which in the nucleus activate and induce FOS family members and phosphorylation of cJun, respectively, which subsequently can auto-regulate their own expression via AP-1 sites [22,26]. TGFβ signaling via membrane-localized TBRI and TBRII triggers phosphorylation and activation of Smad2 and 3, and thereby can both directly and indirectly induce TGFβ target genes controlled by Smad and/or AP-1 sites [1,2]. less

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TGF-β family ligands exhibit distinct signalling dynamics that are driven by receptor localisation.

In J Cell Sci on 15 July 2019 by Miller, D. S. J., Schmierer, B., et al.

Fig.3.B

Fig.3.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: TGF-β family ligands exhibit distinct signalling dynamics that are driven by receptor localisation.

BMP4 and activin do not induce refractory behaviour. (A) Left, a schematic of the experimental set-up. NIH-3T3 cells were untreated (a) or treated with BMP4 for 1 h (b) or 3 h (c). After 1 h of BMP4 stimulation, the signal was brought down to baseline through treatment with noggin for 2 h (d), wh... more

BMP4 and activin do not induce refractory behaviour. (A) Left, a schematic of the experimental set-up. NIH-3T3 cells were untreated (a) or treated with BMP4 for 1 h (b) or 3 h (c). After 1 h of BMP4 stimulation, the signal was brought down to baseline through treatment with noggin for 2 h (d), which was then washed out and cells were re-stimulated with BMP4 for 1 h (e). The efficacy of noggin washout was confirmed (f), as was its inhibitory ability by adding the ligand and antagonist simultaneously (g). To confirm that BMP4 was not depleted from the medium in the time period of these experiments, cells were stimulated with BMP4 for 3 h, then the medium transferred to naïve cells for 1 h (h). Right, western blotting for PSMAD1/5, SMAD1 and tubulin, as a loading control, was performed. Quantifications are means±s.d. of densitometry measurements from three independent experiments, which are normalised to measurements in cells treated with BMP4 for 1 h. (B) Left, a schematic of the experimental set-up. P19 cells were untreated (a), treated overnight with SB-431542 (b) or treated with activin for 1 h (c) or 3 h (d). After 1 h of activin stimulation, the signal was brought down to baseline through treatment with follistatin for 2 h (e), which was then washed out and cells were re-stimulated with activin for 1 h (f). The efficacy of follistatin washout was confirmed (g), as was its inhibitory ability (g). To confirm that activin was not depleted from the medium in the time period of these experiments, cells were stimulated with activin for 3 h, then the medium transferred to naïve cells for 1 h (i). Right, western blotting for PSMAD2, SMAD2 and tubulin, as a loading control, was performed. Quantifications are means±s.d. of densitometry measurements from three independent experiments, which are normalised to measurements in cells treated with activin for 1 h. less

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The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development.

In Nat Commun on 23 March 2018 by Wojciech, S., Ahmad, R., et al.

Fig.2.C

Fig.2.C showing Western Blotting from the publication: The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development.

GPR50 promotes ligand-independent activation of TβRI signaling. a HEK293T cells expressing myc-Smad3, and GPR50Δ4 or GPR50wt were starved overnight and stimulated with TGFβ (2 ng/mL; 1 h). Smad3 phosphorylation was checked. Similar results were obtained in at least two additional experiments. b p... more

GPR50 promotes ligand-independent activation of TβRI signaling. a HEK293T cells expressing myc-Smad3, and GPR50Δ4 or GPR50wt were starved overnight and stimulated with TGFβ (2 ng/mL; 1 h). Smad3 phosphorylation was checked. Similar results were obtained in at least two additional experiments. b p-Smad2 detection in NCI-H520 cells following the silencing of GPR50 and TβRI Control si-RNA (si-Ctrl) with and without TGFβ stimulation (2 ng/mL; 1 h) served as control. Densitometric analysis of three independent experiments (Mean ± s.e.m., n = 3 independent experiments, ***p < 0,001, one-way ANOVA with Dunnett’s post hoc test). c, d Detection of p-Smad3, p-Stat-3, p-Jak2 (c) and p-Smad2 (d) in lysates of hypothalamus and cortex of wt and GPR50ko mice. p-Smad2 was detected after precipitation of total Smad2/3. Quantification is shown on the right side of panel c. H hypothalamus, C cortex; Densitometric analysis of three independent experiments (Mean ± s.e.m., n = 3 independent experiments, **p < 0.01, two-tailed unpaired t-test). e Confocal images of HeLa cells expression GPR50 and TβRI alone or together showing TβRI colocalization with early endosome marker (EEA1) with TGFβ stimulation (scale: 10 µm). f (Left panel) Nuclear extracts of HEK293T cells treated with TGFβ (2 ng/mL, 1 h) and SB431542 (10 µM; O/N) and expressing indicated proteins. (Right) Densitometric analysis of three independent experiments (Mean ± s.e.m., n = 3 independent experiments, *p < 0.05, **p < 0.01, one-way ANOVA with Dunnett’s post hoc test). g HeLa cells were transfected with a a Firefly-Luciferase-coupled ARE- (together with FAST-2) reporter gene construct and Renilla Luciferase for normalization. The cells were transfected with empty (Mock± TGFβ; 0.5 ng/mL, 8 h) or 10, 50 and 100 ng of GPR50Δ4 and GPR50wt constructs (Mean ± s.e.m., n = 3 independent experiments, *p < 0.05, ***p < 0.001 one-way ANOVA with Dunnett’s post hoc test). h 4T1 cells stably expressing either empty plasmid (Mock) or GPR50∆4 were stimulated with TGFβ (2 ng/mL; 0, 8, 24 h). Snail expression was analyzed by immunoblotting. i HEK293T cells expressing indicated plasmids as in (a) and stimulated with TGFβ (2ng/mL, 1 h) to reveal p-p38 protein. Representative results are shown for a, d, e, h, i. Similar results were obtained in at least two additional experiments. See also Supplementary Fig. 2 less

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The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development.

In Nat Commun on 23 March 2018 by Wojciech, S., Ahmad, R., et al.

Fig.2.D

Fig.2.D showing Western Blotting from the publication: The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development.

GPR50 promotes ligand-independent activation of TβRI signaling. a HEK293T cells expressing myc-Smad3, and GPR50Δ4 or GPR50wt were starved overnight and stimulated with TGFβ (2 ng/mL; 1 h). Smad3 phosphorylation was checked. Similar results were obtained in at least two additional experiments. b p... more

GPR50 promotes ligand-independent activation of TβRI signaling. a HEK293T cells expressing myc-Smad3, and GPR50Δ4 or GPR50wt were starved overnight and stimulated with TGFβ (2 ng/mL; 1 h). Smad3 phosphorylation was checked. Similar results were obtained in at least two additional experiments. b p-Smad2 detection in NCI-H520 cells following the silencing of GPR50 and TβRI Control si-RNA (si-Ctrl) with and without TGFβ stimulation (2 ng/mL; 1 h) served as control. Densitometric analysis of three independent experiments (Mean ± s.e.m., n = 3 independent experiments, ***p < 0,001, one-way ANOVA with Dunnett’s post hoc test). c, d Detection of p-Smad3, p-Stat-3, p-Jak2 (c) and p-Smad2 (d) in lysates of hypothalamus and cortex of wt and GPR50ko mice. p-Smad2 was detected after precipitation of total Smad2/3. Quantification is shown on the right side of panel c. H hypothalamus, C cortex; Densitometric analysis of three independent experiments (Mean ± s.e.m., n = 3 independent experiments, **p < 0.01, two-tailed unpaired t-test). e Confocal images of HeLa cells expression GPR50 and TβRI alone or together showing TβRI colocalization with early endosome marker (EEA1) with TGFβ stimulation (scale: 10 µm). f (Left panel) Nuclear extracts of HEK293T cells treated with TGFβ (2 ng/mL, 1 h) and SB431542 (10 µM; O/N) and expressing indicated proteins. (Right) Densitometric analysis of three independent experiments (Mean ± s.e.m., n = 3 independent experiments, *p < 0.05, **p < 0.01, one-way ANOVA with Dunnett’s post hoc test). g HeLa cells were transfected with a a Firefly-Luciferase-coupled ARE- (together with FAST-2) reporter gene construct and Renilla Luciferase for normalization. The cells were transfected with empty (Mock± TGFβ; 0.5 ng/mL, 8 h) or 10, 50 and 100 ng of GPR50Δ4 and GPR50wt constructs (Mean ± s.e.m., n = 3 independent experiments, *p < 0.05, ***p < 0.001 one-way ANOVA with Dunnett’s post hoc test). h 4T1 cells stably expressing either empty plasmid (Mock) or GPR50∆4 were stimulated with TGFβ (2 ng/mL; 0, 8, 24 h). Snail expression was analyzed by immunoblotting. i HEK293T cells expressing indicated plasmids as in (a) and stimulated with TGFβ (2ng/mL, 1 h) to reveal p-p38 protein. Representative results are shown for a, d, e, h, i. Similar results were obtained in at least two additional experiments. See also Supplementary Fig. 2 less

Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license

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The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development.

In Nat Commun on 23 March 2018 by Wojciech, S., Ahmad, R., et al.

Fig.2.F

Fig.2.F showing Western Blotting from the publication: The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development.

GPR50 promotes ligand-independent activation of TβRI signaling. a HEK293T cells expressing myc-Smad3, and GPR50Δ4 or GPR50wt were starved overnight and stimulated with TGFβ (2 ng/mL; 1 h). Smad3 phosphorylation was checked. Similar results were obtained in at least two additional experiments. b p... more

GPR50 promotes ligand-independent activation of TβRI signaling. a HEK293T cells expressing myc-Smad3, and GPR50Δ4 or GPR50wt were starved overnight and stimulated with TGFβ (2 ng/mL; 1 h). Smad3 phosphorylation was checked. Similar results were obtained in at least two additional experiments. b p-Smad2 detection in NCI-H520 cells following the silencing of GPR50 and TβRI Control si-RNA (si-Ctrl) with and without TGFβ stimulation (2 ng/mL; 1 h) served as control. Densitometric analysis of three independent experiments (Mean ± s.e.m., n = 3 independent experiments, ***p < 0,001, one-way ANOVA with Dunnett’s post hoc test). c, d Detection of p-Smad3, p-Stat-3, p-Jak2 (c) and p-Smad2 (d) in lysates of hypothalamus and cortex of wt and GPR50ko mice. p-Smad2 was detected after precipitation of total Smad2/3. Quantification is shown on the right side of panel c. H hypothalamus, C cortex; Densitometric analysis of three independent experiments (Mean ± s.e.m., n = 3 independent experiments, **p < 0.01, two-tailed unpaired t-test). e Confocal images of HeLa cells expression GPR50 and TβRI alone or together showing TβRI colocalization with early endosome marker (EEA1) with TGFβ stimulation (scale: 10 µm). f (Left panel) Nuclear extracts of HEK293T cells treated with TGFβ (2 ng/mL, 1 h) and SB431542 (10 µM; O/N) and expressing indicated proteins. (Right) Densitometric analysis of three independent experiments (Mean ± s.e.m., n = 3 independent experiments, *p < 0.05, **p < 0.01, one-way ANOVA with Dunnett’s post hoc test). g HeLa cells were transfected with a a Firefly-Luciferase-coupled ARE- (together with FAST-2) reporter gene construct and Renilla Luciferase for normalization. The cells were transfected with empty (Mock± TGFβ; 0.5 ng/mL, 8 h) or 10, 50 and 100 ng of GPR50Δ4 and GPR50wt constructs (Mean ± s.e.m., n = 3 independent experiments, *p < 0.05, ***p < 0.001 one-way ANOVA with Dunnett’s post hoc test). h 4T1 cells stably expressing either empty plasmid (Mock) or GPR50∆4 were stimulated with TGFβ (2 ng/mL; 0, 8, 24 h). Snail expression was analyzed by immunoblotting. i HEK293T cells expressing indicated plasmids as in (a) and stimulated with TGFβ (2ng/mL, 1 h) to reveal p-p38 protein. Representative results are shown for a, d, e, h, i. Similar results were obtained in at least two additional experiments. See also Supplementary Fig. 2 less

Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license

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The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development.

In Nat Commun on 23 March 2018 by Wojciech, S., Ahmad, R., et al.

Fig.2.B

Fig.2.B showing Western Blotting from the publication: The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development.

GPR50 promotes ligand-independent activation of TβRI signaling. a HEK293T cells expressing myc-Smad3, and GPR50Δ4 or GPR50wt were starved overnight and stimulated with TGFβ (2 ng/mL; 1 h). Smad3 phosphorylation was checked. Similar results were obtained in at least two additional experiments. b p... more

GPR50 promotes ligand-independent activation of TβRI signaling. a HEK293T cells expressing myc-Smad3, and GPR50Δ4 or GPR50wt were starved overnight and stimulated with TGFβ (2 ng/mL; 1 h). Smad3 phosphorylation was checked. Similar results were obtained in at least two additional experiments. b p-Smad2 detection in NCI-H520 cells following the silencing of GPR50 and TβRI Control si-RNA (si-Ctrl) with and without TGFβ stimulation (2 ng/mL; 1 h) served as control. Densitometric analysis of three independent experiments (Mean ± s.e.m., n = 3 independent experiments, ***p < 0,001, one-way ANOVA with Dunnett’s post hoc test). c, d Detection of p-Smad3, p-Stat-3, p-Jak2 (c) and p-Smad2 (d) in lysates of hypothalamus and cortex of wt and GPR50ko mice. p-Smad2 was detected after precipitation of total Smad2/3. Quantification is shown on the right side of panel c. H hypothalamus, C cortex; Densitometric analysis of three independent experiments (Mean ± s.e.m., n = 3 independent experiments, **p < 0.01, two-tailed unpaired t-test). e Confocal images of HeLa cells expression GPR50 and TβRI alone or together showing TβRI colocalization with early endosome marker (EEA1) with TGFβ stimulation (scale: 10 µm). f (Left panel) Nuclear extracts of HEK293T cells treated with TGFβ (2 ng/mL, 1 h) and SB431542 (10 µM; O/N) and expressing indicated proteins. (Right) Densitometric analysis of three independent experiments (Mean ± s.e.m., n = 3 independent experiments, *p < 0.05, **p < 0.01, one-way ANOVA with Dunnett’s post hoc test). g HeLa cells were transfected with a a Firefly-Luciferase-coupled ARE- (together with FAST-2) reporter gene construct and Renilla Luciferase for normalization. The cells were transfected with empty (Mock± TGFβ; 0.5 ng/mL, 8 h) or 10, 50 and 100 ng of GPR50Δ4 and GPR50wt constructs (Mean ± s.e.m., n = 3 independent experiments, *p < 0.05, ***p < 0.001 one-way ANOVA with Dunnett’s post hoc test). h 4T1 cells stably expressing either empty plasmid (Mock) or GPR50∆4 were stimulated with TGFβ (2 ng/mL; 0, 8, 24 h). Snail expression was analyzed by immunoblotting. i HEK293T cells expressing indicated plasmids as in (a) and stimulated with TGFβ (2ng/mL, 1 h) to reveal p-p38 protein. Representative results are shown for a, d, e, h, i. Similar results were obtained in at least two additional experiments. See also Supplementary Fig. 2 less

Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license

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Image 1 of 33