Journal: Oncogenesis

Article Title: Deficiency of Erbin induces resistance of cervical cancer cells to anoikis in a STAT3-dependent manner

doi: 10.1038/oncsis.2013.18

Figure Lengend Snippet: Loss of Erbin induces STAT3 activation in cervical cancer cells. ( a , b ) HeLa/NC and HeLa/Erbin-sh cells were cultured in the media containing 1% or 10% or no serum. The phosphorylations of FAK ( a ) and Src ( b ) were analyzed by western blot. ( c ) Parental HeLa (HeLa/WT), HeLa/NC and HeLa/Erbin-sh cells were cultured in the media containing 1% or no serum. The phosphorylation of STAT3 was analyzed by western blot. The relative STAT3 phosphorylation levels were determined by densitometry and normalized with protein levels. ( d ) HeLa/NC and HeLa/Erbin-sh cells were cultured in the media containing 1% serum in the plates coated with PolyHEMA. The phosphorylation of STAT3 was analyzed at the indicated time points. The relative STAT3 phosphorylation levels were determined by densitometry and normalized with protein levels. ( e ) HeLa/NC and HeLa/Erbin-sh cells were cultured in serum-free media. The phosphorylation of STAT3 was analyzed at the indicated time points. ( f ) HeLa/NC and HeLa/Erbin-sh cells were cultured in serum-free media overnight and then treated with 10 μℳ ISO. The phosphorylation of STAT3 was analyzed at the indicated time points. ** P <0.01.

Article Snippet: The HeLa cells were treated with 10 ng/ml IL-6 for different time periods and then labeled with the rabbit monoclonal antibody against phosphor-STAT3 (Tyr705, Cell Signaling).

Techniques: Activation Assay, Cell Culture, Western Blot, Phospho-proteomics

Journal: Oncogenesis

Article Title: Deficiency of Erbin induces resistance of cervical cancer cells to anoikis in a STAT3-dependent manner

doi: 10.1038/oncsis.2013.18

Figure Lengend Snippet: Loss of Erbin expression confers resistance of cervical cancer cells to anoikis in a STAT3-dependent manner. ( a , b ) HeLa cells were transfected with the plasmid expressing STAT3C (HeLa/STAT3C) or the empty vector (HeLa/Vector). The transfected cells were cultured in the medium containing 1% serum in the plates coated with PolyHEMA. After incubation for 48 and 72 h, the anoikis rates were determined by FACS. ( c , d ) HeLa/NC and HeLa/Erbin-sh cells cultured in suspension were treated with WP1066. The anoikis rates were determined by FACS. ( e , f ) HeLa/NC and HeLa/Erbin-sh cells under conventional culture were treated with WP1066. The apoptotic rates were determined by FACS. ( g , h ) HeLa/NC and HeLa/Erbin-sh cells were treated with WP1066, U0126 or PD98059. The anoikis rates were determined by FACS. ** P <0.01. DMSO, dimethyl sulfoxide; PI, propidium iodide.

Article Snippet: The HeLa cells were treated with 10 ng/ml IL-6 for different time periods and then labeled with the rabbit monoclonal antibody against phosphor-STAT3 (Tyr705, Cell Signaling).

Techniques: Expressing, Transfection, Plasmid Preparation, Cell Culture, Incubation, Suspension

Journal: Oncogenesis

Article Title: Deficiency of Erbin induces resistance of cervical cancer cells to anoikis in a STAT3-dependent manner

doi: 10.1038/oncsis.2013.18

Figure Lengend Snippet: Erbin negatively regulates IL-6/STAT3 pathway. ( a ) HeLa/NC and HeLa/Erbin-sh cells under conventional culture were treated with 10 ng/ml IL-6. The activation of STAT3 was analyzed by western blot at the indicated time points. ( b ) HeLa/NC and HeLa/Erbin-sh cells were treated with 10 ng/ml IL-6 and then labeled with the anti-phoaphor-STAT3 antibody and Alexa fluor 549-labeled secondary antibody. Nuclei were stained with 1 μg/ml DAPI. Nuclear translocation of activated STAT3 was observed under a laser scanning confocal microscope. ( c ) HeLa/NC and HeLa/Erbin-sh cells grown in 24-well plates were transiently cotransfected by the STAT3 reporter and pRL-TK vectors. The transfected cells were treated with or without 10 ng/ml IL-6. The luciferase activities were measured using a dual luciferase assay kit. ( d , e ) HeLa/NC and HeLa/Erbin-sh cells were cultured in the media containing 1% serum in the plates coated with PolyHEMA for 24 h and treated with or without 10 ng/ml IL-6. The phosphorylation of STAT3 was analyzed ( d ) and the luciferase activities were measured ( e ). ( f ) HeLa cells were treated with or without IL-6, and the expression of Erbin mRNA was analyzed by real-time reverse transcriptase–PCR (RT-PCR). ( g ) HeLa cells were transfected with pRc/CMV-Stat3C-Flag (HeLa/STAT3C) or the empty vector (HeLa/Vector). The expression of Erbin mRNA was examined by real-time RT-PCR. ( h ) The HeLa/STAT3C and HeLa/Vector cells were transfected with Erbin siRNA or control siRNA. The expression of Erbin protein was examined by western blot. ( i ) HeLa cells were starved overnight and then treated with AG490. IL-6 was added into the cell culture after 1 h of the treatment. The phosphorylation of STAT3 was analyzed by western blot at the indicated time points. ** P <0.01.

Article Snippet: The HeLa cells were treated with 10 ng/ml IL-6 for different time periods and then labeled with the rabbit monoclonal antibody against phosphor-STAT3 (Tyr705, Cell Signaling).

Techniques: Activation Assay, Western Blot, Labeling, Staining, Translocation Assay, Microscopy, Transfection, Luciferase, Cell Culture, Phospho-proteomics, Expressing, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Quantitative RT-PCR, Control

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-Phospho-Histone H3 , Cell Signaling Technology , Cat# 53348; RRID: AB_2799431.

Techniques: Virus, Recombinant, Control, Enzyme-linked Immunosorbent Assay, Isolation, Membrane, Protein Extraction, Chromatin Immunoprecipitation, Bicinchoninic Acid Protein Assay, Sircol Collagen Assay, Luciferase, RNA Sequencing Assay, Sequencing, Expressing, Real-time Polymerase Chain Reaction, shRNA, Plasmid Preparation, Software, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: EDIL3/Del-1-Dependent Induction of AMPKβ Phosphorylation Regulates the Progression of Mesenchymal Stem-like Triple-Negative Breast Cancer

doi: 10.3390/ijms27062679

Figure Lengend Snippet: AMPK family members are downregulated in Del-1-silenced breast cancer cells. AMPK, AMP-activated protein kinase; Del-1, developmental endothelial locus-1.

Article Snippet: The following reagents and antibodies against the following proteins were used: A769662 (3336; Tocris, Bristol, United Kingdom); AMPKβ1/2 (4150), total AMPK (2532), and phosphorylated AMPKβ (p-AMPKβ; S108) (23021) from Cell Signaling Technology (Danvers, MA, USA); GAPDH (sc-47724) from Santa Cruz Biotechnology (Dallas, TX, USA); Del-1 (PA5-27994) from Thermo Fisher Scientific (Rockford, IL, USA); and HA (ab236632) from Abcam (Waltham, MA, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: EDIL3/Del-1-Dependent Induction of AMPKβ Phosphorylation Regulates the Progression of Mesenchymal Stem-like Triple-Negative Breast Cancer

doi: 10.3390/ijms27062679

Figure Lengend Snippet: Increased AMPKβ is associated with TNBC ( A ) Western blot analysis of AMPK subunit protein levels in the indicated cells at 24 h. n ≥ 5. Data are representative of three independent experiments. ( B ) Real-time PCR analysis reveals the mRNA level of AMPK subunits AMPKβ, AMPKα, and γ ( C ) in human breast cancer tissues (HER2, n = 4; TNBC, n = 28). ( C ) Kaplan–Meier survival analysis for patients with breast cancer with high (red line) or low (black line) levels of AMPK β mRNA expression. ( D ) Survival curves based on AMPKβ expression in a cohort of 100 patients. ( n = 100). Multivariate analysis is provided in . ( E ) AMPKβ cytoplasmic staining was evaluated using a semi-quantitative approach: positivity was defined as ≥1% of tumor cells showing cytoplasmic staining, and staining intensity was graded as 0–3 (0, none; 1+, faint; 2+, intermediate; 3+, moderate-to-strong). GAPDH served as a loading control. DFS, disease-free survival; DDFS, distant disease-free survival; OS, overall survival; AMPK, AMP-activated protein kinase; HER2, human epidermal growth factor receptor 2; TNBC, triple-negative breast cancer; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PCR, polymerase chain reaction.

Article Snippet: The following reagents and antibodies against the following proteins were used: A769662 (3336; Tocris, Bristol, United Kingdom); AMPKβ1/2 (4150), total AMPK (2532), and phosphorylated AMPKβ (p-AMPKβ; S108) (23021) from Cell Signaling Technology (Danvers, MA, USA); GAPDH (sc-47724) from Santa Cruz Biotechnology (Dallas, TX, USA); Del-1 (PA5-27994) from Thermo Fisher Scientific (Rockford, IL, USA); and HA (ab236632) from Abcam (Waltham, MA, USA).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Expressing, Staining, Control, Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: EDIL3/Del-1-Dependent Induction of AMPKβ Phosphorylation Regulates the Progression of Mesenchymal Stem-like Triple-Negative Breast Cancer

doi: 10.3390/ijms27062679

Figure Lengend Snippet: Pharmacological activation of AMPKβ promotes proliferation and invasion in MDA-MB-231 cells ( A ) Western blot analysis of protein expression in MDA-MB-231 cells treated with DMSO (0 nM) or the AMPKβ activator (10–100 nM) for 24 h. n ≥ 5 ( B ) BrdU proliferation assays reveal the effects of AMPKβ activator (100 nM) after 48 h. ( C ) MTS assays evaluated the proliferative effect of AMPKβ activator in MDA-MB-231 breast cancer cells. ( D ) Migration assays validated the migratory capacity of MDA-MB-231 cells exposed to the AMPKβ activator. ( E ) Transwell invasion assays measured invasiveness after 24 h of exposure to the AMPKβ activator (100 nM). GAPDH served as a loading control. Data are representative of three independent experiments. * p < 0.05 and ** p < 0.01. AMPK, AMP-activated protein kinase; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The following reagents and antibodies against the following proteins were used: A769662 (3336; Tocris, Bristol, United Kingdom); AMPKβ1/2 (4150), total AMPK (2532), and phosphorylated AMPKβ (p-AMPKβ; S108) (23021) from Cell Signaling Technology (Danvers, MA, USA); GAPDH (sc-47724) from Santa Cruz Biotechnology (Dallas, TX, USA); Del-1 (PA5-27994) from Thermo Fisher Scientific (Rockford, IL, USA); and HA (ab236632) from Abcam (Waltham, MA, USA).

Techniques: Activation Assay, Western Blot, Expressing, Migration, Control

Journal: International Journal of Molecular Sciences

Article Title: EDIL3/Del-1-Dependent Induction of AMPKβ Phosphorylation Regulates the Progression of Mesenchymal Stem-like Triple-Negative Breast Cancer

doi: 10.3390/ijms27062679

Figure Lengend Snippet: Del-1 overexpression enhances AMPKβ expression and promotes proliferation, migration, and invasion ( A ) ELISA measured Del-1 protein levels after overexpression in MDA-MB-231 and Hs 578T cells. ( B , C ) Western blot analysis reveals protein expression in MDA-MB-231 ( B ) and Hs 578T ( C ) cells following Del-1 overexpression. n ≥ 5 ( D ) BrdU proliferation assays demonstrate the effect of Del-1 overexpression compared with the control vector in MDA-MB-231 and Hs 578T cells after 48 h. ( E ) MTS assays evaluated the proliferative effect of Del-1 overexpression. ( F , G ) Wound-healing assays show the effects of Del-1 overexpression on cell migration for 72 h in MDA-MB-231 ( F ) and Hs 578T ( G ) cells. ( H ) Transwell matrix invasion assays illustrate the invasive capacity of MDA-MB-231 and Hs 578T cells after 48 h of Del-1 overexpression. GAPDH served as a loading control. Data are representative of 3 independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The following reagents and antibodies against the following proteins were used: A769662 (3336; Tocris, Bristol, United Kingdom); AMPKβ1/2 (4150), total AMPK (2532), and phosphorylated AMPKβ (p-AMPKβ; S108) (23021) from Cell Signaling Technology (Danvers, MA, USA); GAPDH (sc-47724) from Santa Cruz Biotechnology (Dallas, TX, USA); Del-1 (PA5-27994) from Thermo Fisher Scientific (Rockford, IL, USA); and HA (ab236632) from Abcam (Waltham, MA, USA).

Techniques: Over Expression, Expressing, Migration, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: EDIL3/Del-1-Dependent Induction of AMPKβ Phosphorylation Regulates the Progression of Mesenchymal Stem-like Triple-Negative Breast Cancer

doi: 10.3390/ijms27062679

Figure Lengend Snippet: Del-1 knockdown reduces AMPKβ levels. ( A , C ) ELISA confirmed the Del-1 protein levels of CRISPR-Cas9-mediated Del1 knockout MDA-MB-231 ( A ) and Hs 578T cells ( C ). ( B , D ) Western blot analysis of related protein expression in MDA-MB-231 ( B ) and Hs 578T ( D ) cells with CRISPR-Cas9-mediated Del1 knockout. GAPDH served as a loading control. Data are representative of three independent experiments. ** p < 0.01 and *** p < 0.001. AMPK, AMP-activated protein kinase; Del-1, developmental endothelial locus-1; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. n ≥ 5.

Article Snippet: The following reagents and antibodies against the following proteins were used: A769662 (3336; Tocris, Bristol, United Kingdom); AMPKβ1/2 (4150), total AMPK (2532), and phosphorylated AMPKβ (p-AMPKβ; S108) (23021) from Cell Signaling Technology (Danvers, MA, USA); GAPDH (sc-47724) from Santa Cruz Biotechnology (Dallas, TX, USA); Del-1 (PA5-27994) from Thermo Fisher Scientific (Rockford, IL, USA); and HA (ab236632) from Abcam (Waltham, MA, USA).

Techniques: Knockdown, Enzyme-linked Immunosorbent Assay, CRISPR, Knock-Out, Western Blot, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: EDIL3/Del-1-Dependent Induction of AMPKβ Phosphorylation Regulates the Progression of Mesenchymal Stem-like Triple-Negative Breast Cancer

doi: 10.3390/ijms27062679

Figure Lengend Snippet: Del-1-mediated AMPK β phosphorylation at S108 is crucial for TNBC cell metastasis ( A ) Schematic diagram showing the genetic alteration of the phosphorylation site. ( B ) Western blot analysis of protein levels in MDA-MB-231 and Hs 578T cells expressing AMPKβ WT or its phosphomimetic form (SE). ( C ) MTS assays reveal the effects of AMPKβ WT and SE overexpression in MDA-MB-231 and Hs 578T cells. Data are representative of three independent experiments. GAPDH served as a loading control. * indicates a nonspecific band. The arrow represents both HA-AMPKβ WT and S1083E vectors. * p < 0.05 and ** p < 0.01. DEL-1, developmental endothelial locus-1; TNBC, triple-negative breast cancer; S108, serine 108 site; AMPK, AMP-activated protein kinase; GAPDH, glyceraldehyde 3-phosphate; WT, wild-type; SE, AMPKβ phosphomimetic form. n ≥ 5.

Article Snippet: The following reagents and antibodies against the following proteins were used: A769662 (3336; Tocris, Bristol, United Kingdom); AMPKβ1/2 (4150), total AMPK (2532), and phosphorylated AMPKβ (p-AMPKβ; S108) (23021) from Cell Signaling Technology (Danvers, MA, USA); GAPDH (sc-47724) from Santa Cruz Biotechnology (Dallas, TX, USA); Del-1 (PA5-27994) from Thermo Fisher Scientific (Rockford, IL, USA); and HA (ab236632) from Abcam (Waltham, MA, USA).

Techniques: Phospho-proteomics, Western Blot, Expressing, Over Expression, Control

Journal: Journal of Biological Chemistry

Article Title: The T-cell Lymphokine Interleukin-26 Targets Epithelial Cells through the Interleukin-20 Receptor 1 and Interleukin-10 Receptor 2 Chains

doi: 10.1074/jbc.m405000200

Figure Lengend Snippet: FIG. 2. IL-26-mediated induction of STAT1 and STAT3 phos- phorylation. A, IL-26 titration for induction of STAT3 phosphoryla- tion. Colo-205 carcinoma cells were treated with purified recombinant His-IL-26 (up to 30 ng/ml) for 20 min in 24-well plates. Whereas no signal was detectable at 0.3 ng/ml, 1 ng/ml was sufficient to induce STAT3 phosphorylation as determined by Western blot specific for tyrosine-phosphorylated STAT3 (pSTAT3). As a control, a lysate of the HVS-transformed T-cell line CB-15 was used that constitutively ex- presses phosphorylated STAT3. B, kinetics of IL-26-induced STAT3 phosphorylation. Colo-205 carcinoma cells were treated with 10 ng/ml purified recombinant His-IL-26 for 0.5, 1.0, 5, and 10 min. STAT3 phosphorylation was determined by pSTAT3-specific Western blotting. A faint signal was already detectable after 1 min, whereas 5 min were sufficient for strong STAT3 phosphorylation signals. The lower panel shows total STAT3 protein amounts irrespective of phosphorylation (restained membrane). C, IL-26-induced phosphorylation of STAT1 and STAT3. Colo-205 carcinoma cells were treated with a low concentration of recombinant His-IL-26 (2 ng/ml) for 0.5, 1.0, 5, 10, 20, 30, and 60 min. Phosphorylation of STAT1 and STAT3 were determined by Western blotting and staining with antibodies against the phosphorylated forms. After pSTAT1 staining, the same membrane was reprobed for pSTAT3. The phosphorylation of both factors occurred synchronously.

Article Snippet: STAT factor signaling was investigated with the following rabbit antibodies: STAT1 (9172), pSTAT1 (against phosphorylated tyrosine residue 701 of STAT1, 9171), STAT3 (9132), pSTAT3 (against phosphorylated tyrosine residue 705 or STAT3, 9131, all from Cell Signaling Technology, Frankfurt).

Techniques: Titration, Purification, Recombinant, Phospho-proteomics, Western Blot, Control, Transformation Assay, Membrane, Concentration Assay, Staining

Journal: Journal of Biological Chemistry

Article Title: The T-cell Lymphokine Interleukin-26 Targets Epithelial Cells through the Interleukin-20 Receptor 1 and Interleukin-10 Receptor 2 Chains

doi: 10.1074/jbc.m405000200

Figure Lengend Snippet: FIG. 3. Inhibition of IL-26-mediated STAT3 phosphorylation by antiserum and heparin. A, antibody blockade of IL-26-mediated STAT3 phosphorylation. In order to demonstrate the specificity of the IL-26-mediated STAT3 phosphorylation, a rabbit anti-IL-26 antiserum and the preimmune serum as a negative control were preincubated with 10 ng/ml purified recombinant His-IL-26 and then applied to Colo-205 cells. Just 5% (v/v) of rabbit anti-IL-26 antiserum was sufficient to inhibit STAT3 phosphorylation, whereas the preimmune serum did not show an effect. In the lanes that resulted from antisera treatment, an additional band corresponding to immunoglobulin heavy chains (IgG hc) is visible. The bottom panel shows a restain of the same membrane as a control for the presence of equal total STAT3 amounts. B, inhibition of IL-26-mediated STAT3 phosphorylation by heparin. Purified recombinant IL-26 (10 ng/ml) was preincubated for 15 min with heparin, and Colo-205 cells were then incubated for 20 min before lysis. A Western blot for phosphorylated STAT3 indicated that already 0.1 unit/ml heparin considerably reduced STAT3 phosphorylation in Colo-205. At the bottom, total STAT3 Western blots of the same membranes are shown as control for equal protein loading. C, inhibitory activity of various glycosaminoglycans on IL-26. After preincubation with the glycosaminoglycans heparin, heparan sulfate, and chondroitin sulfates A, B, and C (100 g/ml each, corresponding to 20 units of heparin/ml) for 30 min, the effect of purified recombinant His-IL-26 (10 ng/ml) on Colo-205 cells was analyzed by Western blotting for phosphorylated STAT3. As a control, a total STAT3 Western blot from the same lysates is shown. Whereas heparin completely blocked STAT3 phosphorylation, a weaker inhibitory effect was observed for chondroitin B sulfate (dermatan sulfate). In contrast, heparan sulfate and chondroitin A and C sulfate had little influence on the STAT3 phosphorylation levels.

Article Snippet: STAT factor signaling was investigated with the following rabbit antibodies: STAT1 (9172), pSTAT1 (against phosphorylated tyrosine residue 701 of STAT1, 9171), STAT3 (9132), pSTAT3 (against phosphorylated tyrosine residue 705 or STAT3, 9131, all from Cell Signaling Technology, Frankfurt).

Techniques: Inhibition, Phospho-proteomics, Negative Control, Purification, Recombinant, Membrane, Control, Incubation, Lysis, Western Blot, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: The T-cell Lymphokine Interleukin-26 Targets Epithelial Cells through the Interleukin-20 Receptor 1 and Interleukin-10 Receptor 2 Chains

doi: 10.1074/jbc.m405000200

Figure Lengend Snippet: FIG. 4. IL-26-responsive colon carcinoma cells and keratino- cytes. The human colon carcinoma cell lines SW-403 and Lovo (A) and the human keratinocyte cell line HaCaT (B), which is known to react to IL-20, responded to treatment with purified, recombinant His-IL-26 (10 ng/ml; 20 min) with STAT3 phosphorylation as detected by phospho- STAT3 specific staining in Western blots. The STAT3 phosphorylation could be blocked by preincubation of the cytokine with heparin (10 units/ml) and by a rabbit antiserum (10% v/v) against IL-26. Lysates from HVS-transformed CB-15 cells served as a positive control. In contrast to the differential pSTAT3 signals, the total STAT3 control Western blots demonstrated equal protein loadings.

Article Snippet: STAT factor signaling was investigated with the following rabbit antibodies: STAT1 (9172), pSTAT1 (against phosphorylated tyrosine residue 701 of STAT1, 9171), STAT3 (9132), pSTAT3 (against phosphorylated tyrosine residue 705 or STAT3, 9131, all from Cell Signaling Technology, Frankfurt).

Techniques: Purification, Recombinant, Phospho-proteomics, Staining, Western Blot, Transformation Assay, Positive Control, Control

Journal: Journal of Biological Chemistry

Article Title: The T-cell Lymphokine Interleukin-26 Targets Epithelial Cells through the Interleukin-20 Receptor 1 and Interleukin-10 Receptor 2 Chains

doi: 10.1074/jbc.m405000200

Figure Lengend Snippet: FIG. 6. Cytokine receptor molecules involved in IL-26 responsiveness. A, expression patterns of cytokine receptor family type II genes. A series of cell types were tested by RT-PCR for the transcription of various members of the cytokine receptor type II family (IL-10R1, IL-10R2, IL-20R1, IL-20R2, IL-22R, IL-28R, and glyceraldehyde-3-phosphate dehydrogenase as positive control): Dendritic cells (DC, differentiated in vitro from CD14 monocytes), lipopolysaccharide-activated monocytes, SW-403 (colon carcinoma), Lovo (colon carcinoma), HepG2 (hepatoma), Colo-320 (colon carcinoma), Colo-205 (colon carcinoma), HeLa (cervical carcinoma), KMH-2 (Hodgkin’s disease), Panc-I (pancreatic carcinoma), and HaCaT (keratinocytes). The IL-26 responsiveness (cell line designations in boxes) strictly correlated with the expression of IL-20R1. The ubiquitous expression pattern of IL-10R2 is compatible with its possible role in forming the IL-26R. B, involvement of IL-10R2 in the IL-26 response. Colo-205 cells were treated for 5 min with purified, recombinant His-IL-26 (1–2 ng/ml) without or with preincubation for 6 h with blocking antibodies to IL-10R1 and IL-10R2, respectively. Whereas a blocking monoclonal antibody against the IL-10R1 chain did not influence STAT3 phosphorylation levels of IL-26-treated Colo-205 cells, a dose-dependent (1–100 g/ml) inhibition was achieved using a blocking monoclonal antibody against IL-10R2. Equal total STAT3 protein amounts were demonstrated by restaining the same membrane (lower panel). C, participation of IL-20R1 in the IL-26 response. Whereas IL-10R1 antibodies (10 g/ml) did not influence and IL-10R2 (10 g/ml) antibodies reduced STAT3 phosphorylation levels of His-IL-26-stimulated (1 ng/ml) Colo-205 cells, a polyclonal murine antiserum raised against IL-20R1 (diluted 1:500) completely blocked STAT3 activation. Equal total STAT3 protein amounts were demonstrated by restaining the same membrane (lower panel). D, solid-phase interaction test between IL-20R1 and IL-26. Polysterene microtiter plate wells were coated with supernatants from 293T cells (200 l), which had been transfected with either a control vector (gray bars) or an expression construct for the extracellular region of IL-20R1 (gray boxed bars). Purified recombinant His-tagged IL-26 was detected via His tag-specific antibodies. 293T-cell supernatants expressing low amounts of FLAG- tagged IL-20, which was detected using a FLAG tag specific antibody, served as a control. Results of a triplicate experiment are shown. n.d., not detectable. E, reconstitution of functional IL-26 receptors by transient cotransfection of a non-responsive cell line. Chinese hamster ovary cells were transfected with expression constructs for either IL-10R2 or IL-20R1 or both chains in combination. An IL-26-dependent activation of STAT3 phosphorylation was observed only if both chains were transiently expressed. In contrast to the regulated pSTAT3 signals, the total STAT3 control Western blot shows constant signal intensities (lower panel).

Article Snippet: STAT factor signaling was investigated with the following rabbit antibodies: STAT1 (9172), pSTAT1 (against phosphorylated tyrosine residue 701 of STAT1, 9171), STAT3 (9132), pSTAT3 (against phosphorylated tyrosine residue 705 or STAT3, 9131, all from Cell Signaling Technology, Frankfurt).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, In Vitro, Purification, Recombinant, Blocking Assay, Phospho-proteomics, Inhibition, Membrane, Activation Assay, Transfection, Control, Plasmid Preparation, Construct, FLAG-tag, Functional Assay, Cotransfection, Western Blot

Journal: British Journal of Pharmacology

Article Title: Molecular mechanisms underlying the anti-proliferative and anti-migratory effects of folate on homocysteinechallenged rat aortic smooth muscle cells

doi: 10.1111/bph.12130

Figure Lengend Snippet: Molecular mechanisms underlying the effects of folate on p21/p27 up-regulation and the resulting anti-proliferative effect on RASMC. Cells were treated with folate for the indicated time points. The expression of p21/p27 was determined (A), and related signalling pathways of Src and Erk1/2 were analysed (B) using GAPDH as an internal control in RT-PCR or Western blot analysis. (C) Cells were pretreated with PP2 (a Src inhibitor) or U0126 (an ERK inhibitor) for 1 h, or transfected with DNERK2 overnight, followed by the addition of folate or homocysteine (Hcy). The indicated molecules were analysed by Western blots. A representative result for three separate experiments is shown and values are presented as the mean ± SD (*P < 0.05 vs. the control; #P < 0.05 vs. the folate- or homocysteine-treated group). (D) Cells were transfected with antisense (AS) p21/p27 oligonucleotides (40 nM) overnight and then treated with 20 μM folate for an additional 24 h. Effects of p21 and p27 AS oligonucleotides and DNERK2 overexpression in inducing the anti-proliferative effect of folate in RASMC were examined using an MTT assay. Six samples were analysed in each group, and values are presented as the mean ± SD. (E) Cells were transfected with DNERK2 plasmid overnight to reduce the levels of p21 and p27; thereafter, cells cultured at 0.5% FBS medium were challenged with 50 μM homocysteine (Hcy) for 30 min, followed by 20 μM folate treatment for 18 h. Cell migration was observed using a microscope with a CCD camera attached. Magnification 100×; graphs on the right indicate the migration normalized relative to the cell counts of the control. Data are expressed as mean ± SD from five randomly selected fields. *P < 0.05 vs. the control; #P < 0.05 vs. pCDNA alone.

Article Snippet: Western blotting was conducted using the following antibodies: p21, p27, phospho-ERK1/2 and phospho-190RhoGAP from BD Biosciences (San Jose, CA, USA); AKT1/AKT2, phospho-focal adhesion kinase (FAK), phospho-p27(Ser 10 ), phospho-p21(Ser 146 ), RhoA, GAPDH, Lamin A/C and pan-cadherin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); FAK, phospho-paxillin, paxillin and phosphoserine from Millipore; Src (Sigma); and phospho-AKT1 from GeneTex (Irvine, CA, USA).

Techniques: Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Over Expression, MTT Assay, Plasmid Preparation, Cell Culture, Migration, Microscopy

Journal: British Journal of Pharmacology

Article Title: Molecular mechanisms underlying the anti-proliferative and anti-migratory effects of folate on homocysteinechallenged rat aortic smooth muscle cells

doi: 10.1111/bph.12130

Figure Lengend Snippet: Folate prevented homocysteine-mediated increases in p21/p27 cytosolic and RhoA localization by inactivating AKT1. Cells were treated as described for Figure ​Figure3A,3A, but with an increased duration (6 or 9 h) of homocysteine challenge (A), or were transfected with AKT constructs as described in Figure ​Figure3B.3B. The resulting cell lysates were fractionated and analysed for the cytosolic-nuclear distribution of p21 and p27 and cytosolic-membrane RhoA by Western blotting. Data were derived from three independent experiments and are presented as the mean ± SD *P < 0.05 vs. the control; #P < 0.05 vs. the homocysteine-challenged or CAAKT1 group.

Article Snippet: Western blotting was conducted using the following antibodies: p21, p27, phospho-ERK1/2 and phospho-190RhoGAP from BD Biosciences (San Jose, CA, USA); AKT1/AKT2, phospho-focal adhesion kinase (FAK), phospho-p27(Ser 10 ), phospho-p21(Ser 146 ), RhoA, GAPDH, Lamin A/C and pan-cadherin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); FAK, phospho-paxillin, paxillin and phosphoserine from Millipore; Src (Sigma); and phospho-AKT1 from GeneTex (Irvine, CA, USA).

Techniques: Transfection, Construct, Membrane, Western Blot, Derivative Assay, Control

Journal: British Journal of Pharmacology

Article Title: Molecular mechanisms underlying the anti-proliferative and anti-migratory effects of folate on homocysteinechallenged rat aortic smooth muscle cells

doi: 10.1111/bph.12130

Figure Lengend Snippet: Folate-induced activation of RhoA correlated with decreased cytosolic distribution of p21 and p27, and interactions between p21, p27, RhoA and p190RhoGAP in RASMC. (A) Cells cultured on coverslips were challenged with homocysteine for 30 min followed by folate treatment for 6 h. Cells were then immunostained using an anti-p21 or anti-p27 antibody, and were then incubated with a second antibody conjugated with Texas red. Red spots represent p21- or p27-positive staining in the cytosol or nuclei. Fields stained with p21 or p27 were also stained with DAPI to reveal the positions of cell nuclei. Micrographs of representative fields were recorded. (B) Cells receiving similar treatments were immunoprecipitated using an anti-RhoA antibody. The pulled-down complex was detected using anti-p21, anti-p27 and anti-p190RhoGAP antibodies; an IgG heavy chain was used as an internal control for normalization. Bar charts show the band intensities of the indicated molecules according to densitometry. Data were derived from three independent experiments and are presented as the mean ± SD. *P < 0.05 vs. the control; #P < 0.05 vs. the homocysteine-challenged group. (C) Cells were pre-challenged with 50 μM homocysteine for 30 min, followed by incubation with 20 μM folic acid for 6 h. Cell morphology was captured using a fluorescent confocal microscope with a CCD camera attached. Red filaments represent stress fibre formation. Fields stained for the formation of stress fibres were also stained using DAPI to reveal the positions of cell nuclei. Magnification 630×.

Article Snippet: Western blotting was conducted using the following antibodies: p21, p27, phospho-ERK1/2 and phospho-190RhoGAP from BD Biosciences (San Jose, CA, USA); AKT1/AKT2, phospho-focal adhesion kinase (FAK), phospho-p27(Ser 10 ), phospho-p21(Ser 146 ), RhoA, GAPDH, Lamin A/C and pan-cadherin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); FAK, phospho-paxillin, paxillin and phosphoserine from Millipore; Src (Sigma); and phospho-AKT1 from GeneTex (Irvine, CA, USA).

Techniques: Activation Assay, Cell Culture, Incubation, Staining, Immunoprecipitation, Control, Derivative Assay, Microscopy

Journal: British Journal of Pharmacology

Article Title: Molecular mechanisms underlying the anti-proliferative and anti-migratory effects of folate on homocysteinechallenged rat aortic smooth muscle cells

doi: 10.1111/bph.12130

Figure Lengend Snippet: Schematic representation of the signalling pathways and molecular mechanisms involved in the anti-proliferative and anti-migratory effects of folate on homocysteine-treated RASMC. Both homocysteine and folate treatment increased p21 and p27 induction through a Src-Erk-dependent pathway, whereas their differential effects on AKT1 (de)phosphorylation caused different outcomes in the cytosol-nuclear distribution of p21 and p27. The increased nuclear fraction of p21 and p27 induced by AKT1 inactivation resulting from folate treatment contributed to the anti-proliferative effect in RASMC. By contrast, activation of AKT1 following homocysteine treatment increased p21 and p27 phosphorylation, resulting in their increased cytosolic sequestration and increased phosphorylation of p190RhoGAP. This contributed to RhoA inactivation, leading to the pro-migratory effect of homocysteine on RASMC. Folate treatment prevented the effects of homocysteine by activating RhoA through the inhibition of AKT1-mediated signalling pathways.

Article Snippet: Western blotting was conducted using the following antibodies: p21, p27, phospho-ERK1/2 and phospho-190RhoGAP from BD Biosciences (San Jose, CA, USA); AKT1/AKT2, phospho-focal adhesion kinase (FAK), phospho-p27(Ser 10 ), phospho-p21(Ser 146 ), RhoA, GAPDH, Lamin A/C and pan-cadherin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); FAK, phospho-paxillin, paxillin and phosphoserine from Millipore; Src (Sigma); and phospho-AKT1 from GeneTex (Irvine, CA, USA).

Techniques: De-Phosphorylation Assay, Activation Assay, Phospho-proteomics, Inhibition

Journal: PLoS ONE

Article Title: Crosstalk between Nuclear Factor I-C and Transforming Growth Factor-β1 Signaling Regulates Odontoblast Differentiation and Homeostasis

doi: 10.1371/journal.pone.0029160

Figure Lengend Snippet: MDPC-23 cells were cultured in differentiation medium for up to 3 weeks. ( A ) The expression of DSP was evaluated by western blot analysis. GAPDH was used as a loading control. ( B ) Mineralized nodules stained with alizarin red-S were photographed. ( C ) Four stages of differentiation were identified: confluent (preodontoblast; 0 days); early odontoblast differentiation (∼7 days); late odontoblast differentiation (7∼14 days); and mineralization (14∼21 days). Solid gray bars indicate the periods of elevated expression of genes indicated during culture. ( D ) The expression of NFI-C was evaluated by western blot analysis and the results were quantified using ImageJ. ( E ) TGFβ-RI, TGFβ-RII, p-Smad2/3, Runx2, Osx, and p21 were evaluated by western blot analysis.

Article Snippet: All other antibodies against TGFβ-RI (sc-398), TGFβ-RII (sc-400), p-Smad2/3 (sc-11769), p21 (sc-6246), and Osterix (sc-22538) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Cell Culture, Expressing, Western Blot, Control, Staining

Journal: PLoS ONE

Article Title: Crosstalk between Nuclear Factor I-C and Transforming Growth Factor-β1 Signaling Regulates Odontoblast Differentiation and Homeostasis

doi: 10.1371/journal.pone.0029160

Figure Lengend Snippet: ( A ) The protein levels of NFI-C, p-Smad2/3, and p21 protein in TGF-β1-treated MDPC-23 cells were analyzed by western blot (left panel), and the results were quantified using ImageJ (right panel). GAPDH used as a loading control. ( B ) MDPC-23 cells were incubated with TGF-β1 (10 ng/ml) in the presence or absence of the proteasome inhibitor, MG132 (10 µM) for 1 hr. Cytoplasmic and nuclear fractions were isolated and subjected to western blot analysis. GAPDH and lamin B served as cell fractionation controls. ( C ) The subcellular localization of NFI-C was analyzed by immunostaining with NFI-C specific antibody. DAPI staining was used to detect nuclei.

Article Snippet: All other antibodies against TGFβ-RI (sc-398), TGFβ-RII (sc-400), p-Smad2/3 (sc-11769), p21 (sc-6246), and Osterix (sc-22538) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Control, Incubation, Isolation, Cell Fractionation, Immunostaining, Staining

Journal: PLoS ONE

Article Title: Crosstalk between Nuclear Factor I-C and Transforming Growth Factor-β1 Signaling Regulates Odontoblast Differentiation and Homeostasis

doi: 10.1371/journal.pone.0029160

Figure Lengend Snippet: ( A ) MDPC-23 cells were transfected with empty vector (pCMV empty vector, control), Smad2, or Smad3 expression vectors in the presence or absence of MG132. NFI-C protein levels were analyzed by western blot 48 hr post-transfection. GAPDH was used as a loading control. ( B ) HEK293T cells were co-transfected with HA-tagged NFI-C and FLAG-tagged Smad3 (F-Smad3) expression vectors for 48 hr and then lysed. The whole cell lysates (WCL) and NFI-C immunoprecipitates were analyzed by western blot with anti-FLAG antibody. ( C ) Co-localization of NFI-C (red) and FLAG-Smad3 (green). MDPC-23 cells were co-transfected with NFI-C and FLAG-tagged Smad3 expression vector for 48 hr. After 48 hr, transfected cells were stimulated with TGF-β1 for 1 hr in the presence of MG132 (10 µM). Double immunostaining was performed with anti-NFI-C and anti-FLAG antibodies, followed by the secondary antibodies conjugated to goat anti-rabbit rhodamine (red, NFI-C) and goat anti-mouse FITC (green, FLAG-Smad3), respectively. The merged panel shows co-localization (yellow) of NFI-C and FLAG-Smad3 in the cytoplasm. DAPI staining was used to detect nuclei. ( D ) MDPC-23 cells were stimulated with TGF-β1 (10 ng/ml) in the presence of the proteasome inhibitor, MG132 (10 µM) for 1 hr. Cytoplasmic and nuclear fractions were isolated and NFI-C immunoprecipitates were analyzed by western blot with the anti-NFI-C or anti-pSmad3 antibody. GAPDH and lamin B served as cell fractionation controls. (E) MDPC-23 cells were transfected with shRNA Smad3 expression vector or control empty vector. Knockdown of Smad3 was evaluated by RT-PCR (left panel). Cell lysates were analyzed by western blot with NFI-C antibody, and the results were quantified using ImageJ (right panel; *P<0.01).

Article Snippet: All other antibodies against TGFβ-RI (sc-398), TGFβ-RII (sc-400), p-Smad2/3 (sc-11769), p21 (sc-6246), and Osterix (sc-22538) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Double Immunostaining, Staining, Isolation, Cell Fractionation, shRNA, Knockdown, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Crosstalk between Nuclear Factor I-C and Transforming Growth Factor-β1 Signaling Regulates Odontoblast Differentiation and Homeostasis

doi: 10.1371/journal.pone.0029160

Figure Lengend Snippet: ( A ) MDPC-23 cells were co-transfected with NFI-C, Smurf1, Smurf2, Nedd4, and Praja1 expression vectors for 48 hr. After 48 hr, transfected cells were incubated with or without TGF-β1 (10 ng/ml) for 1 hr. NFI-C protein levels were analyzed by western blot. GAPDH was used as a loading control. ( B ) MDPC-23 cells were co-transfected with NFI-C and Smurf1 expression vector for 48 hr. After 48 hr, transfected cells were incubated with or without TGF-β1 (10 ng/ml) in the presence or absence of MG132 for 1 hr. NFI-C protein levels were analyzed by western blot. ( C ) MDPC-23 cells were transfected with shRNA Smurf1 expression vector or control empty vector. Forty-eight hours post-transfection cells were stimulated with TGF-β1 for 1 hr. Cell lysates were analyzed by western blot with NFI-C antibody (left panel), and the results were quantified using ImageJ. Knockdown of Smurf1 was evaluated by real time PCR (right panel). ( D ) HEK293T cells were co-transfected with HA-tagged NFI-C, FLAG-tagged ubiquitin (Ub), and Smurf1, and then treated with MG132 (5 µM) for 48 hr. Forty-eight hours post-transfection, cells were stimulated with TGF-β1 for 1 hr. The NFI-C immunoprecipitates or whole cell lysates (WCL) were analyzed by western blot with anti-FLAG or anti-HA antibody.

Article Snippet: All other antibodies against TGFβ-RI (sc-398), TGFβ-RII (sc-400), p-Smad2/3 (sc-11769), p21 (sc-6246), and Osterix (sc-22538) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Expressing, Incubation, Western Blot, Control, Plasmid Preparation, shRNA, Knockdown, Real-time Polymerase Chain Reaction, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Crosstalk between Nuclear Factor I-C and Transforming Growth Factor-β1 Signaling Regulates Odontoblast Differentiation and Homeostasis

doi: 10.1371/journal.pone.0029160

Figure Lengend Snippet: ( A ) MDPC-23 cells were treated with TGF-β1 (10 ng/ml) for 1 hr and then lysed. The NFI-C immunoprecipitates or whole cell lysates (WCL) were subjected to western blot analysis with the anti-Smurf1 or anti-NFI-C antibody. GAPDH was used as a loading control. ( B ) HEK293T cells were co-transfected with HA-tagged NFI-C and FLAG-tagged Smurf1 expression vectors for 48 hr. WCL and NFI-C immunoprecipitates were analyzed by western blot with anti-FLAG or anti-HA antibody. ( C ) MDPC-23 cells were incubated with TGF-β1 (1 hr, 10 ng/ml) in the presence or absence of the MEK inhibitor, U0126 (10 µM), added 1 hr prior to TGF-β1 addition. Cells were lysed, and NFI-C, p-ERK, and GAPDH levels were analyzed by western blot. ( D ) MDPC-23 cells were stimulated with TGF-β1 (10 ng/ml) for 1 hr in the presence or absence of the MEK inhibitor, U0126 (10 µM). WCL and NFI-C immunoprecipitates were analyzed by western blot. ( E ) WCL and anti-phospho-Ser/Thr-Pro immunoprecipitates were analyzed by western blot with anti-NFI-C antibody. ( F ) HA-tagged NFI-C protein was metabolically labeled with [γ- 32 P]-ATP in HEK293T cells. Bound Smurf1 proteins were eluted from the beads and detected by western blot analysis with the indicated antibody. The incorporation of 32 P was detected by autoradiography, and the amount of HA-NFI-C was detected by western blot analysis.

Article Snippet: All other antibodies against TGFβ-RI (sc-398), TGFβ-RII (sc-400), p-Smad2/3 (sc-11769), p21 (sc-6246), and Osterix (sc-22538) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Control, Transfection, Expressing, Incubation, Metabolic Labelling, Labeling, Autoradiography

Journal: PLoS ONE

Article Title: Crosstalk between Nuclear Factor I-C and Transforming Growth Factor-β1 Signaling Regulates Odontoblast Differentiation and Homeostasis

doi: 10.1371/journal.pone.0029160

Figure Lengend Snippet: During early odontoblast differentiation, p-Smad2/3 induced by TGF-β1 signaling increased binding to NFI-C in the cytoplasm. On the other hand, MAPK activation by TGF-β1 signaling increased the interaction of phosphorylated NFI-C and Smurf1/2. Collectively, TGF-β1 and MAPK activation enhanced the interaction and formation of Smad2/3-NFI-C-Smurf1/2 complex and resulted in the degradation of NFI-C. In contrast, during late odontoblast differentiation and mineralization, NFI-C signaling resulted in the dephosphorylation of p-Smad2/3. Consequently, TGF-β1 induces odontoblast differentiation through the Smad signaling pathway in early odontoblast differentiation, while NFI-C signaling modulates late odontoblast differentiation and mineralization.

Article Snippet: All other antibodies against TGFβ-RI (sc-398), TGFβ-RII (sc-400), p-Smad2/3 (sc-11769), p21 (sc-6246), and Osterix (sc-22538) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Activation Assay, De-Phosphorylation Assay

Journal: Genes & Development

Article Title: MYC/MAX control ERK signaling and pluripotency by regulation of dual-specificity phosphatases 2 and 7

doi: 10.1101/gad.211300.112

Figure Lengend Snippet: MYC transcriptionally regulates DUSP2 and DUSP7. (A) Whole-cell lysates from R1 mESCs grown in differentiating conditions (−LIF) were collected over 5 d in 24 h increments and then immunoblotted and probed with antibodies as indicated. (B) Transcript levels were assayed in R1 ESCs and 4 d following withdrawal of LIF. Fold changes in transcript levels were determined in triplicate after normalization to untreated cells. Error bars represent standard deviation. (C) DUSP2 and DUSP7 transcript levels were assayed in MYCfl/fl MYCNfl/fl- or MYC−/− MYCN−/−-deleted cells. CreGR expression was induced by addition of Dex (+Dex) for 48 h. (D) Wild-type R1 mESCs carrying a MYC-ER transgene were induced by addition of 4OHT for 24 h. Transcript levels were assayed as in C. (E) DUSP2 and DUSP7 transcript levels were assayed from MYCfl/fl MYCNfl/fl CreGR MYC-ER cells without or after Dex treatment for 2 d, 3 d, and 3 d + 24 h 4OHT. Fold changes were normalized to untreated cells. (F,G) MYC/MAX specifically bind to the promoter region of DUSP2 (F) and DUSP7 (G). ChIP-immunoprecipitated DNA was amplified by quantitative PCR (qPCR) with primers spanning the DUSP2,7 promoters. Schematic representations of DUSP2,7 are shown with potential MYC-binding sites (E-boxes) indicated by triangles. All E-boxes are canonical with the sequence 5′-CACGTG-3′. (*) P < 0.05. All other P-values are <0.01. “Control” regions are located 2 kb upstream of the transcription start sites of DUSP2 and DUSP7 and do not contain an E-box.

Article Snippet: For immunoblotting, immunostaining, and ChIP experiments, we used commercially available antibodies to MYC (Santa Cruz Biotechnology, sc-764), phospho-ERK1/2 (Cell Signaling, 9102), ERK1/2 (Cell Signaling, 9102), MYCN (Calbiochem, OP13), MAX (Santa Cruz Biotechnology, sc-197), OCT4 (Santa Cruz Biotechnology, sc-5279), DUSP7 (Santa Cruz Biotechnology, sc-47667), ERK1/2 (Santa Cruz Biotechnology, sc-135900), NANOG (CosmoBio, REC-RCAB0002PF), rabbit IGG (Abcam, ab46540), and FOXA2 (Millipore, 07-633).

Techniques: Standard Deviation, Expressing, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Binding Assay, Sequencing, Control

Journal: Genes & Development

Article Title: MYC/MAX control ERK signaling and pluripotency by regulation of dual-specificity phosphatases 2 and 7

doi: 10.1101/gad.211300.112

Figure Lengend Snippet: Down-regulation of MYC, MYCN, DUSP2, and DUSP7 correspond to an increase in ERK activity and differentiation. (A) Tet regulation of HA-DUSP7 ESC line grown in the absence (−) or presence (+) of Tet activator protein. After 24 h induction (+Tet), cells were harvested and subjected to immunoblot analysis, probing with HA monoclonal antibody to detect DUSP7. Load control, HA cross-reacting, nonspecific protein was used as a load control. (B) MYCfl/fl MYCNfl/fl cells containing Tet-inducible DUSP7 were transfected with a CreGFP vector ± Tet and then sorted for GFP 48 h post-transfection. Whole-cell lysates were then immunoblotted and probed with antibodies as indicated. (C) Transcript levels from B were assayed in triplicate. Fold changes were normalized to untreated MYCfl/fl MYCNfl/fl cells. Error bars represent standard deviation. (D) Tet-inducible DUSP7 ESCs were plated on 24-well plates. After LIF withdrawal, Tet-express was added to induce DUSP7 expression. Cells were allowed to grow in differentiating condition for 4 d ±Tet express and then stained for AP activity. (E) The percentage of AP-positive colonies under each condition is shown. All assays were performed in triplicate (N = 164).

Article Snippet: For immunoblotting, immunostaining, and ChIP experiments, we used commercially available antibodies to MYC (Santa Cruz Biotechnology, sc-764), phospho-ERK1/2 (Cell Signaling, 9102), ERK1/2 (Cell Signaling, 9102), MYCN (Calbiochem, OP13), MAX (Santa Cruz Biotechnology, sc-197), OCT4 (Santa Cruz Biotechnology, sc-5279), DUSP7 (Santa Cruz Biotechnology, sc-47667), ERK1/2 (Santa Cruz Biotechnology, sc-135900), NANOG (CosmoBio, REC-RCAB0002PF), rabbit IGG (Abcam, ab46540), and FOXA2 (Millipore, 07-633).

Techniques: Activity Assay, Western Blot, Control, Transfection, Plasmid Preparation, Standard Deviation, Expressing, Staining

Journal: Genes & Development

Article Title: MYC/MAX control ERK signaling and pluripotency by regulation of dual-specificity phosphatases 2 and 7

doi: 10.1101/gad.211300.112

Figure Lengend Snippet: DUSP2/7 expression is necessary for maintenance of mPSCs. (A) R1 mESCs were infected with lentiviruses expressing shRNAs for DUSP2 (TRCN0000028960), DUSP7 (TRCN0000080728), DUSP2 and DUSP7, or GFP (control). Fours days after lentiviral transduction, target knockdown was assayed in triplicate by qPCR. Fold changes in transcripts were determined after normalization to GFP knockdown cells. Error bars represent standard deviation. (B) Colony morphology after DUSP2/7 and GFP knockdown (4 d). Bar, 100 μm. (C) Whole-cell lysates were collected after DUSP2/7 and GFP shRNA knockdown. Samples were immunoblotted and probed with antibodies as indicated. (D) Immunofluorescent staining using FOXA2 antibody after DUSP2/7 and GFP knockdown. Bar, 50 μm. (E) R1 mESCs marked by β-galactosidase activity were infected with lentiviruses expressing shRNAs for DUSP2/7 or GFP control and injected into blastocyst stage C57BL/6 embryos. After transfer into recipient females, embryos were allowed to develop until embryonic day 14.5 (E14.5). LacZ staining was then performed on fixed, whole embryos. The number of blastocysts injected, the number of chimeras generated, and the percentage of chimeras generated are indicated (control, N = 21; DUSP2/7, N = 18). The percentage of embryo contribution for control and DUSP2/7 knockdown cells is graphed on the right. (*) P < 0.05.

Article Snippet: For immunoblotting, immunostaining, and ChIP experiments, we used commercially available antibodies to MYC (Santa Cruz Biotechnology, sc-764), phospho-ERK1/2 (Cell Signaling, 9102), ERK1/2 (Cell Signaling, 9102), MYCN (Calbiochem, OP13), MAX (Santa Cruz Biotechnology, sc-197), OCT4 (Santa Cruz Biotechnology, sc-5279), DUSP7 (Santa Cruz Biotechnology, sc-47667), ERK1/2 (Santa Cruz Biotechnology, sc-135900), NANOG (CosmoBio, REC-RCAB0002PF), rabbit IGG (Abcam, ab46540), and FOXA2 (Millipore, 07-633).

Techniques: Expressing, Infection, Control, Transduction, Knockdown, Standard Deviation, shRNA, Staining, Activity Assay, Injection, Generated

Journal: Genes & Development

Article Title: MYC/MAX control ERK signaling and pluripotency by regulation of dual-specificity phosphatases 2 and 7

doi: 10.1101/gad.211300.112

Figure Lengend Snippet: DUSP7 directly interacts with ERK in PSCs. (A) HA-DUSP7-expressing ESC line was established by transfecting a Tet-inducible HA-DUSP7 expression plasmid into R1 ESCs. After 1 d of Tet-express induction, cell lysate was made for coimmunoprecipitation (IP) experiments. Reciprocal immunoprecipitation of DUSP7 and ERK was detected using ERK1/2 and HA antibodies. (B) R1 mESCs containing Tet-inducible ERK2(D319N) were cultured ±Tet for 2 d. Whole-cell lysates were then immunoblotted and probed with antibodies as indicated. (C) Tet-inducible ERK2(D319N) cells were cultured ±Tet for 2 d, followed by −Tet for 3 d, and then stained for AP. (D) The percentage of AP-positive colonies under each condition was determined from triplicate experiments. Error bars represent standard deviation (N = 74). (*) P < 0.05. All other P-values are <0.01.

Article Snippet: For immunoblotting, immunostaining, and ChIP experiments, we used commercially available antibodies to MYC (Santa Cruz Biotechnology, sc-764), phospho-ERK1/2 (Cell Signaling, 9102), ERK1/2 (Cell Signaling, 9102), MYCN (Calbiochem, OP13), MAX (Santa Cruz Biotechnology, sc-197), OCT4 (Santa Cruz Biotechnology, sc-5279), DUSP7 (Santa Cruz Biotechnology, sc-47667), ERK1/2 (Santa Cruz Biotechnology, sc-135900), NANOG (CosmoBio, REC-RCAB0002PF), rabbit IGG (Abcam, ab46540), and FOXA2 (Millipore, 07-633).

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Cell Culture, Staining, Standard Deviation

Journal: Genes & Development

Article Title: MYC/MAX control ERK signaling and pluripotency by regulation of dual-specificity phosphatases 2 and 7

doi: 10.1101/gad.211300.112

Figure Lengend Snippet: A model for ERK regulation by MYC/MAX complexes in murine pluripotent cells. MYC/MAX activates transcription of DUSP2 and DUSP7 in their E-box-containing promoters. The protein products are characterized by phosphatase activity necessary for dephosphorylation of phospho-ERK. Suppressing ERK activity is necessary for maintenance of a pluripotent state and is necessary in the presence of autocrine FGF signaling.

Article Snippet: For immunoblotting, immunostaining, and ChIP experiments, we used commercially available antibodies to MYC (Santa Cruz Biotechnology, sc-764), phospho-ERK1/2 (Cell Signaling, 9102), ERK1/2 (Cell Signaling, 9102), MYCN (Calbiochem, OP13), MAX (Santa Cruz Biotechnology, sc-197), OCT4 (Santa Cruz Biotechnology, sc-5279), DUSP7 (Santa Cruz Biotechnology, sc-47667), ERK1/2 (Santa Cruz Biotechnology, sc-135900), NANOG (CosmoBio, REC-RCAB0002PF), rabbit IGG (Abcam, ab46540), and FOXA2 (Millipore, 07-633).

Techniques: Activity Assay, De-Phosphorylation Assay

Journal: Cell Genomics

Article Title: A highland-adaptation variant near MCUR1 reduces its transcription and attenuates erythrogenesis in Tibetans

doi: 10.1016/j.xgen.2025.100782

Figure Lengend Snippet:

Article Snippet: Proteins were transferred to BioTraceTM NT Nitrocellulose Transfer Membrane (#P/N66485; PALL, USA) and incubated with antibodies against Human MCUR1 (SAB2100356; Sigma, USA), Mouse MCUR1 (SAB2700722; Sigma, USA), mTOR (#2983; Cell Signaling Technology, USA), phosphorylated mTOR (p-mTOR, Ser2448; #5536; Cell Signaling Technology, USA), p70 S6 kinase (S6K; #9202; Cell Signaling Technology, USA), phosphorylated p70 S6 kinase (p-S6K, Thr389; #9205; Cell Signaling Technology, USA), 4EBP1 (#9644; Cell Signaling Technology, USA), phosphorylated 4EBP1 (p-4EBP1, Thr37/46; #2855; Cell Signaling Technology, USA), ULK1 (#20986-1-AP; Proteintech, China), phosphorylated ULK (p-ULK1, Ser757; #14202; Cell Signaling Technology, USA), LC3 (#12741; Cell Signaling Technology, USA), p62 (#55274-1-AP; Proteintech, China), PGC1α (#66369-1-lg; Proteintech, China), NRF1 (#12482-1-AP; Proteintech, China), YY1 (#66281-1-lg; Proteintech, China), mtTFA (#19998-1-AP; Proteintech, China), AMPKα (#5831; Cell Signaling Technology, USA), phosphorylated AMPKα (p-AMPKα, Thr172; #2535; Cell Signaling Technology, USA), TSC1 (#6935; Cell Signaling Technology, USA), TSC2 (#4308; Cell Signaling Technology, USA), phosphorylated TSC2 (p-TSC2, Ser1387; #23402; Cell Signaling Technology, USA); Raptor (#20984-1-AP; Proteintech, China), phosphorylated Raptor (p-Raptor, Ser792; #89146; Cell Signaling Technology, USA), LKB1 (#3047; Cell Signaling Technology, USA), phosphorylated LKB1 (p-LKB1, Ser428; #3482; Cell Signaling Technology, USA), CAMKK2 (#16810; Cell Signaling Technology, USA), phosphorylated CAMKK2 (p-CAMKK2, Ser495; #16737; Cell Signaling Technology, USA), PU.1 (#2258; Cell Signaling Technology, USA) and β-actin (#60008-1-Ig; Proteintech, China).

Techniques: Recombinant, Modification, Protease Inhibitor, Luminescence Assay, DNA Extraction, Multiplex Assay, Reporter Assay, Reverse Transcription, SYBR Green Assay, Electrophoresis, Mobility Shift, Transfection, Chromatin Immunoprecipitation, Mutagenesis, Enzyme-linked Immunosorbent Assay, Purification, Sequencing, RNA Sequencing, Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Software