Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: Ras (G12V) causes the expression of ST2 gene products in NIH-3T3 murine fibroblasts. (A) NIH-3T3 cells were infected with the empty retrovirus (CTR) or retrovirus harboring Ras (G12V), and infected cells were selected by the treatment with puromycin (7.5 μg/ml) for 72 h. Then, total RNA was extracted from infected cells, and the expression levels of ST2 gene products were analyzed by quantitative PCR. In the graph, error bars = S.D. (n = 3, *P < 0.005). (B) Conditioned medium and whole cell lysates were concentrated, and treated with N-glycosidase F. Then, ST2 and ST2L proteins induced by Ras (G12V) were detected by immunoblot analysis with anti-mouse ST2 antibody, and β-actin was also stained as a loading control for the immunoblot analysis. The intensities of bands were quantified, and shown in the graphs. In the graph, error bars = S.D. (n = 3, *P<0.005). Original blots are shown in supplementary file named as “Original blots for Ras & ST2”.

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

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

Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: Enforced expression of ST2 gene products in NIH-3T3 cells transformed by oncogenic Ras. (A) We diluted retroviruses to forcibly express the various amount Ras (G12V) by adjusting the amounts of retroviruses harboring gene of Ras (G12V), such as 50- (lane 2, 6, and 10), 15- (lane 3, 7, and 11), and 5-fold (lane 4, 8, and 12) dilution, and infected cells were selected by the treatment with puromycin for 72 h. To co-express ST2-FLAG and ST2L-FLAG, we then infected the retroviruses harboring ST2-FLAG and ST2L-FLAG, respectively. After selection with puromycin, cells were treated with serum starvation for 24 h. Then, whole cell lysates (WCL) and conditioned media (CM) were prepared, and analyzed by immunoblotting with the primary antibodies such as anti-FLAG (M2), H-Ras, cyclin D1, Rb, phosphorylated Rb (Ser-807/811), and β-actin antibodies, respectively. To detect secreted ST2, the culture supernatant (conditioned media: CM) was also analyzed. The positions of bands for ST2L and ST2 are indicated. Original blots are shown in supplementary file named as “Original blots for Ras & ST2”. (B) The intensity of the expression of total Rb protein, the ratio between Rb phosphorylation and total Rb, and the expression of cyclin D1 protein were quantitated, calculated, and then results were shown in graphs. In the graph, error bars = S.D. (n = 3, *P<0.005).

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

Techniques: Expressing, Transformation Assay, Infection, Selection, Western Blot, Phospho-proteomics

Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: Effect of enforced expression of ST2 gene products on the expression of Ras protein and cyclin D1 mRNA. (A) In the cells analyzed in , the intensity of the expression of H-Ras protein, which were normalized with the expression of β-actin, and then results were shown in graph. In the graph, error bars = S.D. (n = 3). (B) The expressional ratio between cyclin D1 and H-Ras was calculated, and then results were shown in graph. In the graph, error bars = S.D. (n = 3). (C) Infected cells were utilized for the total RNA extraction, and analyzed by qPCR for evaluating the effect of Ras (G12V) and ST2 gene products on the expression of cyclin D1 mRNA. The results were shown in graph. In the graph, error bars = S.D. (n = 3, *P < 0.005). (D) In the cells analyzed in , infected cells were seeded onto soft agar at 1 × 10 4 cells per 35 mm-diameter dishes, and grown for 2–3 weeks. The colonies were visualized by the treatment with MTT, and the results were shown in graph (magnification: 4 ×). In the graph, error bars = S.D. (n = 3, *P<0.005).

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

Techniques: Expressing, Infection, RNA Extraction

Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: Enforced expression of ST2 gene products accelerates cellular transformation induced by Ras (G12V). (A) NIH-3T3 cells were infected with the indicated combination of retroviruses harboring Ras (G12V), ST2-FLAG, and ST2L-FLAG, and infected cells were selected by the treatment with puromycin for 72 h. Then, a total of 3 × 10 3 infected cells were mixed with 1 × 10 5 uninfected cells and seeded onto 60 mm-diameter dishes. Two weeks later, transformed foci were stained with Giemsa, and then photographed at 4 × magnification. (B) The numbers of foci shown in (A) were counted, and the results were shown in the graph. In the graph, error bars = S.D. (n = 3, *P < 0.01). (C) Infected NIH-3T3 cells were seeded onto soft agar at 1 × 10 4 cells per 35-mm dish, and grown for 2–3 weeks. Grown colonies were photographed at 4 × magnification, and the numbers of colonies were counted. (D) The number of colonies of transformed NIH-3T3 cells counted in (C) is shown in the graph. In the graph, error bars = S.D. (n = 3, *P < 0.005). (E) To evaluate the resistance to anoikis, cells were suspended in DMEM including 1% FBS, and then seeded onto the 24-well plates treated with poly-HEMA. Twenty-hour hours later, cell viability was measured using a WST-1 assay. In the graph, error bars = S.D. (n = 3, *P<0.01).

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

Techniques: Expressing, Transformation Assay, Infection, Staining, WST-1 Assay

Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: Soluble ST2 accelerated the cellular transformation in paracrine- and autocrine-dependent manner. (A) Then infected NIH-3T3 cells analyzed in were seeded on slide glasses, and fixed with paraformaldehyde. Then, the expression of FLAG-tagged ST2 and ST2L were detected by immuno-staining analysis using anti-FLAG (green). Nucleus was also stained with DAPI (blue). In bottom photographs, scale bars were added (length: 153 μm) (B) Conditioned medium was also collected from the infected NIH-3T3 cells analyzed in (A). Then, concentration of ST2 was analyzed by ELISA. In the graph, error bars = S.D. (n = 3, *P < 0.001). (C) Using conditioned medium from control NIH-3T3 and Ras (G12V)-transformed NIH-3T3 cells, ELISA for IL-33 was also performed. Recombinant IL-33 (rIL-33) was utilized as a positive control. In the graph, error bars = S.D. (n = 3, *P < 0.005). N.D. means “Not detected”. The expressions of Ras (G12V) and intracellular IL-33 were detected by immunoblot analysis. The blot for β-tubulin is a loading control. Original blots are shown in supplementary file named as “Original blots for Ras & ST2”. (D) To test the paracrine- and autocrine-dependent effect of ST2 on cellular transformation, 3 × 10 3 untransformed NIH-3T3 cells or transformed cells provoked by Ras (G12V) were mixed with 1 × 10 5 normal NIH-3T3 (top photograph), NIH-3T3 producing soluble ST2 (middle photograph), or NIH-3T3 cells expressing ST2L (bottom photograph), and then cultured in 60-mm diameter dishes during 2 weeks. Foci were visualized by staining with Giemsa reagent, and then photographed at 4 × magnification. (E) The numbers of foci shown in (D) were counted, and the results were shown in the graph. In the graph, error bars = S.D. (n = 3, *P<0.005).

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

Techniques: Transformation Assay, Infection, Expressing, Immunostaining, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Recombinant, Positive Control, Western Blot, Cell Culture

Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: Knockdown of the expression of ST2 gene products in normal and transformed NIH-3T3 cells. (A) NIH-3T3 cells were infected with the indicated retroviruses including Ras (G12V), sh-luciferase (sh-Luc), and sh-ST2/ST2L (Referred to as sh-ST2), and infected cells were selected by the treatment with puromycin for 72 h. And then, total RNA was extracted from infected cells, and the expression levels of ST2 gene products were analyzed by quantitative PCR. In the graph, error bars = S.D. (n = 3, *P < 0.005). (B) The reduction of the protein expression of ST2 and ST2L by sh-RNA was detected by immunoblot analysis. Original blots are shown in supplementary file named as “Original blots for Ras & ST2”. (C) The results of (B) were shown in the graphs, respectively. In the graph, error bars = S.D. (n = 3, *P<0.005).

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

Techniques: Knockdown, Expressing, Transformation Assay, Infection, Luciferase, Real-time Polymerase Chain Reaction, Western Blot

Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: Silencing the expression of ST2 gene products suppresses the cellular transformation induced by Ras (G12V). (A) Infected NIH-3T3 cells were seeded onto soft agar at 1 × 10 4 cells per 35-mm dish, and grown for 2–3 weeks. Grown colonies were photographed at 4 × magnification, and the numbers of colonies were counted. In the labels of photographs, RasV means Ras (G12V) mutant. (B) The number of colonies of transformed NIH-3T3 cells counted in (A) is shown in the graph. In the graph, error bars = S.D. (n = 3, *P < 0.005). N.D. means “Not detected”. (C) Whole cell lysates were prepared from the infected cells and analyzed by immunoblotting with the primary antibodies indicated on the left of the image. Original blots are shown in supplementary file named as “Original blots for Ras & ST2”. (D) The intensity of Rb phosphorylation, total Rb, cyclin D1, and the ratio between phosphorylated Rb and total Rb were quantitated, calculated, and then results were shown in graphs, respectively. In the graph, error bars = S.D. (n = 3, *P<0.005).

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

Techniques: Expressing, Transformation Assay, Infection, Mutagenesis, Western Blot, Phospho-proteomics

Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: ST2 gene products may modulate the regulation of the cell cycle . (A) NIH-3T3 cells were infected with the indicated retroviruses. Infected cells were held in G 0 phase by serum starvation for 24 h. Then, the cells were released into the cell cycle by dividing the cells onto new dishes. At the indicated time points, cells were harvested and stained with propidium iodide. Cell suspension was analyzed by flow cytometry. (B) The percentage of cells entering into S-phase was shown in graph. Significant difference of the cell population entering to S-phase at 20 hr after cell cycle release between ST2-knockdowned cells and control cells was statistically determined by the Student's t -test. In the graph, error bars = S.D. (n = 3, *P < 0.005). (C) At indicated time points, cells were harvested and analyzed by immunoblot with primary antibodies, as indicated at the left of the image. Original blots are shown in supplementary file named as “Original blots for Ras & ST2”.

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

Techniques: Infection, Staining, Suspension, Flow Cytometry, Control, Western Blot

Journal: Heliyon

Article Title: ST2 gene products critically contribute to cellular transformation caused by an oncogenic Ras mutant

doi: 10.1016/j.heliyon.2017.e00436

Figure Lengend Snippet: Working hypothesis for the mechanism how ST2 gene products contribute to the tumor growth and progression.

Article Snippet: The concentrations of murine ST2 and IL-33 were measured by using Quantikine ELISA for mouse ST2 (MST200) and mouse/rat IL-33 (M3300) (R&D system, Minneapolis, MN, USA), respectively.

Techniques: