Journal: International Journal of Molecular Sciences

Article Title: Cytochalasins Suppress 3D Migration of ECM-Embedded Tumoroids at Non-Toxic Concentrations

doi: 10.3390/ijms26147021

Figure Lengend Snippet: The effects of Jasplakinolide, Latruncilin A, Blebbistatin, and GSK-269962 on 2D random migration. ( Left panels ): The proliferation curves of Hs578T cells seeded in 2D, showing confluence after 24 h of exposure to concentration ranges of the indicated inhibitors, normalized to time point 0. The boxes indicate the highest concentrations tolerated without growth suppression. The mean ± SEM is shown for N = 3 biological replicates, each containing 10 technical replicates. ( Right panels ): The random cell migration speed of subconfluent Hs578T cells seeded in 2D and exposed to concentration ranges of inhibitors. The boxes indicate the same maximum tolerated concentrations boxed as in the ( left panels ). Bosutinib was used as a positive control. The mean ± SEM is shown for 2 biological replicates, each containing 4 technical replicates.

Article Snippet: To do so, a subconfluent monolayer of Lenti-X 293T cells (Clontech, 632180, Mountain View, CA, USA) was transfected with a mixture of this plasmid and lentiviral helper vectors pMDLg-RRE, pCMV-VSVG, and pRSV-Rev (Addgene, #12251, #8454, and #12253, Watertown, MA, USA) using polyethylenimine (Polysciences, 23966-2, Warrington, PA, USA).

Techniques: Migration, Concentration Assay, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Cytochalasins Suppress 3D Migration of ECM-Embedded Tumoroids at Non-Toxic Concentrations

doi: 10.3390/ijms26147021

Figure Lengend Snippet: CyB suppresses 2D migration speed and cell stiffness at non-toxic concentrations, without disrupting F-actin. ( A ): The proliferation curves of Hs578T and MV3 cells seeded in 2D, showing confluence after 24 h of exposure to concentration ranges of the indicated inhibitors, normalized to time point 0. The boxes indicate the highest concentrations tolerated without growth suppression. The mean ± SEM is shown for N = 3 biological replicates, each with 10 technical replicates. n.s., non-significant. ( B ): The random cell migration speed of subconfluent Hs578T and MV3 cells seeded in 2D and exposed to concentration ranges of cyB. The boxes indicate the same maximum tolerated concentrations boxed in the left panel. The mean ± SEM is shown for N = 3 biological replicates, each with 8 technical replicates. n.s., non-significant; * p < 0.05. ( C ): F-actin fibers (Phalloidin, green or red) and nuclei (Hoechst, blue) of Hs578T and MV3 cells seeded in 2D in the absence or presence of the indicted cyB concentrations. The black arrows indicate actin aggregates and structures resembling membrane ruffles appearing with increasing inhibitor concentrations and replacing the F-actin stress fibers observed under control conditions (white arrows). Scalebar = 20 µm. ( D ): The stress fiber length (left; indicated in ( C ) with white arrows) and area of membrane ruffles (right; indicated in ( C ) with black arrows). The mean ± SD for one experiment with two replicates is shown. n.s., non-significant; * p < 0.05; ** p < 0.01; *** p < 0.0001. ( E ): A cartoon illustrating the principle of AFS. Acoustic force pushes the bead upwards, thereby stretching the cells, with the amount of stretch inversely related to stiffness. ( F ): AFS measurements for cells exposed for 24 h to a control medium or cyB (741 nM for Hs578T and 247 nM for MV3), normalized to the measurement before exposure. The mean ± SD for one experiment of two, each containing 2 technical replicates, is shown.

Article Snippet: To do so, a subconfluent monolayer of Lenti-X 293T cells (Clontech, 632180, Mountain View, CA, USA) was transfected with a mixture of this plasmid and lentiviral helper vectors pMDLg-RRE, pCMV-VSVG, and pRSV-Rev (Addgene, #12251, #8454, and #12253, Watertown, MA, USA) using polyethylenimine (Polysciences, 23966-2, Warrington, PA, USA).

Techniques: Migration, Concentration Assay, Membrane, Control

Journal: International Journal of Molecular Sciences

Article Title: Cytochalasins Suppress 3D Migration of ECM-Embedded Tumoroids at Non-Toxic Concentrations

doi: 10.3390/ijms26147021

Figure Lengend Snippet: CyD suppresses 2D migration speed and cell stiffness at non-toxic concentrations, without disrupting F-actin. ( A ): Proliferation curves of Hs578T and MV3 cells seeded in 2D showing confluence after 24 h of exposure to concentration ranges of the indicated inhibitors, normalized to time point 0. The boxes indicate the highest concentrations tolerated without growth suppression. The mean ± SEM is shown for N = 3 biological replicates, each with 10 technical replicates. ( B ): The random cell migration speed of subconfluent Hs578T and MV3 cells seeded in 2D and exposed to concentration ranges of cyD. The boxes indicate the same maximum tolerated concentrations boxed in the left panel. The mean ± SEM is shown for N = 3 biological replicates, each with 8 technical replicates. n.s., non-significant; * p < 0.05; ** p < 0.01. ( C ): F-actin fibers (Phalloidin, green or red) and nuclei (Hoechst, blue) of Hs578T and MV3 cells seeded in 2D in the absence or presence of the indicted cyD concentrations. The black arrows indicate actin aggregates and structures resembling membrane ruffles appearing with increasing inhibitor concentrations and replacing the F-actin stress fibers observed under control conditions (white arrows). Scalebar = 20 µm. ( D ): the Stress fiber length (left; indicated in ( C ) with white arrows) and area of membrane ruffles (right; indicated in ( C ) with black arrows). The mean ± SD for one experiment with two replicates is shown. n.s, non-significant; * p < 0.05; ** p < 0.01. ( E ): AFS measurements for cells exposed for 24 h to a control medium or cyD (41.2 nM for Hs578T and 123 nM for MV3), normalized to the measurement before exposure. The mean ± SD for one experiment of two, each containing 2 technical replicates, is shown.

Article Snippet: To do so, a subconfluent monolayer of Lenti-X 293T cells (Clontech, 632180, Mountain View, CA, USA) was transfected with a mixture of this plasmid and lentiviral helper vectors pMDLg-RRE, pCMV-VSVG, and pRSV-Rev (Addgene, #12251, #8454, and #12253, Watertown, MA, USA) using polyethylenimine (Polysciences, 23966-2, Warrington, PA, USA).

Techniques: Migration, Concentration Assay, Membrane, Control

Journal: Molecular Biology of the Cell

Article Title: EphrinA/EphA signal facilitates insulin-like growth factor-I–induced myogenic differentiation through suppression of the Ras/extracellular signal–regulated kinase 1/2 cascade in myoblast cell lines

doi: 10.1091/mbc.E11-03-0183

Figure Lengend Snippet: The ephrinA/EphA signal promotes IGF-I–induced myogenic differentiation. (A) Immunocytochemical analysis of C2C12 myoblasts using anti-MHC antibody. Confluent cells were differentiated into myotubes in DMEM containing 1% FBS and 10 nM IGF-I in the presence of either 6.4 nM Fc (Fc) or ephrinA1-Fc (A1-Fc) for 3 d. The cells were then fixed, immunostained with anti-MHC antibody, and visualized with Alexa Fluor 488–conjugated secondary antibody. The cells were also poststained with Hoechst 33342 to visualize the nuclei. Alexa 488 (MHC), bright-field (BF), and Hoechst 33342 (Hoechst) images are shown as indicated at the left. Experiments were repeated three times with similar results. Scale bars, 100 μm. (B) The fusion index observed in A was determined by dividing the number of nuclei within multinucleated myotubes by the total number of nuclei analyzed. Values are expressed relative to that observed in the cells differentiated in the presence of Fc and shown as means ± SD of three independent experiments. (C) Confluent C2C12 myoblasts were differentiated into myotubes in DMEM containing 1% FBS and 10 nM IGF-I in the presence of either 6.4 nM Fc (Fc) or ephrinA1-Fc (A1-Fc) for time periods (days) indicated at the top. Cell lysates were subjected to Western blot analysis using anti-MHC (MHC) and anti-AKT (AKT) antibodies as indicated at the left. (D) The expression level of MHC observed in C was quantified by normalizing the expression of MHC by that of AKT. Values are expressed relative to that observed in the cells differentiated in the presence of Fc for 3 d and shown as means ± SD of three independent experiments. *p < 0.05, significant differences between two groups. a.u., arbitrary unit(s).

Article Snippet: Mouse C2C12 and rat L6 myoblasts were maintained as subconfluent monolayers in DMEM (Nissui, Tokyo, Japan) supplemented with FBS (20% for C2C12 cells, 10% for L6 cells), 4.5 g/l glucose, 0.58 g/l l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Western Blot, Expressing

Journal: Molecular Biology of the Cell

Article Title: EphrinA/EphA signal facilitates insulin-like growth factor-I–induced myogenic differentiation through suppression of the Ras/extracellular signal–regulated kinase 1/2 cascade in myoblast cell lines

doi: 10.1091/mbc.E11-03-0183

Figure Lengend Snippet: The ephrinA/EphA signal promotes IGF-I–dependent myogenic differentiation through p120RasGAP. (A) C2C12 myoblasts transfected with either control siRNA (C) or two independent siRNAs targeting p120RasGAP (1 and 2) were differentiated into myotubes in DMEM containing 1% FBS and 10 nM IGF-I in the presence of 6.4 nM ephrinA1-Fc for 2 d. The cells were immunostained with anti-MHC antibody and visualized with Alexa Fluor 488–conjugated secondary antibody. The cells were also poststained with Hoechst 33342 to visualize the nuclei. Alexa 488 (MHC), bright-field (BF), and Hoechst 33342 (Hoechst) images are shown as indicated at the left. Experiments were repeated three times with similar results. Scale bars, 100 μm. (B) The fusion index observed in A was determined as described in the legend of . Values are expressed relative to that observed in the cells transfected with control siRNA and shown as means ± SD of three independent experiments. (C) siRNA-transfected C2C12 myoblasts were differentiated into myotubes as described in A for time periods (days) indicated at the top. Cell lysates were subjected to Western blot analysis using anti-MHC (MHC), anti-p120RasGAP (p120RasGAP), and anti-AKT (AKT) antibodies as indicated at the left. (D) Expression level of MHC observed in C was quantified by normalizing the expression of MHC by that of AKT. Values are expressed relative to that observed in the control siRNA-transfected cells differentiated for 2 d and shown as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, significant differences between two groups. a.u., arbitrary unit(s).

Article Snippet: Mouse C2C12 and rat L6 myoblasts were maintained as subconfluent monolayers in DMEM (Nissui, Tokyo, Japan) supplemented with FBS (20% for C2C12 cells, 10% for L6 cells), 4.5 g/l glucose, 0.58 g/l l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Transfection, Control, Western Blot, Expressing

Journal: Molecular Biology of the Cell

Article Title: EphrinA/EphA signal facilitates insulin-like growth factor-I–induced myogenic differentiation through suppression of the Ras/extracellular signal–regulated kinase 1/2 cascade in myoblast cell lines

doi: 10.1091/mbc.E11-03-0183

Figure Lengend Snippet: Constitutive activation of the ERK1/2 pathway inhibits the promotion of IGF-I–mediated myogenic differentiation by the ephrinA/EphA signal. (A) Serum-starved C2C12 myoblasts infected with adenoviruses encoding either β-gal (β-gal) or constitutive active mutant of MEK1 (caMEK1) were stimulated with or without 10 nM IGF-I for 10 min in the presence of 1.7 nM Fc (−) or ephrinA1-Fc (A1-Fc) as indicated at the top. Cell lysates were subjected to Western blot analysis with anti–phospho-ERK1/2 (p-ERK1/2), anti-ERK1/2 (ERK1/2), anti–phospho-AKT (p-AKT), anti-AKT (AKT), and MEK1 (MEK1) antibodies as indicated at the left. (B) C2C12 myoblasts infected without (–) or with adenoviruses encoding either β-gal (β-gal) or constitutive active mutant of MEK1 (caMEK1) were differentiated into myotubes in DMEM containing 1% FBS and 10 nM IGF-I in the presence of 6.4 nM Fc (–) or ephrinA1-Fc (A1-Fc) for 2 d. The cells were immunostained with anti-MHC antibody, and visualized with Alexa Fluor 488–conjugated secondary antibody. The cells were also poststained with Hoechst 33342 to visualize the nuclei. Alexa 488 (MHC), bright-field (BF), and Hoechst 33342 (Hoechst) images are shown as indicated at the left. Experiments were repeated three times with similar results. Scale bars, 100 μm. (C) The fusion index observed in B was determined as described in the legend of . Values are expressed relative to that observed in the uninfected cells differentiated in the presence of Fc and shown as means ± SD of three independent experiments. (D) Adenovirus-infected C2C12 myoblasts were differentiated into myotubes as described in B for time periods (days) indicated at the top. Cell lysates were subjected to Western blot analysis using anti-MHC (MHC), anti-MEK1 (MEK1), and anti-AKT (AKT) antibodies as indicated at the left. (E) The expression level of MHC observed in D was quantified by normalizing the expression of MHC by that of AKT. Values are expressed relative to that observed in the uninfected cells differentiated in the presence of Fc for 2 d and shown as means ± SD of three independent experiments. **p < 0.01, significant differences between two groups. a.u., arbitrary unit(s).

Article Snippet: Mouse C2C12 and rat L6 myoblasts were maintained as subconfluent monolayers in DMEM (Nissui, Tokyo, Japan) supplemented with FBS (20% for C2C12 cells, 10% for L6 cells), 4.5 g/l glucose, 0.58 g/l l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Activation Assay, Infection, Mutagenesis, Western Blot, Expressing

Journal: Molecular Biology of the Cell

Article Title: EphrinA/EphA signal facilitates insulin-like growth factor-I–induced myogenic differentiation through suppression of the Ras/extracellular signal–regulated kinase 1/2 cascade in myoblast cell lines

doi: 10.1091/mbc.E11-03-0183

Figure Lengend Snippet: IGF-I–induced myogenic differentiation is blunted by blocking the ephrinA/EphA signal. (A) C2C12 myoblasts infected without (−) or with adenoviruses encoding either β-gal or EphA2Δcyto were differentiated into myotubes in DMEM containing 1% FBS and 10 nM IGF-I for 3 d. The cells were immunostained with anti-MHC antibody and visualized with Alexa Fluor 488–conjugated secondary antibody. The cells were also poststained with Hoechst 33342 to visualize the nuclei. Alexa 488 (MHC), bright-field (BF), and Hoechst 33342 (Hoechst) images are shown as indicated at the left. Experiments were repeated three times with similar results. Scale bars, 100 μm. (B) The fusion index observed in A was determined as described in the legend of . Values are expressed relative to that observed in the uninfected cells and shown as means ± SD of three independent experiments. (C) Adenovirus-infected C2C12 myoblasts were differentiated into myotubes as described in A for time periods (days) indicated at the top. Cell lysates were subjected to Western blot analysis using anti-MHC (MHC), anti-HA (EphA2Δcyto), and anti-AKT (AKT) antibodies as indicated at the left. (D) The expression level of MHC observed in C was quantified by normalizing the expression of MHC by that of AKT. Values are expressed relative to that observed in the uninfected cells differentiated for 3 d and shown as means ± SD of three independent experiments. **p < 0.01, significant differences between two groups. a.u., arbitrary unit(s).

Article Snippet: Mouse C2C12 and rat L6 myoblasts were maintained as subconfluent monolayers in DMEM (Nissui, Tokyo, Japan) supplemented with FBS (20% for C2C12 cells, 10% for L6 cells), 4.5 g/l glucose, 0.58 g/l l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Blocking Assay, Infection, Western Blot, Expressing

Journal: Molecular Biology of the Cell

Article Title: EphrinA/EphA signal facilitates insulin-like growth factor-I–induced myogenic differentiation through suppression of the Ras/extracellular signal–regulated kinase 1/2 cascade in myoblast cell lines

doi: 10.1091/mbc.E11-03-0183

Figure Lengend Snippet: Inhibition of IGF-I–induced myogenic differentiation by a dominant-negative EphA receptor mutant is canceled by inhibiting the ERK1/2 pathway. (A) C2C12 myoblasts infected without (–) or with adenoviruses encoding either β-gal or EphA2Δcyto were differentiated into myotubes in DMEM containing 1% FBS and 10 nM IGF-I in the presence of vehicle (dimethyl sulfoxide [DMSO]) or 3 μM U0126 for 3 d. The cells were immunostained with anti-MHC antibody and visualized with Alexa Fluor 488–conjugated secondary antibody. The cells were also poststained with Hoechst 33342 to visualize the nuclei. Alexa 488 (MHC), bright-field (BF), and Hoechst 33342 (Hoechst) images are shown as indicated at the left. Experiments were repeated three times with similar results. Scale bars, 100 μm. (B) The fusion index observed in A was determined as described in the legend of . Values are expressed relative to that observed in the uninfected cells differentiated in the presence of DMSO and shown as means ± SD of three independent experiments. (C) Adenovirus-infected C2C12 myoblasts were differentiated into myotubes as described in A for time periods (days) indicated at the top. Cell lysates were subjected to Western blot analysis using anti-MHC (MHC), anti-HA (EphA2Δcyto), and anti-AKT (AKT) antibodies as indicated at the left. (D) The expression level of MHC observed in C was quantified by normalizing the expression of MHC by that of AKT. Values are expressed relative to that observed in the uninfected cells differentiated in the presence of DMSO for 3 d and shown as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, significant differences between two groups. N.S., no significance between two groups. a.u., arbitrary unit(s).

Article Snippet: Mouse C2C12 and rat L6 myoblasts were maintained as subconfluent monolayers in DMEM (Nissui, Tokyo, Japan) supplemented with FBS (20% for C2C12 cells, 10% for L6 cells), 4.5 g/l glucose, 0.58 g/l l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Inhibition, Dominant Negative Mutation, Mutagenesis, Infection, Western Blot, Expressing