[PMC free article] [PubMed] [Google Scholar]Pavlath GK. ephrinA1-Fc failed to suppress the Ras-ERK1/2 cascade by IGF-I and to promote IGF-ICmediated myogenesis. EphrinA1-Fc did not promote IGF-ICdependent myogenesis when the ERK1/2 was constitutively activated. Furthermore, a dominant-negative EphA receptor blunted IGF-ICinduced myogenesis in C2C12 and L6 myoblasts. However, the inhibition of IGF-ICmediated myogenesis by down-regulation of ephrinA/EphA signal was canceled by inactivation of the ERK1/2 pathway. Collectively, these findings demonstrate that this ephrinA/EphA signal facilitates IGF-ICinduced myogenesis by suppressing the Ras-ERK1/2 cascade through p120RasGAP in myoblast cell lines. INTRODUCTION Skeletal myogenesis is usually a complex process that begins with the commitment of multipotent mesodermal precursor cells to the muscle fate (Andres and Walsh, 1996 ; Taylor, 2002 ). These committed cellsthe myoblastssubsequently withdraw from the cell cycle, differentiate, and fuse into multinucleated myotubes. In culture, most skeletal muscle cell lines proliferate under high serum conditions, whereas the cells placed under low serum conditions spontaneously undergo differentiation into myotubes (Florini with Cdo, a cell surface receptor of the immunoglobulin (Ig) superfamily in C2C12 myoblasts (Lu and Krauss, 2010 ). On N-cadherin ligation, the Cdo intracellular region binds to Bnip-2, a scaffold protein for Cdc42 small GTPase, and to JLP, a scaffold protein for the p38/ MAPK, which results in Cdc42-dependent activation of p38/ (Takaesu and subjected to Western blot analysis with anti-Ras antibody (GTP-Ras). Aliquots of cell lysates were also subjected to Western blot analysis with anti-Ras (Ras), anti-p120RasGAP (p120RasGAP), and antiC-tubulin (-tubulin) antibodies as indicated at the left. (B) Serum-starved C2C12 myoblasts transfected with siRNA as described in A were stimulated for 10 min with or without 10 nM IGF-I in the absence or presence of 1 1.7 or 3.4 nM ephrinA1-Fc (A1-Fc) as indicated at the top. Cell lysates were subjected to Western blot analysis with antiCphospho-ERK1/2 (p-ERK1/2), anti-ERK1/2 (ERK1/2), antiCphospho-AKT (p-AKT), anti-AKT (AKT), and anti-p120RasGAP (p120RasGAP) antibodies as indicated at the left. (C) Phosphorylation levels of ERK1/2 (top) and AKT (bottom) observed in B were quantified and represented by the ratio of phospho-ERK1/2 or phospho-AKT to total ERK1/2 or total AKT, respectively. Values are expressed as explained in the legend of Physique 1B. Values are expressed relative to the ratio obtained from the IGF-ICtreated cells transfected with control siRNA. *p 0.05, **p 0.01, significant differences between two groups. N.S., no significance between two groups. a.u., arbitrary unit(s). EphrinA/EphA signal enhances IGF-ICinduced myogenic differentiation Myogenic differentiation is usually negatively and positively regulated by the ERK1/2 and AKT cascades, respectively (Kaliman for 20 min at 4C. The supernatant was used as precleared total-cell lysate. For the immunoprecipitation of EphA2, the cells were lysed in lysis buffer made up of 20 mM Tris, pH 7.5, 150 mM NaCl, 3 mM EDTA, 1% Nonidet P-40, and 1 protease inhibitor cocktail. EphA2 was immunoprecipitated from the cleared lysates by incubation with anti-EphA2 antibody for 2 h at 4C. Immunocomplexes were recovered with the aid of protein ACSepharose beads (GE Healthcare Life Sciences). Aliquots of cell lysate and the immunoprecipitates were subjected to SDSCPAGE and Western blot analysis with the antibodies as indicated in the physique legends. Immunocytochemistry Cells plated on 3.5-cm collagen type ICcoated plastic dishes (Iwaki Asahi Glass, Tokyo, Japan) were fixed with 2% formaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with PBS containing 4% bovine serum albumin for 1 h. The cells were then stained with anti-MHC antibody for 1 h at room heat. Protein reacting with the antibody was visualized with Alexa Fluor 488Cconjugated secondary antibody. The nucleus was also poststained with Hoechst 33342 nuclear dye. Fluorescent pictures of Alexa.Mol Biol Cell. IGF-ICmediated myogenesis. EphrinA1-Fc didn’t promote IGF-ICdependent myogenesis when the ERK1/2 was constitutively triggered. Furthermore, a dominant-negative EphA receptor blunted IGF-ICinduced myogenesis in C2C12 and L6 myoblasts. Nevertheless, the inhibition of IGF-ICmediated myogenesis by down-regulation of ephrinA/EphA sign was canceled by inactivation from the ERK1/2 pathway. Collectively, these results demonstrate how the ephrinA/EphA sign facilitates IGF-ICinduced myogenesis by suppressing the Ras-ERK1/2 cascade through p120RasGAP in myoblast cell lines. Intro Skeletal myogenesis can be a complex procedure that begins using the dedication of multipotent mesodermal precursor cells towards the muscle tissue destiny (Andres and Walsh, 1996 ; Taylor, 2002 ). These dedicated cellsthe myoblastssubsequently withdraw through the cell routine, differentiate, and fuse into multinucleated myotubes. In tradition, most skeletal muscle tissue cell lines proliferate under high serum circumstances, whereas the cells placed directly under low serum circumstances spontaneously go through differentiation into myotubes (Florini with Cdo, a cell surface area receptor from the immunoglobulin (Ig) superfamily in C2C12 myoblasts (Lu and Krauss, 2010 ). On N-cadherin ligation, the Cdo intracellular area binds to Bnip-2, a scaffold proteins for Cdc42 little GTPase, also to JLP, a scaffold proteins for the p38/ MAPK, which leads to Cdc42-reliant activation of p38/ (Takaesu and put through Western blot evaluation with anti-Ras antibody (GTP-Ras). Aliquots of cell lysates had been also put through Western blot evaluation with anti-Ras (Ras), anti-p120RasGAP (p120RasGAP), and antiC-tubulin (-tubulin) antibodies as indicated in the remaining. (B) Serum-starved C2C12 myoblasts transfected with siRNA as referred to in A had been activated for 10 min with or without 10 nM IGF-I in the lack or presence of just one 1.7 or 3.4 nM ephrinA1-Fc (A1-Fc) as indicated at the very top. Cell lysates had been subjected to Traditional western blot evaluation with antiCphospho-ERK1/2 (p-ERK1/2), anti-ERK1/2 (ERK1/2), antiCphospho-AKT (p-AKT), anti-AKT (AKT), and anti-p120RasGAP (p120RasGAP) antibodies as indicated in the remaining. (C) Phosphorylation degrees of ERK1/2 (best) and AKT (bottom level) seen in B had been quantified and displayed by the percentage of phospho-ERK1/2 or phospho-AKT to total ERK1/2 or total AKT, respectively. Ideals are indicated as described in the tale of Shape 1B. Ideals are expressed in accordance with the percentage from the IGF-ICtreated cells transfected with control siRNA. *p 0.05, **p 0.01, significant variations between two organizations. N.S., no significance between two organizations. a.u., arbitrary device(s). EphrinA/EphA sign enhances IGF-ICinduced myogenic differentiation Myogenic differentiation can be adversely and positively controlled from the ERK1/2 and AKT cascades, respectively (Kaliman for 20 min at 4C. The supernatant was utilized as precleared total-cell lysate. For the immunoprecipitation of EphA2, the cells had been lysed in lysis buffer including 20 mM Tris, pH 7.5, 150 mM NaCl, 3 mM EDTA, 1% Nonidet P-40, and 1 protease inhibitor cocktail. EphA2 was immunoprecipitated through the cleared lysates by incubation with anti-EphA2 antibody for 2 h at 4C. Immunocomplexes had been recovered using proteins ACSepharose beads (GE Health care Existence Sciences). Aliquots of cell lysate as well as the immunoprecipitates had been put through SDSCPAGE and Traditional western blot analysis using the antibodies as indicated in the shape legends. Immunocytochemistry Cells plated on 3.5-cm collagen type ICcoated plastic material dishes (Iwaki Asahi Glass, Tokyo, Japan) were set with 2% formaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with PBS containing 4% bovine serum albumin for 1 h. The cells had been after that stained with anti-MHC antibody for 1 h at space temperature. Protein responding using the antibody was visualized with Alexa Fluor 488Cconjugated supplementary antibody. The nucleus was also poststained with Hoechst 33342 nuclear dye. Fluorescent pictures of Alexa Fluor 488 and Hoechst 33342 and phase-contrast pictures had been documented with an Olympus IX-81 inverted fluorescence microscope (Olympus Company, Tokyo, Japan) as referred to previously (Noda em et al. /em , 2010 ). Change transcription-PCR Total RNA was ready from C2C12 myoblasts using TRIzol reagent (Invitrogen), and reverse-transcribed by arbitrary hexamer primers using Superscript II (Invitrogen) based on the manufacturer’s instructions. PCR was performed using the gene-specific primers detailed in the Supplemental Desk S1. Amplification of glyceraldehyde-3-phosphate dehydrogenase was performed in parallel like a control also. Recognition of GTP-bound type of Ras Ras activation was evaluated utilizing a pull-down technique. Cells had been lysed at 4C inside a pull-down lysis buffer including 20 mM Tris, pH 7.5, 100 mM NaCl, 10 AN3199 mM MgCl2, 1% Triton X-100, 1.EphA4/ephrin-A5 interactions in muscle precursor cell migration in the avian forelimb. neither ERK1/2 nor AKT triggered by IGF-I. IGF-ICdependent myogenic differentiation of C2C12 myoblasts was potentiated by ephrinA1-Fc. In p120RasGAP-depleted cells, ephrinA1-Fc didn’t suppress the Ras-ERK1/2 cascade by IGF-I also to promote IGF-ICmediated myogenesis. EphrinA1-Fc didn’t promote IGF-ICdependent myogenesis when the ERK1/2 was constitutively triggered. Furthermore, a dominant-negative EphA receptor blunted IGF-ICinduced myogenesis in C2C12 and L6 myoblasts. Nevertheless, the inhibition of IGF-ICmediated myogenesis by down-regulation of ephrinA/EphA sign was canceled by inactivation from the ERK1/2 pathway. Collectively, these results demonstrate how the ephrinA/EphA sign facilitates IGF-ICinduced myogenesis by suppressing the Ras-ERK1/2 cascade through p120RasGAP in myoblast cell lines. Intro Skeletal myogenesis can be a complex procedure that begins using the dedication of multipotent mesodermal precursor cells towards the muscle tissue destiny AN3199 (Andres and Walsh, 1996 ; Taylor, 2002 ). These dedicated cellsthe myoblastssubsequently withdraw through the cell routine, differentiate, and fuse into multinucleated myotubes. In tradition, most skeletal muscle tissue cell lines proliferate under high serum circumstances, whereas the cells placed directly under low serum circumstances spontaneously go through differentiation into myotubes (Florini with Cdo, a cell surface area receptor of the immunoglobulin (Ig) superfamily in C2C12 myoblasts (Lu and Krauss, 2010 ). On N-cadherin ligation, the Cdo intracellular region binds to Bnip-2, a scaffold protein for Cdc42 small GTPase, and to JLP, a scaffold protein for the p38/ MAPK, which results in Cdc42-dependent activation of p38/ (Takaesu and subjected to Western blot analysis with anti-Ras antibody (GTP-Ras). Aliquots of cell lysates were also subjected to Western blot analysis with anti-Ras (Ras), anti-p120RasGAP (p120RasGAP), and antiC-tubulin (-tubulin) antibodies as indicated in the remaining. (B) Serum-starved C2C12 myoblasts transfected with siRNA as explained in A were stimulated for 10 min with or without 10 nM IGF-I in the absence or presence of 1 1.7 or 3.4 nM ephrinA1-Fc (A1-Fc) as indicated at the top. Cell lysates were subjected to Western blot analysis with antiCphospho-ERK1/2 (p-ERK1/2), anti-ERK1/2 (ERK1/2), antiCphospho-AKT (p-AKT), anti-AKT (AKT), and anti-p120RasGAP (p120RasGAP) antibodies as indicated in the remaining. (C) Phosphorylation levels of ERK1/2 (top) and AKT (bottom) observed in B were quantified and displayed by the percentage of phospho-ERK1/2 or phospho-AKT to total ERK1/2 or total AKT, respectively. Ideals are indicated as explained in the story of Number 1B. Ideals are expressed relative to the percentage from the IGF-ICtreated cells transfected with control siRNA. *p 0.05, **p 0.01, significant variations between two organizations. N.S., no significance between two organizations. a.u., arbitrary unit(s). EphrinA/EphA transmission enhances IGF-ICinduced myogenic differentiation Myogenic differentiation is definitely negatively and positively controlled from the ERK1/2 and AKT cascades, respectively (Kaliman for 20 min at 4C. The supernatant was used as precleared total-cell lysate. For the immunoprecipitation of EphA2, the cells were lysed in lysis buffer comprising 20 mM Tris, pH 7.5, 150 mM NaCl, 3 mM EDTA, 1% Nonidet P-40, and 1 protease inhibitor cocktail. EphA2 was immunoprecipitated from your cleared lysates by incubation with anti-EphA2 antibody for 2 h at 4C. Immunocomplexes were recovered with the aid of protein ACSepharose beads (GE Healthcare Existence Sciences). Aliquots of cell lysate and the immunoprecipitates were subjected to SDSCPAGE and Western blot analysis with the antibodies as indicated in the number legends. Immunocytochemistry Cells plated on 3.5-cm collagen type ICcoated plastic dishes (Iwaki Asahi Glass, Tokyo, Japan) were fixed with 2% formaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with PBS containing 4% bovine serum albumin for 1 h. The cells were then stained with anti-MHC antibody for 1 h at space temperature. Protein reacting with the antibody was visualized with Alexa Fluor 488Cconjugated secondary antibody. The nucleus was also poststained with Hoechst 33342 nuclear dye. Fluorescent images of Alexa Fluor 488 and Hoechst 33342 and phase-contrast images were recorded with an Olympus IX-81 inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan) as explained previously (Noda em et al. /em , 2010 ). Reverse transcription-PCR Total RNA was prepared from C2C12 myoblasts using TRIzol reagent (Invitrogen), and reverse-transcribed by random hexamer primers using Superscript II (Invitrogen) according to the manufacturer’s teaching. PCR was performed using the gene-specific primers outlined in the Supplemental Table S1. Amplification of glyceraldehyde-3-phosphate dehydrogenase was also performed in parallel like a control. Detection of GTP-bound form of Ras Ras activation was assessed using a pull-down technique. Cells were lysed at 4C inside a pull-down lysis buffer comprising 20 mM Tris, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1% Triton X-100, 1 mM ethylene glycol tetraacetic acid, 1 mM dithiothreitol, 1 mM sodium orthovanadate, and 1 protease inhibitor cocktail. GTP-bound.[PMC free article] [PubMed] [Google Scholar]Gutierrez J, Brandan E. pathway. Collectively, these findings demonstrate the ephrinA/EphA transmission facilitates IGF-ICinduced myogenesis by suppressing the Ras-ERK1/2 cascade through p120RasGAP in myoblast cell lines. Intro Skeletal myogenesis is definitely a complex process that begins with the commitment of multipotent mesodermal precursor cells to the muscle mass fate (Andres and Walsh, 1996 ; Taylor, 2002 ). These committed cellsthe myoblastssubsequently withdraw from your cell cycle, differentiate, and fuse into multinucleated myotubes. In tradition, most skeletal muscle mass cell lines proliferate under high serum conditions, whereas the cells placed under low serum conditions spontaneously undergo differentiation into myotubes (Florini with Cdo, a cell surface AN3199 receptor of the immunoglobulin (Ig) superfamily in C2C12 myoblasts (Lu and Krauss, 2010 ). On N-cadherin ligation, the Cdo intracellular region binds to Bnip-2, a scaffold protein for Cdc42 small GTPase, and to JLP, a scaffold protein for the p38/ MAPK, which results in Cdc42-dependent activation of p38/ (Takaesu and subjected to Western blot analysis with anti-Ras antibody (GTP-Ras). Aliquots of cell lysates were also subjected to Western blot analysis with anti-Ras (Ras), anti-p120RasGAP (p120RasGAP), and antiC-tubulin (-tubulin) antibodies as indicated in the remaining. (B) Serum-starved C2C12 myoblasts transfected with siRNA as explained in A were stimulated for 10 min with or without 10 nM IGF-I in the absence or presence of 1 1.7 or 3.4 nM ephrinA1-Fc (A1-Fc) as indicated at the top. Cell lysates were subjected to Western blot analysis with antiCphospho-ERK1/2 (p-ERK1/2), anti-ERK1/2 (ERK1/2), antiCphospho-AKT (p-AKT), anti-AKT (AKT), and anti-p120RasGAP (p120RasGAP) antibodies as indicated in the remaining. (C) Phosphorylation levels of ERK1/2 (top) and AKT (bottom) observed in B were quantified and displayed by the percentage of phospho-ERK1/2 or phospho-AKT to total ERK1/2 or total AKT, respectively. Ideals are indicated as explained in the story of Number 1B. Ideals are expressed relative to the percentage from the IGF-ICtreated cells transfected with control siRNA. *p 0.05, **p 0.01, significant variations between two organizations. N.S., no significance between two organizations. a.u., arbitrary unit(s). EphrinA/EphA transmission enhances IGF-ICinduced myogenic differentiation Myogenic differentiation is definitely negatively and positively governed with the ERK1/2 and AKT cascades, respectively (Kaliman for 20 min at 4C. The supernatant was utilized as precleared total-cell lysate. For the immunoprecipitation of EphA2, the cells had been lysed in lysis buffer formulated with 20 mM Tris, AN3199 pH 7.5, 150 mM NaCl, 3 mM EDTA, 1% Nonidet P-40, and 1 protease inhibitor cocktail. EphA2 was immunoprecipitated in the cleared lysates by incubation with anti-EphA2 antibody for 2 h at 4C. Immunocomplexes had been recovered using proteins ACSepharose beads (GE Health care Lifestyle Sciences). Aliquots of cell lysate as well as the immunoprecipitates had been put through SDSCPAGE and Traditional western blot analysis using the antibodies as indicated in the body legends. Immunocytochemistry Cells plated on 3.5-cm collagen type ICcoated plastic material dishes (Iwaki Asahi Glass, Tokyo, Japan) were set with 2% formaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with PBS containing 4% bovine serum albumin for 1 h. The cells had been after that stained with anti-MHC antibody for 1 h at area temperature. Protein responding using the antibody was visualized with Alexa Fluor 488Cconjugated supplementary antibody. The nucleus was also poststained with Hoechst 33342 nuclear dye. Fluorescent pictures of Alexa Fluor 488 and Hoechst 33342 and phase-contrast pictures had been documented with an Olympus IX-81 inverted fluorescence microscope (Olympus Company, Tokyo, Japan) as defined previously (Noda em et al. /em , 2010 ). Change transcription-PCR Total RNA was ready from C2C12 myoblasts using TRIzol reagent (Invitrogen), and reverse-transcribed by arbitrary hexamer primers using Superscript II (Invitrogen) based on the manufacturer’s instructions. PCR was performed using the gene-specific primers shown in the Supplemental Desk S1. Amplification of glyceraldehyde-3-phosphate dehydrogenase was also performed in parallel being a control. Recognition of GTP-bound type of Ras Ras activation was evaluated utilizing a pull-down technique. Cells had been lysed at 4C within a pull-down lysis buffer formulated with 20 mM Tris, pH 7.5, 100 mM NaCl,.2010;316:3042C3049. in C2C12 myoblasts, whereas ephrinB1-Fc affected neither ERK1/2 nor AKT turned on by IGF-I. IGF-ICdependent myogenic differentiation of C2C12 myoblasts was potentiated by ephrinA1-Fc. In p120RasGAP-depleted cells, ephrinA1-Fc didn’t suppress the Ras-ERK1/2 cascade by IGF-I also to promote IGF-ICmediated myogenesis. EphrinA1-Fc didn’t promote IGF-ICdependent myogenesis when the ERK1/2 was constitutively turned on. Furthermore, a dominant-negative EphA receptor blunted IGF-ICinduced myogenesis in C2C12 and L6 myoblasts. Nevertheless, the inhibition of IGF-ICmediated myogenesis by down-regulation of ephrinA/EphA indication was canceled by inactivation from the ERK1/2 pathway. Collectively, these results demonstrate the fact that ephrinA/EphA indication facilitates IGF-ICinduced myogenesis by suppressing Rabbit polyclonal to ATS2 the Ras-ERK1/2 cascade through p120RasGAP in myoblast cell lines. Launch Skeletal myogenesis is certainly a complex procedure that begins using the dedication of multipotent mesodermal precursor cells towards the muscles destiny (Andres and Walsh, 1996 ; Taylor, 2002 ). These dedicated cellsthe myoblastssubsequently withdraw in the cell routine, differentiate, and fuse into multinucleated myotubes. In lifestyle, most skeletal muscles cell lines proliferate under high serum circumstances, whereas the cells placed directly under low serum circumstances spontaneously go through differentiation into myotubes (Florini with Cdo, a cell surface area receptor from the immunoglobulin (Ig) superfamily in C2C12 myoblasts (Lu and Krauss, 2010 ). On N-cadherin ligation, the Cdo intracellular area binds to Bnip-2, a scaffold proteins for Cdc42 little GTPase, also to JLP, a scaffold proteins for the p38/ MAPK, which leads to Cdc42-reliant activation of p38/ (Takaesu and put through Western blot evaluation with anti-Ras antibody (GTP-Ras). Aliquots of cell lysates had been also put through Western blot evaluation with anti-Ras (Ras), anti-p120RasGAP (p120RasGAP), and antiC-tubulin (-tubulin) antibodies as indicated on the still left. (B) Serum-starved C2C12 myoblasts transfected with siRNA as defined in A had been activated for 10 min with or without 10 nM IGF-I in the lack or presence of just one 1.7 or 3.4 nM ephrinA1-Fc (A1-Fc) as indicated at the very top. Cell lysates had been subjected to Traditional western blot evaluation with antiCphospho-ERK1/2 (p-ERK1/2), anti-ERK1/2 (ERK1/2), antiCphospho-AKT (p-AKT), anti-AKT (AKT), and anti-p120RasGAP (p120RasGAP) antibodies as indicated on the still left. (C) Phosphorylation degrees of ERK1/2 (best) and AKT (bottom level) seen in B had been quantified and symbolized by the proportion of phospho-ERK1/2 or phospho-AKT to total ERK1/2 or total AKT, respectively. Beliefs are portrayed as described in the star of Body 1B. Beliefs are expressed in accordance with the proportion extracted from the IGF-ICtreated cells transfected with control siRNA. *p 0.05, **p 0.01, significant distinctions between two groupings. N.S., no significance between two groupings. a.u., arbitrary device(s). EphrinA/EphA indication enhances IGF-ICinduced myogenic differentiation Myogenic differentiation is negatively and positively regulated by the ERK1/2 and AKT cascades, respectively (Kaliman for 20 min at 4C. The supernatant was used as precleared total-cell lysate. For the immunoprecipitation of EphA2, the cells were lysed in lysis buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 3 mM EDTA, 1% Nonidet P-40, and 1 protease inhibitor cocktail. EphA2 was immunoprecipitated from the cleared lysates by incubation with anti-EphA2 antibody for 2 h at 4C. Immunocomplexes were recovered with the aid of protein ACSepharose beads (GE Healthcare Life Sciences). Aliquots of cell lysate and the immunoprecipitates were subjected to SDSCPAGE and Western blot analysis with the antibodies as indicated in the figure legends. Immunocytochemistry Cells plated on 3.5-cm collagen type ICcoated plastic dishes (Iwaki Asahi Glass, Tokyo, Japan) were fixed with 2% formaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with PBS containing 4% bovine serum albumin for 1 h. The cells were then stained with anti-MHC antibody for 1 h at room temperature. Protein reacting with the antibody was visualized with Alexa Fluor 488Cconjugated secondary antibody. The nucleus was also poststained with Hoechst 33342 nuclear dye. Fluorescent images of Alexa Fluor 488 and Hoechst 33342 and phase-contrast images were recorded with an Olympus IX-81 inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan) as described previously (Noda em et al. /em , 2010 ). Reverse transcription-PCR Total RNA.