Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 1. Expression of signal transducers and activators of transcription 3 (STAT3) in SW1990 cells and HeLa cells. (a) STAT3 expression was determined by Western blot analysis. Detection of β-actin was used as a loading control. HeLa cell line served as a positive control of STAT3 expression. (b) STAT3 distribution in SW1990 cells was examined by immunocytochemistry (400 ×), the buffy particle corresponded for STAT3 (shown by arrow).

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: Expressing, Western Blot, Control, Positive Control, Immunocytochemistry

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 2. Effects of signal transducers and activators of transcription 3 (STAT3) specific shRNA expression vector on STAT3 expression. (a) Reverse transcription-polymerase chain reaction (RT-PCR) analysis. The RNA samples (2 µg in each) extracted from SW1990 cells, SW1990 cells transfected with pRNAT-Con, pRNAT-STAT3-siRNA-I, pRNAT-STAT3- siRNA-II and pRNAT-STAT3-siRNA-III were subjected to RT-PCR for STAT3 and β-actin mRNAs as described in Materials and Methods. RT- PCR for β-actin was carried out in parallel to show an equal amount of total RNA in the sample. (b) Western blot analysis. Cellular whole and nuclear protein extracts (100 µg in each) were prepared from SW1990 cells, SW1990 cells transfected with pRNAT-Con, pRNAT-STAT3-siRNA-I, pRNAT-STAT3-siRNA-II and pRNAT-STAT3-siRNA-III. The expression of STAT3 protein was determined using Western blot analysis with an anti-STAT3 antibody. The expression of p-STAT3 protein was deter- mined by hybridizing the same membrane filter with an antip-STAT3 antibody. The levels of β-actin expression were determined as a control for equivalent protein loading. Results shown are for one represent- ative experiment of three.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: shRNA, Expressing, Plasmid Preparation, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot, Membrane, Control

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 3. Effects of stable transfection of pRNAT-STAT3-siRNA-II vector on signal transducers and activators of transcription 3 (STAT3) expres- sion. (a) Reverse transcription-polymerase chain reaction (RT-PCR) analysis. The RNA samples (2 µg in each) extracted from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b) were subjected to RT-PCR for STAT3 and β-actin mRNAs as described in Materials and Methods. RT-PCR for β-actin was carried out in parallel to show an equal amount of total RNA in the sample. (b) Western blot analysis. Cellular whole and nuclear protein extracts (100 µg in each) were prepared from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b). The expression of STAT3 protein was determined using Western blot analysis with an anti-STAT3 antibody. The expression of p-STAT3 protein was determined by hybridizing the same membrane filter with an antip-STAT3 antibody. The levels of β-actin expression were determined as a control for equivalent protein loading. (c) STAT3 DNA-binding activity. Cell nuclear protein extracts (10 µg in each) were prepared from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b) and subjected to electrophoretic mobility shift analysis (EMSA) using biotin end-labeled oligonucleotide probes containing a consensus-binding motif for STAT3. Results shown are for one representative experiment of three.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: Stable Transfection, Plasmid Preparation, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Western Blot, Expressing, Membrane, Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Labeling

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 4. Effects of silence of signal transducers and activators of transcription 3 (STAT3) gene on SW1990 cell invasion in vitro. SW1990-RNAi-a, SW1990-RNAi-b, SW1990-Con and the parental cell lines were seeded into the upper compart- ments of invasion chambers. Cells were allowed to invade for 48 h at 37°C. The tumor cells that invaded the ECMatrix and migrated through the polycarbonate membrane were stained by the staining solution and dissolved in 10% acetic acid. The dye/solution mixture was transferred onto a 96-well plate for colorimetric reading of optical density (OD) at 560 nm. The number of migrated cells that penetrated through the ECMatrix-coated filters was expressed as the OD at 560 nm. The standard deviation bars represent replicates within the assay. *P < 0.001 compared with that of parental SW1990 cells. Results shown are for one representative experiment of three.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: In Vitro, Membrane, Staining, Standard Deviation

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 5. Effects of silence of signal transducers and activators of transcription 3 (STAT3) gene on SW1990 cell metastasis in vivo. In total, 5 × 105 SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b) were injected i.v. into groups of nude mice (n = 6). The mice were killed on day 21 or when mice became moribund. The number of experimental lung metastases was determined with the aid of a dissecting microscope. Results were expressed as the median number and range of lung metastasis nodules. *P < 0.001 compared with that of parental SW1990 cells. Results shown here are for one representative experiment of three. (a) H&E staining of formalin-fixed, paraffin-embedded lung tissue of nude mice (100 ×). The metastasis nodus are indicated by arrows. (b) H&E staining of formalin-fixed, paraffin-embedded metastasis nodus of lung tissue of nude mice (400 ×). (c) In SW1990 cells injected nude mice, STAT3, matrix metalloproteinases-2 (MMP-2) and vascular endothelial growth factor (VEGF) distribution in metastasis nodus of lung tissue was examined by immunohistochemistry (400 ×), the buffy particle corresponded for STAT3, MMP-2 and VEGF. Immunohistochemistry showed that STAT3, MMP-2 and VEGF were distributed in cytoplasm.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: In Vivo, Transfection, Control, Plasmid Preparation, Injection, Microscopy, Staining, Formalin-fixed Paraffin-Embedded, Immunohistochemistry

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 6. Effects of silence of signal transducers and activators of transcription 3 (STAT3) gene on the expression of matrix metalloproteinases-2 (MMP-2) and vascular endothelial growth factor (VEGF). (a) Reverse transcription-polymerase chain reaction (RT-PCR) analysis. The RNA samples (2 µg in each) extracted from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3- RNAi (SW1990-RNAi-a and SW1990-RNAi-b) were subjected to RT-PCR for MMP-2, VEGF, and β-actin mRNAs as described in Materials and Methods. RT-PCR for β-actin was carried out in parallel to show an equal amount of total RNA in the sample. (b) Western blot analysis. Cellular whole protein extracts (100 µg in each) were prepared from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b). The expression of MMP-2 protein was determined using Western blot analysis with an anti-MMP-2 antibody. The expression of VEGF protein was determined by hybridizing the same membrane filter with an anti-VEGF antibody. The levels of β-actin expression were determined as a control for the equivalent protein loading. Results shown are for one representative experiment of three.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Plasmid Preparation, Western Blot, Membrane

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: Vascular endothelial growth factor (VEGF)- and epoetin alpha (EPOα)-mediated STAT-5 phosphorylation. Human umbilical vein endothelial cells (HUVECs) were treated with 25 ng/mL VEGF (10 min) or 1 IU/mL EPOα (5–10 and 30 min) under three different cell culture conditions. White bar: replacement of cell culture medium for 24 h with M199 without fetal bovine serum (FBS) and growth factors supplemented with 0.5% bovine serum albumin (BSA) and 0.2 mM orthovanadate. Gray bars: replacement of cell culture medium for 24 h with M199 without FBS and growth factors. Black bars: replacement of cell culture medium for 12 h with M199 medium without FBS and growth factors supplemented with 0.2 mM orthovanadate 30 min before the experiments. In each experimental condition, cell responsiveness was evaluated by assessing whether VEGF or EPOα stimulated STAT-5 phosphorylation. The data are expressed as the percent STAT-5 phosphorylation compared to the untreated control cells (set to 100%) and represent the mean ± SEM of three different experiments. *** p < 0.001; ** p < 0.01 vs. basal value.

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques: Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: Concentration-dependence of EPOα-, darbepoetin alpha (DarbEPO)- and continuous erythropoietin receptors activator (CERA)-mediated STAT-5 phosphorylation. HUVECs were treated with different epoetin concentrations (0.5–10 IU/mL) for 5 min. STAT-5 phosphorylation levels were quantified using an enzyme-linked immunosorbent assay (ELISA) kit (see Materials and Methods). The data are expressed as the percent STAT-5 phosphorylation compared to the untreated control cells (set to 100%) and represent the mean ± SEM of three different experiments.

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: Time-dependence of EPOα-, DarbEPO- and CERA-mediated STAT-5 phosphorylation. HUVECs were treated with medium alone (control) or 1 IU/mL EPOα, 1 IU/mL DarbEPO or 5 IU/mL CERA for different periods of time ranging from 5 to 30 min ( A ). Aliquots of cells were treated with VEGF (25 ng/mL) or granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 IU/mL) for the same time intervals as above ( B ). STAT-5 phosphorylation levels were quantified using an ELISA kit (see Materials and Methods). The data are expressed as the percent STAT-5 phosphorylation compared to the untreated control cells (set to 100%) and represent the mean ± SEM of three different experiments.

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: Effect of the STAT-5 inhibitor on EPOα-, DarbEPO- and CERA-mediated STAT-5 phosphorylation. HUVECs were treated with medium alone (basal) or 1 IU/mL EPOα, 1 IU/mL DarbEPO or 5 IU/mL CERA for 5–30 min or VEGF for 10 min in the absence (gray bar) or presence of the STAT-5 inhibitor (80 μM, black bar). STAT-5 phosphorylation levels were quantified using an ELISA kit (see Materials and Methods). The data are expressed as the per cent STAT-5 phosphorylation compared to the untreated control cells (set to 100%) and represent the mean ± SEM of three different experiments. *** p < 0.001; ** p < 0.01: * p < 0.05 vs . basal value. ### p < 0.001; ## p < 0.01 vs. control (in the absence of inhibitor).

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: Effect of erythropoiesis-stimulating agents (ESAs) on HUVEC viability. The cells were treated for 72 h with EPOα (1 IU/mL), DarbEPO (1 IU/mL) or CERA (5 IU/mL) in the absence or presence of 80 μM STAT-5 inhibitor. Cell viability was determined by an MTS assay, as described in the Methods section. The data are expressed as the percent cell viability compared to the untreated basal cells (set to 100%) and represent the mean ± SEM of three different experiments. ** p < 0.01; * p < 0.05 vs. basal value.

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques: MTS Assay

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: Effect of ESAs on HUVEC angiogenesis. The cells were treated with medium alone (basal) or ESAs ( 1 IU/mL EPOα, 1 IU/mL DarbEPO or 5 IU/mL CERA) for 24 h before seeding onto Matrigel with fresh medium. Capillary-like tube formation was observed by microscopy and quantified using the ImageJ program. ( A ) Representative pictures of HUVEC tubule formation on Matrigel after 24 h drug incubation. ( a ) Basal; ( b ) STAT-5 inhibitor; ( c ) 1 IU/mL EPOα; ( d ) 1 IU/mL DarbEPO; ( e ) 5 IU/mL CERA (original magnification = 10×). ( B ) The number of mesh-like structures were quantified and expressed as a percent of the control sample. The data are expressed as the mean ± SEM of three independent experiments performed in duplicate. *** p < 0.001; ** p < 0.01; * p < 0.05 vs. basal value.

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques: Microscopy, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: EPOR desensitization. ( A ) Time-dependence: cells were pre-treated with medium alone (control) or 1 IU/mL EPOα, 1 IU/mL DarbEPO or 5 IU/mL CERA for different periods of time ranging from 5 to 120 min. ( B ) Concentration-dependence: cells were pre-treated with medium alone (control) or different EPOα, DarbEPO or CERA concentrations (0.5–10 IU/mL) for 60 min. The cells were then washed and stimulated by the addition of 1 IU/mL EPOα, 1 IU/mL DarbEPO or 5 IU/mL CERA for 5 min. STAT-5 phosphorylation levels were quantified using an ELISA kit (see Materials and Methods). The data are expressed as the percent STAT-5 phosphorylation compared to the untreated control cells (set to 100%) and represent the mean ± SEM of three different experiments.

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: EPOR resensitization following 1 IU/mL EPOα- or DarbEPO-induced receptor desensitization. HUVECs were pre-treated with medium alone (control) or 1 IU/mL EPOα ( A ) or 1 IU/mL DarbEPO ( B ) for 18 or 54 min to induce receptor desensitization. Then, cells were the washed and replaced in medium alone for different periods of time (2 to 24 h) to allow for receptor resensitization. All samples were then stimulated by the addition of 1 IU/mL EPOα or 1 IU/mL DarbEPO for 5 min and STAT-5 phosphorylation levels were quantified. The data are expressed as the percent STAT-5 phosphorylation compared to the untreated control cells (set to 100%) and represent the mean ± SEM of three different experiments. ** p < 0.01; * p < 0.05 vs. DES’.

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: EPOR resensitization following 3 IU/mL EPOα-, 3 IU/mL DarbEPO- or 5 IU/mL CERA-induced receptor desensitization. HUVECs were pre-treated with medium alone (control) or 3 IU/mL EPOα ( A ), 3 IU/mL DarbEPO ( B ) or 5 IU/mL CERA ( C ) for 18 or 54 min to induce receptor desensitization. The cells were then washed and replaced in medium alone for different periods of time (2 to 24 h) to allow for receptor resensitization. All samples were then stimulated by the addition of 3 IU/mL EPOα, 3 IU/mL DarbEPO or 5 IU/mL CERA for 5 min, and STAT-5 phosphorylation levels were quantified. The data are expressed as the percent STAT-5 phosphorylation compared to the untreated control cells (set to 100%) and represent the mean ± SEM of three different experiments. ** p < 0.01; * p < 0.05 vs. DES’.

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques:

Journal: PLoS ONE

Article Title: Nogo-66 Promotes the Differentiation of Neural Progenitors into Astroglial Lineage Cells through mTOR-STAT3 Pathway

doi: 10.1371/journal.pone.0001856

Figure Lengend Snippet: (A) Gel imagine of RT-PCR for NgR mRNA expression in NPCs. (B) Immunocytochemistry for NgR (green) expression in NPCs. Nuclei were stained by PI (shown in red). (C) PI-PLC and NEP1-40 rescued the astroglial induction by Nogo-66. After PI-PLC or NEP1-40 treatment for 2 hours, the proportion of GFAP positive cells induced by Nogo-66 was significantly lower than that induced only by Nogo-66 treatment. ( p <0.01). (D) The statistical result of GFAP positive cells percentage after Y27632 (RhoA-ROCK inhibitor) treatment. The astroglial induction of Nogo-66 was not rescued by Y27632. (E) Rapamycin and AG490 rescued the astroglial induction by Nogo-66. After rapamycin or AG490 treatment for 2 hours, the proportion of GFAP positive cells induced by Nogo-66 was recovered. (F) Nogo-66 activated the phosphorylation of STAT3 at Ser727 and Tyr705 and phosphorylation of mTOR. After starved for 24 hours in serum-free DMEM medium, total cell lysates from NPCs treated with GST (G) or Nogo-66 (N) (100 nM) for the indicated time were immunoblotted and probed with the indicated antibodies. (G) After starved for 24 hours in serum- free DMEM medium, total cell lysates from NPCs not treated (−) or treated (+) with PI-PLC (1 U/ml) or NEP1-40 for 2 hours and then stimulated with Nogo-66 (100 nM) for 30 minutes were immunoblotted and probed with the indicated antibodies. PI-PLC and NEP1-40 could rescue the phosphorylation of STAT3 activated by Nogo-66. (H) Y27632 did not alter the phosphorylation of STAT3 activated by Nogo0-66. After starved for 24 hours in serum-free DMEM medium, total cell lysates from NPCs not treated (−) or treated (+) with Y27632 (10 uM) for 2 hours and then stimulated with Nogo-66 (100 nM) for 30 minutes were immunoblotted and probed with the indicated antibodies. (I) Rapamycin could inhibit the activated phosphorylation of STAT3 induced by Nogo-66. AG490 strongly inhibited phosphorylation of STAT3 at Tyr705. After starved for 24 hours in serum-free DMEM medium, total cell lysates from NPCs not treated (−) or treated (+) with rapamycin (50 uM) or AG 490 (3ug/ml) for 2 hours and then stimulated with Nogo-66 (100 nM) for 30 minutes were immunoblotted and probed with the indicated antibodies. (J) Rapamycin treatment decreased the complex formation between mTOR and Stat3 in NPCs at the presence of Nogo-66. Nondenatured whole cell lysates were immunoprecipitated with an mTOR antibody before western blot analysis using anti-STAT3 and mTOR, respectively. Data are mean±S.E. Error bars indicate SE. * p <0.05 ** p <0.01 (n = 3).

Article Snippet: For immunoblotting, primary antibodies were diluted as follow: NeuN, β III tubulin, α-tubulin, P-STAT3 (Ser727), P-STAT3 (Tyr 705), STAT3 (CST), P-mTOR and mTOR (CST) at 1∶1000; Nogo-A at 1∶300, MBP at 1∶200, GFAP 1∶100.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Immunocytochemistry, Staining, Phospho-proteomics, Immunoprecipitation, Western Blot