Journal: The Journal of Neuroscience

Article Title: Generation of Functional Radial Glial Cells by Embryonic and Adult Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-37-11587.2003

Figure Lengend Snippet: Regulation of RGC differentiation by growth factor signaling. A-D, Neurospheres grown (G) in FGF-2 (F), EGF (E), or TGFα (T) were differentiated (D) in the presence of FGF-2, EGF, or TGFα, and the percentage of neurospheres extending at least one RGC process was determined. Neurospheres grown in the presence of FGF-2 and differentiated in the presence of FGF-2 gave rise to almost no RGC processes relative to differentiation in the presence of either EGF or TGFα (**p < 0.0001; n = 3; arrows in images indicate RGC processes). Scale bar, 100 μm. Neurospheres grown in EGF and differentiated in FGF-2 also gave rise to very few RGC processes relative to differentiation in EGF (*p = 0.017, paired t test; n = 3); similarly TGFα-grown neurospheres gave rise to very few RGC processes in FGF-2 compared with TGFα (*p = 0.015, paired t test; n = 3). E-G, P-STAT3 was not expressed by RC2-expressing radial cell population of the E14 lateral ganglionic eminence (LGE). Scale bar, 50 μm. H, P-STAT3 expression was observed in the developing cortical plate. I-L, P-MAPK was heavily expressed by RC2-expressing radial cells in the E14 LGE. Scale bar: in MERGE, 20μm. CTX, Cortex. M, Western blot analysis revealed that EGF-derived neurospheres, rinsed in basal media for 6 hr (6 hr time point is time 0) and then pulsed with either FGF-2 or EGF for 15 min, displayed decreased P-MAPK levels in the FGF-2 condition relative to EGF (similar results were obtained in 3 independent experiments). N, The inhibition of MAPK activation by UO126 (an ERK1/2-specific inhibitor) during neurosphere differentiation significantly reduced the percentage that extended RGC processes (**p = 0.0084; paired t test; n = 3). O, P, Of the RC2-expressing cells (green) from dissociated EGF-derived E14 neurospheres, 29.6 ± 6.1% adopted a radial morphology in the presence of EGF+DMSO after 14-16 hr of differentiation (O, arrows). Scale bar, 20 μm. Only 5.63 ± 1.6%, however, adopted a radial morphology in the presence of EGF+U0126 (P; examples of nonradial cells are indicated by arrowheads; Hoechst is shown in blue; paired t test, p = 0.035; n = 3).

Article Snippet: Primary antibodies used were as follows: mouse anti-RC2 (neat; Developmental Hybridoma Bank; 1:100, a generous gift from Dr. P. LePrince, University of Liege, Belgium), mouse anti-nestin (neat; Developmental Hybridoma Bank), rabbit anti-nestin (1:100; a generous gift from Dr. U. Lendahl, Karolinska Institute, Sweden); rabbit anti-GFAP [1:400 (Biomedical Technologies, Stoughton, MA); 1:800 (Sigma)], mouse anti-TuJ1 (1:500; Sigma), mouse anti-Tau (1:100; PharMingen, San Diego, CA), mouse anti-Hu (1:20; Molecular Probes, Eugene, OR), rabbit anti-astrocyte-specific glutamate transporter (GLAST) (1:100; generous gift from M. Watanabe, Hokkaido University School of Medicine, Japan), mouse anti-Vimentin (1:100; Boehringer Mannheim, Mannheim, Germany), rabbit anti-brain lipid binding protein (BLBP) (1:800; generous gift from N. Heintz, Rockefeller University, New York, NY), rabbit anti-phospho signal transducer and activator of transcription-3 (STAT3) (1:100; Cell Biology Technologies), rabbit anti-phospho MAPK (1:100; Cell Biology Technologies), mouse anti-s100β (1:1000; Sigma), rat anti-BrdU (1:50; Harlan Seralab), rabbit anti-FGF receptor-1 (FGFR1) (1:50; Santa Cruz Biotechnology), rabbit anti-FGFR2 (1:50; Santa Cruz Biotechnology), sheep anti-EGFR (1:50, Biodesign), and goat anti-doublecortin (1:200; Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Derivative Assay, Inhibition, Activation Assay

Journal: Oncotarget

Article Title: Adipose-derived mesenchymal stem cells promote osteosarcoma proliferation and metastasis by activating the STAT3 pathway

doi: 10.18632/oncotarget.15866

Figure Lengend Snippet: (A) The transfection efficiency of the siRNA was analysed in OS cells at different time points after transfection using flow cytometry. (B) Western blot analysis of STAT3 and pSTAT3 expression in OS cells treated with ADSC-conditioned medium. OS cells were pretreated with the STAT3 siRNA (50 nM) for 48 h before treatment with ADSC-conditioned medium for an additional 12 h. STAT3 expression levels were detected by western blotting. OS cells treated with Lipofectamine 2000 alone served as negative controls. (C) OS cell invasion was analysed using transwell assay. OS cells were transfected with the siRNA and treated with ADSC-conditioned medium. OS cells treated with ADSC-conditioned medium alone served as positive controls, and OS cells treated with Lipofectamine 2000 alone served as negative controls. (D) The MMP2 and MMP9 levels in the supernatants were detected using ELISAs. (E) OS cells were treated with the STAT3 siRNA or ADSC-conditioned medium, and MMP2/9 and E-cadherin expression in OS cells was examined by western blotting. OS cells treated with Lipofectamine 2000 alone served as negative controls. The results are expressed as mean±SD. Abbreviation: C Control; L Lipofectamine 2000; S siRNA. * P<0.05, ** P<0.01, *** P<0.001.

Article Snippet: Antibody staining with 100 μL of specific antibodies against human STAT3 (1:100 dilution; R&D Systems) and rabbit monoclonal antibodies against human Ki67, MMP2 and MMP9 (1:100 dilution; ProteinTech groups inc, Chicago, IL) was applied to the cells, which were incubated overnight at 4°C.

Techniques: Transfection, Flow Cytometry, Western Blot, Expressing, Transwell Assay, Control

Journal: Oncotarget

Article Title: Adipose-derived mesenchymal stem cells promote osteosarcoma proliferation and metastasis by activating the STAT3 pathway

doi: 10.18632/oncotarget.15866

Figure Lengend Snippet: (A-B) Cell proliferation was evaluated using the CCK8 assay and EdU analysis. OS cells were transfected with the siRNA and treated with ADSC-conditioned medium. OS cells treated with ADSC-conditioned medium alone served as positive controls, and OS cells treated with Lipofectamine 2000 alone served as negative controls. (C-D) OS cells were transfected with the STAT3 siRNA or treated with ADSC-conditioned medium, and apoptosis rates were determined using flow cytometry. OS cells treated with Lipofectamine 2000 alone served as negative controls. The percentages of Annexin V-positive cells are presented in bar charts. *** P<0.001.

Article Snippet: Antibody staining with 100 μL of specific antibodies against human STAT3 (1:100 dilution; R&D Systems) and rabbit monoclonal antibodies against human Ki67, MMP2 and MMP9 (1:100 dilution; ProteinTech groups inc, Chicago, IL) was applied to the cells, which were incubated overnight at 4°C.

Techniques: CCK-8 Assay, Transfection, Flow Cytometry

Journal: Oncotarget

Article Title: Adipose-derived mesenchymal stem cells promote osteosarcoma proliferation and metastasis by activating the STAT3 pathway

doi: 10.18632/oncotarget.15866

Figure Lengend Snippet: (A) An in vivo imaging system was used to monitor OS xenograft luminescence activity, which represented tumour growth and metastasis. (B) Living Image Software was used to analyse tumour bioluminescence intensity weekly. The quantitation of the normalized image counts is shown. (C) Lungs of the tumour-bearing mice were excised, and the bioluminescence intensity was analysed to determine the level of tumour metastasis in the lungs. (D) Survival curves of the three groups are shown, and the median survival of the OS group was 43 days, which was significantly longer than the survival of the OS + conditioned-medium group (25 days, P<0.01). (E) The immunohistochemical analysis of Ki67, STAT3, MMP2 and MMP9 expression in the orthotopic tumour xenografts is shown. (F) Quantitation of the intensity of Ki67, STAT3 and MMP2/9 staining in the xenografts. Scale bar: 25 μm. * P<0.05, ** P<0.01, *** P<0.001.

Article Snippet: Antibody staining with 100 μL of specific antibodies against human STAT3 (1:100 dilution; R&D Systems) and rabbit monoclonal antibodies against human Ki67, MMP2 and MMP9 (1:100 dilution; ProteinTech groups inc, Chicago, IL) was applied to the cells, which were incubated overnight at 4°C.

Techniques: In Vivo Imaging, Activity Assay, Software, Quantitation Assay, Immunohistochemical staining, Expressing, Staining

Journal: Kidney international

Article Title: Lipoxin A4 inhibits TNF-alpha-induced production of interleukins and proliferation of rat mesangial cells.

doi: 10.1111/j.1523-1755.2005.00379.x

Figure Lengend Snippet: Fig. 8. Semiquantitative analysis using UVP-gel densitometry (A) of electrophroretic mobility shift assay (EMSA) for activated signal tran- ducers and activators of transcription (STAT3) (B) in rat mesangial cells. Cultured and serum-deprived glomerular mesangial cells were treated with tumor necrosis factor-a (TNF-a) (10 ng/mL) for 1 hour, with or without preincubation with lipoxin A4 (LXA4) at the indicated concentrations. Thirty micrograms of nuclear protein extracts were pre- pared for detection of STAT3 activity by EMSA with c-[32P]-labeled double-stranded oligonucleotide probe of STAT3. In (B),the upper ar- row denotes the specific STAT3-DNA complexes. In (A), arbitrary unit (AU) = (ATNF−a or ALXA4 or ATNF−a+LXA4/A0.5%FCS) × 100%. Data were mean ± SEM of four independent experiments. ∗P < 0.05 com- pared to the cells treated with TNF-a and 0.5% fetal calf serum (FCS) without LXA4. #P < 0.05 compared to the cells treated with 0.5% FCS alone.

Article Snippet: The 2 lL of nuclear extracts containing 30 lg of total proteins was preincubated with 2 lL of gel shift binding 5× buffer for 10 minutes, followed by the addition of 1 lL of c-[32P]-labeled double-stranded oligonucleotide probes of STAT3 or NF-jB (Santa Cruz Bioechnology) and further incubation for 20 minutes at room temperature.

Techniques: Mobility Shift, Cell Culture, Activity Assay, Labeling

Journal: Kidney international

Article Title: Lipoxin A4 inhibits TNF-alpha-induced production of interleukins and proliferation of rat mesangial cells.

doi: 10.1111/j.1523-1755.2005.00379.x

Figure Lengend Snippet: Fig. 10. Semiquantitative analysis using UVP-gel densitometry (A) of electrophroretic mobility shift assay (EMSA) for activated nuclear factor-jB (NF-jB) (B) in rat mesangial cells. Gel supershift assay (C) demonstrated the specialty of activation of NF-jB by tumor necrosis factor-a (TNF-a). Cultured and growth-arrested glomerular mesangial cells were treated with TNF-a (10 ng/mL) for 1 hour, with or with- out preincubation with lipoxin A4 (LXA4) at the indicated concen- trations. Thirty micrograms of nuclear protein extracts were prepared for detection of NF-jB activity by EMSA with c-[32P]-labeled double- stranded oligonucleotide probe of NF-jB. In (B), the upper arrow de- notes the specific NF-jB-DNA complexes. In (A), arbitrary unit (AU) = (ATNF−a or ALXA4 or ATNF−a+LXA4 or ATNF−a+PDTC/A0.5%FCS) × 100%. Data were mean ± SEM of four independent experiments. ∗P < 0.05 compared to the cells treated with TNF-a and 0.5% fetal calf serum (FCS) without LXA4 and pyrrolidine dithio-carbamate (PDTC). #P < 0.05 compared to the cells treated with 0.5% FCS alone.

Article Snippet: The 2 lL of nuclear extracts containing 30 lg of total proteins was preincubated with 2 lL of gel shift binding 5× buffer for 10 minutes, followed by the addition of 1 lL of c-[32P]-labeled double-stranded oligonucleotide probes of STAT3 or NF-jB (Santa Cruz Bioechnology) and further incubation for 20 minutes at room temperature.

Techniques: Mobility Shift, Activation Assay, Cell Culture, Activity Assay, Labeling

Journal: Cancer biology & therapy

Article Title: Lack of AKT activation in lung cancer cells with EGFR mutation is a novel marker of cetuximab sensitivity.

doi: 10.4161/cbt.19238

Figure Lengend Snippet: Figure 2. Phosphorylation of EGFR, ERK, AKT and STAT3, and expression and mutation status of PTEN in EGFR mutant and wild-type NSCLC cell lines. (A–D) Cells were incubated with/without serum for 24 h, and immunoblotting was done with antibod- ies targeting phosphorylated (p) or total EGFR, ERK and AKT. Densitometric analysis was performed with TotaLab software (Nonlinear Dynamics), and the rela- tive pEGFR/EGFR, pERK/ERK, pAKT/AKT, pSTAT3/STAT3 ratios are indicated as bar graphs under each blot. Experiments were repeated three times and represen- tative results are shown. (E) PTEN protein status in each cell line was determined by immunoblotting in the presence or absence of serum. The β-actin was used as loading control. Experiments were repeated three times and representative results are shown. The muta- tion status of PTEN in each cell line is also indicated. WT, wild type.

Article Snippet: The following primary antibodies were used: pEGFR (Tyr 1068), pAKT (Ser 473), AKT, pSTAT3 (Tyr 705), STAT3, PTEN (Cell Signaling, MA), EGFR (1005) (Santa Cruz), β-ACTIN (Sigma), pERK and ERK (Promega).

Techniques: Phospho-proteomics, Expressing, Mutagenesis, Incubation, Western Blot, Software, Control