Journal: Endocrine-Related Cancer

Article Title: Anosmin-1 contributes to brain tumor malignancy through integrin signal pathways

doi: 10.1530/ERC-13-0181

Figure Lengend Snippet: Effects of anosmin-1 in tumor cell motility. (A) Serum-starved cells were treated with either SFM (negative control), 10 nM recombinant anosmin-1, or FBS (positive control). The average moving distance (μm) of 20 random cells tracked over 20 h are shown. Error bars indicate s.e.m. from five independent experiments. The P values calculated by two-way ANOVA between the SFM and anosmin-1-treated groups in each cell line are 0.0175 (LN229), 0.0037 (A172), and 0.0399 (U87MG), where * P ≤0.05 or ** P ≤0.01 is considered significant. (B) Effects of KAL1 knockdown on A172 cell motility. The P values obtained from three independent experiments are 0.0395 (for shRNA 673), 0.0253 (for shRNA 675), and 0.0015 (for shRNA 676) when compared with the nontargeting control shRNA. (C) As indicated, LN229 cells were pretreated with chemical inhibitors or specific antibodies for 30 min before addition of anosmin-1 (labeled A). Only anosmin-1 treatment alone or with nonspecific IgG resulted in a significant increase in motility. Error bars indicate s.e.m . from three independent experiments. (D) LN229, A172, and U87MG cells endogenously express anosmin-1, uPA, and FGFR1 proteins at variable levels. See Supplementary Table 3 for the mRNA levels of each gene. (E) KAL1 -shRNAs significantly knocked down the endogenous anosmin-1 protein as assessed by two different anti-anosmin-1 (mouse or rabbit polyclonal) antibodies. (F) Knockdown efficacy of each shRNA is indicated as the percentage of the remaining KAL1 mRNA assessed by qRT-PCR, compared with control shRNA, which was significant (*** P ≤0.0001) in all three shRNAs. qRT-PCR was performed in triplicates, from four independent experiments. Error bars indicate the s.e.m .

Article Snippet: The serum-starved cells were incubated with various treatments for 18 h, and cell movements were recorded for further 20 h. The cells were treated with 5% fetal bovine serum (FBS), 50 μM amiloride, 25 μM SU5402, anti-uPA or anti-FGFR1 ectodomain antibodies, and nonspecific mouse IgG (Santa Cruz Biotechnology) at 10 μg/ml.

Techniques: Negative Control, Recombinant, Positive Control, Knockdown, shRNA, Control, Labeling, Quantitative RT-PCR

Journal: Endocrine-Related Cancer

Article Title: Anosmin-1 contributes to brain tumor malignancy through integrin signal pathways

doi: 10.1530/ERC-13-0181

Figure Lengend Snippet: Interaction of anosmin-1 with β1 integrin activates downstream signal pathways. (A) Anosmin-1 co-immunoprecipitated with β1 integrin was identified by probing with anti-His or anti-GFP antibody in LN229 cells transfected with pHis-KAL, pKAL-GFP (+), or empty vector (−). Integrin β1 precipitated by anti-β1 antibody or nonspecific mouse IgG is shown as positive and negative control respectively. (B) Immunofluorescence staining of active integrin β1 (red) in LN229 cells expressing EGFP-tagged anosmin-1 (green). Nuclei were labeled with Hoechst (blue). The colocalization points are also shown (white). A display color-scatter plot is shown of red intensities (Ch1) vs green intensities (Ch2), with the pixels representing the actual color in the image and yellow indicating colocalization. Manders Overlap Coefficient was 0.83, where 1 represents perfect colocalization and 0 represents no colocalization ( Manders et al . 1992 ). On the right panel, an independent image demonstrating anosmin-1 localization at the leading edge of a polarized migrating cell. Scale bar is shown. (C) Induction of p-FAK, p-AKT, and p-ERK upon anosmin-1 treatment in serum-starved LN229 cells at the time points indicated. A172 lysate is included as a positive control for constitutive anosmin-1 expression. The ratio of phosphorylated vs total protein determined by densitometry is shown as fold induction compared with the control. All western blots were repeated twice. (D) Effects of FAK inhibitor (PF-228) on anosmin-1-induced motility. Anosmin-1 significantly increased LN229 cell motility (* P =0.0302 in anosmin-1 recombinant protein treated, and * P =0.0208 in HisKAL-transfected). The pretreatment with increasing concentrations of PF-228, but not with the solvent (DMSO), inhibited the effect of anosmin-1 in a dose-dependent manner. PF-228 alone reduced the basal level motility in LN229, as similarly reported in other cancer cell lines ( Slack-Davis et al . 2007 ). Error bars indicate s.e.m. from four independent experiments (**, P ≤0.01; ***, P ≤0.001).

Article Snippet: The serum-starved cells were incubated with various treatments for 18 h, and cell movements were recorded for further 20 h. The cells were treated with 5% fetal bovine serum (FBS), 50 μM amiloride, 25 μM SU5402, anti-uPA or anti-FGFR1 ectodomain antibodies, and nonspecific mouse IgG (Santa Cruz Biotechnology) at 10 μg/ml.

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Negative Control, Immunofluorescence, Staining, Expressing, Labeling, Positive Control, Control, Western Blot, Recombinant, Solvent

Journal: Endocrine-Related Cancer

Article Title: Anosmin-1 contributes to brain tumor malignancy through integrin signal pathways

doi: 10.1530/ERC-13-0181

Figure Lengend Snippet: Effects of anosmin-1 on cell adhesion and survival. (A) The percentage of cells adhered to the fibronectin-coated plates after 1 h at 37 °C was quantified and normalized to the nonspecific total adhesion on the poly- l -lysine-coated plate. LN229 His-KAL cells show 23% reduction (** P =0.0044) and U87MG KAL-GFP show 21% reduction (** P =0.0011) in cell adhesion, compared with the empty vector control. Experiments were performed in quintuplicate and repeated five times. Expression of the transfected anosmin-1 constructs is confirmed by anti-His or anti-GFP antibodies. (B) Effects of KAL1 knockdown on apoptosis. Caspase3/7 activity of A172 cells infected with shRNA was measured in relative light units and the average fold induction from four independent experiments is shown. P values are 0.0504 (for shRNA 673), 0.0086 (for shRNA 675), and 0.0002 (for shRNA 676). (C) Effects of KAL1 knockdown in phosphorylation status of FAK, AKT, and ERK in A172 cells. The relative ratio of phosphorylated vs total protein assessed by densitometry is shown as fold induction compared with the control shRNA, normalized to β-actin loading control. (D) Induction of PARP protein cleavage by KAL1 knockdown. The full length PARP protein is assessed by western blot in shRNA-infected A172 cells before (FBS) and after serum-starvation (SFM). The densitometry ratio is shown as fold induction compared with the control shRNA, normalized to the β-actin loading controls.

Article Snippet: The serum-starved cells were incubated with various treatments for 18 h, and cell movements were recorded for further 20 h. The cells were treated with 5% fetal bovine serum (FBS), 50 μM amiloride, 25 μM SU5402, anti-uPA or anti-FGFR1 ectodomain antibodies, and nonspecific mouse IgG (Santa Cruz Biotechnology) at 10 μg/ml.

Techniques: Plasmid Preparation, Control, Expressing, Transfection, Construct, Knockdown, Activity Assay, Infection, shRNA, Phospho-proteomics, Western Blot