Journal: Journal of cellular physiology

Article Title: Novel role of Giα2 in cell migration: Downstream of PI3-kinase-AKT and Rac1 in prostate cancer cells.

doi: 10.1002/jcp.26894

Figure Lengend Snippet: FIGURE 1 Knockdown of endogenous Giα2 and RGS2 overexpression negatively regulate migration and invasion of PC3 cells in response to diverse stimuli. (a) Western blot analysis of Giα2 in PC3 cells lysates to validate the knockdown of endogenous Giα2 protein using siRNA. β‐Actin was used as an internal control. (b) Cell migration of PC3 cells after transfection with control siRNA or Giα2 siRNA in response to SDF‐1α (100 nM), TGFβ1 (5 ng/ml), or EGF (10 ng/ml). Results are expressed as migration index defined as the average number of cells per field for the ligand tested/the average number of cells per field for the vehicle control. Each bar represents mean ± SEM (n = 3). Significant differences (p < 0.05) among different groups are represented with different lowercase letters. (c) Invasive behavior of PC3 cells transfected with control siRNA and Giα2 siRNA in response to SDF‐1α (100 nM), TGFβ1 (5 ng/ml), or EGF (10 ng/ml). Results are expressed as invasion index defined as average number of cells per field for the ligand tested/the average number of cells per field for the vehicle control. Each bar represents mean ± SEM (n = 3). Significant differences (p < 0.05) among different groups are represented with different lowercase letters. 5% FBS was used as a positive control. (d) The upper panel shows western blot analysis of HA‐tag and RGS2 in cell lysates to validate the overexpression of RGS2 protein. β‐Actin was used as an internal control. Cell migration in PC3 cells transfected with control or RGS2 overexpression plasmids in response to stimulation with OXT (100 nmol/L), TGFβ1 (5 ng/ml), or EGF (10 ng/ml). Results are expressed as migration index. Each bar represents mean ± SEM (n = 3). *Significantly different (p < 0.05) when compared with appropriate controls. EGF: epidermal growth factor; FBS: fetal bovine serum; OXT: oxytocin; siRNA: small interfering RNA; SEM: standard error of the mean; TGFβ1: transforming growth factor β1

Article Snippet: Control (sc‐37007) and Giα2‐targeting (sc‐41752) small interfering RNAs (siRNAs), transfection reagent (sc‐29528), rabbit polyclonal anti‐Giα2 antibody (sc‐7276), and mouse β‐actin antibody were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Knockdown, Over Expression, Migration, Western Blot, Control, Transfection, Positive Control, Small Interfering RNA

Journal: Journal of cellular physiology

Article Title: Novel role of Giα2 in cell migration: Downstream of PI3-kinase-AKT and Rac1 in prostate cancer cells.

doi: 10.1002/jcp.26894

Figure Lengend Snippet: FIGURE 4 Knockdown of endogenous Giα2 has differential effects on PI3‐kinase activation and cell migration in response to diverse ligands. (a) Western blot analysis of p‐AKT and p‐EGFR in PC3 cells lysates, transfected with control siRNA or Giα2 siRNA in response to OXT (100 nmol/L), TGFβ1 (5 ng/ml), or EGF (3 ng/ml). Total AKT and EGFR served as loading and normalization controls. (b) Quantitative analysis of p‐AKT activation in PC3 cells. Data is presented as mean ± SEM (n = 3) and analyzed by ANOVA and Duncan’s modified range tests. Significant differences between groups in a given category (p < 0.05) are designated with different lowercase letters. (c) Cell migration in PC3 cells after transfection with control siRNA or Giα2 siRNA transfected cells in response to OXT (100 nmol/L), TGFβ1 (5 ng/ml), or EGF (10 ng/ml). Results are expressed as migration index. Each bar represents mean ± SEM (n = 3). Significant differences (p < 0.05) among different groups are represented with different lowercase letters. ANOVA: analysis of variance; EGF: epidermal growth factor; OXT: oxytocin; siRNA: small interfering RNA; SEM: standard error of the mean; TGFβ1: transforming growth factor β1

Article Snippet: Control (sc‐37007) and Giα2‐targeting (sc‐41752) small interfering RNAs (siRNAs), transfection reagent (sc‐29528), rabbit polyclonal anti‐Giα2 antibody (sc‐7276), and mouse β‐actin antibody were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Knockdown, Activation Assay, Migration, Western Blot, Transfection, Control, Modification, Small Interfering RNA