Fig 1.
Gross structure of human dynAP and its expression in NIH3T3 cells.
(A) Structure of dynAP showing the transmembrane domain (TM), N-glycosylation site (N-gly), threonine/serine rich region (T/S-rich), and the antigen peptide (aa. 20–33) used for raising the rabbit antibody. (B) Immunoblot analyses of dynAP expressed in NIH3T3 cells. Lane 1, NIH3T3LacZ cell lysate and lane 2, NIH3T3dynAP cell lysate. In lane 3, the NIH3T3dynAP cell lysate was analyzed with an antibody that had been pretreated with 10 nM of the antigen peptide at 37°C for 30 minutes. *non-specific.
Fig 2.
In vitro properties of NIH3T3 cells transformed by dynAP.
(A) Proliferation in 2D culture. Cell proliferation was evaluated by the MTT method (left panel) and counting cell numbers (right panel). Error bars show the means ± SD (n = 4). NIH3T3dynAP vs NIH3T3LacZ cells; *p<0.05, p**<0.01. (B) Focus formation. The cells (5 × 104/2 ml/well in 6-well plates) were cultured for 15 days and then stained with crystal violet to visualize foci. (C) Colony formation on soft agar. Colonies with diameters larger than 50 μm were counted on day 18 after plating 2 × 104 cells/dish. Error bars show the means ± SD (n = 3). (D) Spheroid formation in 3D culture. Scanner images (left panels) indicate vigorous formation of spheroids by NIH3T3dynAP cells. Spheroid formation was quantified by measuring the pseudo-volume (area × optical density) (right panel). Error bars show the means ± SD (n = 3). NIH3T3dynAP vs NIH3T3LacZ cells; p < 0.01 at all points. Active spheroid growth of NIH3T3dynAP cells was also observed by fluorescence emitted from EGFP that was expressed in the parental NIH3T3 cells.
Fig 3.
(A) Tumor weights in nude mice that received NIH3T3 cells expressing dynAP, H-Ras, or H-RasdynAP. Tumor weights were measured at the indicated days after injection of the cells. Data were analyzed by the Mann-Whitney test. (B) Gross characteristics of tumors. Tumor tissues derived from NIH3T3dynAP and NIH3T3H-RasdynAP cells were highly vascularized compared with those derived from NIH3T3H-Ras cells.
Fig 4.
Tumor tissues were obtained from mice in the first experiment in Fig 3A. (A) Hematoxylin and eosin staining of tumors derived from NIH3T3dynAP, NIH3T3H-Ras, and NIH3T3H-RasdynAP cells. Arrows indicate capillaries filled with erythrocytes. Cell-cell adhesion appeared to be weak in NIH3T3dynAP cell-derived tumor tissue. (B) Sections stained with an antibody against CD34 (endothelial cell marker) and control sections stained without the antibody. The control sections were sections consecutive to those stained with the antibody. (C) Quantification of the CD34-positive area (left panel) and CD34-positive number (right panel). Data are the means ± SD (n = 9). Tumors obtained from 27 mice (nine mice for NIH3T3dynAP cells, nine for NIH3T3H-RasdynAP cells, and nine for NIH3T3H-Ras cells) were processed to prepare sections. One section of each tumor was randomly selected and two fields of each section were analysed.
Fig 5.
Molecular mechanisms of dynAP-induced transformation of NIH3T3 cells.
(A) Immunoblots of key signaling molecules. Numbers on the right sides indicate relative band intensities of NIH3T3dynAP cells when those of NIH3T3LacZ cells were defined as 1. *non-specific band because its mobility differed from the FOXO3a band. (B) DynAP-induced increases of rictor protein and mRNA expression. β-Actin was used as a control. (C) Rictor knockdown in NIH3T3dynAP cells decreases AktSer473 phosphorylation. Apparent molecular masses of proteins on immunoblots were as follows. dynAP, 42 kDa; Akt, 56 kDa; p53, 53 kDa; S6K, 70 kDa; 4E-BP1, 18 kDa; FOXO3a, 97 kDa; rictor, 200 kDa, β-actin, 45 kDa.
Fig 6.
Rictor knockdown in NIH3T3dynAP cells represses dynAP-induced transformation.
(A) Focus formation in 2D culture. (B) Colony formation on soft agar. (C) Spheroid formation in 3D culture. Left panel shows microscopic images of spheroids, and right panel shows quantification of spheroids. All data are the means ± SD (n = 3).
Fig 7.
Expression of dynAP in NIH3T3 reduces E-cadherin expression and promotes cell motility.
Cell motility was quantified by wound healing assays. Data are the means ± SD (n = 6).