Figure 3.

β1 Integrins from PrCa Cells Are Required for EV-Mediated Stimulation of Anchorage-Independent Growth

(A) Quantification of anchorage-independent growth of shβ1 or mock PC3 cells with no other treatments. The total number of colonies were counted per field (325 × 250 μm) after 4 weeks in soft agar. p < 0.0000013.

(B) NTA profiles of the EV population isolated from PC3 cells transfected either with shRNA to β1 (shβ1) or an empty vector (mock). Three technical replicates were performed on each sample. The area under the curve represents all detected particles in the sample and an average of three independent readings.

(C) Quantification of anchorage-independent growth of PC3 cells after treatment with EVs from either shβ1 or mock PC3 cells. Data are represented as mean ± SEM. *p < 0.045, **p < 0.0045 (Student's t test).

(D) Representative images of the data quantified in (C) are shown. Two technical and biological replicates were performed.

(E) Immunoblotting of both EVs and total cell lysates (TCL) from shβ1 and mock PC3 cells for β1, TSG101, and calnexin (left panel); and for α5 (right panel); 15 μg protein was loaded per lane, and results were obtained in reducing conditions.

(F) Immunoblotting of EVs from shβ1 or mock PC3 cells for FAK and TSG101; 15 μg protein was loaded per lane.

(G) Immunoblotting for FAK and ERK of TCL from PC3 cells incubated with EVs from shβ1 PC3 cells, mock PC3 cells, or PBS; 40 μg protein was loaded per lane.

See also Figure S1.