Figure 8. Cortactin-dependent oncogenic... | Rockefeller University Press

Figure 8.

$Figure 8. Cortactin-dependent oncogenic phenotypes are controlled by exosomes. (A) Cells were cultured in serum-free medium (SFM) or SFM supplemented with equal concentrations (50 µg/ml, calculated to be 35 × 108 exosomes per well) of control (+SC Exo or +shLacZ Exo) or cortactin-KD or Rab27a-KD UC exosomes, as indicated, for 4 d. Relative number of cells compared with control cells (SFM condition) shown as mean ± SE, n = 3 independent experiments with triplicate wells. (B) 48 h invasion across Matrigel-coated invasion chambers in the presence or absence of equal concentrations (50 µg/ml) control or KD UC exosomes. Cells per image were averaged for each replicate. n = 3 independent experiments with duplicate wells. (C) WB of exosome markers in control and cortactin-KD total cell lysates (TCL), conditioned medium (CM), microvesicles (MV), UC exosomes, or density gradient (DG) fractions 1–12. Note the presence of exosome markers in fraction 7. (D) Representative (from n = 3) NTA size distribution traces of UC exosomes and DG exosome fraction 7. (E) Representative TEM images of DG exosome fraction 7 preparation from scrambled control (Sc) cells. (F) Table outlining exosome doses used in experiments G–I and K. (G) Serum-independent growth of control (Sc) and cortactin-KD1 cells cultured in the presence of various concentrations (12–120 × 106, indicated below) of UC or DG exosomes, or MVs derived from control cells. n = 3 independent experiments with triplicate wells. (H) 48 h invasion across Matrigel-coated invasion chambers in the presence of various concentrations of control cell UC or DG exosomes, or MV. n = 3 independent experiments with duplicate wells. (I) Serum-independent growth of control (Sc) and cortactin-KD1 cells cultured in the presence or absence of exosomes purified by 70 min ultracentrifugation from Sc cells cultured in growth factor–free medium (GFFM). n = 3 independent experiments with triplicate wells. (J) WBs of whole-cell lysate and UC exosomes derived from SCC61 cells shLacZ (control) and MT1-MMP knockdown (MT1-sh1 and sh3). Note active MT1-MMP at ∼55 kD; *, nonspecific bands; #, degradation forms (K). Bar graphs represent mean ± SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001; determined by Student’s t test.$

Cortactin-dependent oncogenic phenotypes are controlled by exosomes. (A) Cells were cultured in serum-free medium (SFM) or SFM supplemented with equal concentrations (50 µg/ml, calculated to be 35 × 10⁸ exosomes per well) of control (+SC Exo or +shLacZ Exo) or cortactin-KD or Rab27a-KD UC exosomes, as indicated, for 4 d. Relative number of cells compared with control cells (SFM condition) shown as mean ± SE, n = 3 independent experiments with triplicate wells. (B) 48 h invasion across Matrigel-coated invasion chambers in the presence or absence of equal concentrations (50 µg/ml) control or KD UC exosomes. Cells per image were averaged for each replicate. n = 3 independent experiments with duplicate wells. (C) WB of exosome markers in control and cortactin-KD total cell lysates (TCL), conditioned medium (CM), microvesicles (MV), UC exosomes, or density gradient (DG) fractions 1–12. Note the presence of exosome markers in fraction 7. (D) Representative (from n = 3) NTA size distribution traces of UC exosomes and DG exosome fraction 7. (E) Representative TEM images of DG exosome fraction 7 preparation from scrambled control (Sc) cells. (F) Table outlining exosome doses used in experiments G–I and K. (G) Serum-independent growth of control (Sc) and cortactin-KD1 cells cultured in the presence of various concentrations (12–120 × 10⁶, indicated below) of UC or DG exosomes, or MVs derived from control cells. n = 3 independent experiments with triplicate wells. (H) 48 h invasion across Matrigel-coated invasion chambers in the presence of various concentrations of control cell UC or DG exosomes, or MV. n = 3 independent experiments with duplicate wells. (I) Serum-independent growth of control (Sc) and cortactin-KD1 cells cultured in the presence or absence of exosomes purified by 70 min ultracentrifugation from Sc cells cultured in growth factor–free medium (GFFM). n = 3 independent experiments with triplicate wells. (J) WBs of whole-cell lysate and UC exosomes derived from SCC61 cells shLacZ (control) and MT1-MMP knockdown (MT1-sh1 and sh3). Note active MT1-MMP at ∼55 kD; *, nonspecific bands; #, degradation forms (K). Bar graphs represent mean ± SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001; determined by Student’s t test.