Figure 3

CCN5 RNAi reduces heparin sensitivity of VSMC. 8 × 10³ VSMC/well, previously untreated or treated with control siRNA or CCN5 siRNA, were washed with RPMI and placed in RPMI containing 0.4% FCS for 72 h. Cells were released from quiescence by replacing the low serum medium with normal growth medium, RPMI-1640 containing 10% FCS. A range of heparin doses was added to the medium (10–300 μg/ml). After 4 days of growth, cell proliferation was determined by direct cell counting using a Coulter Counter. The net growth of VSMC was obtained by subtracting the starting cell number from the final count results (at the time of release from G₀).