Fig. 5.

Osteoblasts mechanosensitivity during FSS is retained by blocking RhoA GTPase signaling and siRNA knockdown of P2Y₂R. The intracellular Ca²⁺ ([Ca²⁺]_i) in MC3T3-E1 cells was measured in response to two consecutive sessions of FSS. A: representative trace of the Ca²⁺ response to FSS showing the peak [Ca²⁺]_i increased within 1 min after the onset of the first bout of FSS (12 dyn/cm²), but no rise was observed during the second bout after a 5-min rest period. B: representative [Ca²⁺]_i trace indicating that when cells were pretreated with 10 μM Y27632 before FSS, two distinct [Ca²⁺]_i spikes were observed during both loading bouts of FSS (12 dyn/cm²). C: bar graph showing that Y27632 inhibition of ROCK and siRNA knockdown of P2Y₂R increased the peak [Ca²⁺]_i during the first and second FSS compared with the control cells first and second peak values, respectively. D: similar to the magnitude of response, the percentage of cells responding to the second application of FSS was significantly higher when treated with 10 μM Y27632 or siRNA knockdown of P2Y₂R. ^aP < 0.05 compared with control cells second response, ^bP < 0.05 compared with control cells during first response.