Figure 5. Sustained ROS accumulation induced through CCN1-α₆β₁ interaction is required for senescence.

(a) BJ cells were pretreated with NAC (2.5 mM) for 1 h followed by the addition of CCN1. NAC was replenished daily and cell numbers were counted, and (b) SA-β-gal expression was evaluated after 3 days. (c) Cells were pre-treated with function-blocking mAbs against α_vβ₃ or α₆ (50 μg/ml each) for 1 h, followed by CCN1 treatment for an additional 1 h and stained with H₂DCFDA (10 μM). ROS was quantified by fluorescence measurements. (d) Cells were adhered to surfaces coated with PLL or ECM proteins including FN, VN, LN, Collagen I (Col1; 10 μg/ml), and CCN1 as in Fig. 3a, and ROS levels were measured after 1 or 4 h as above. (e) Cells were treated with CCN1 and SA β-gal expression was assayed after 3 days. NAC (2.5 mM) was added either 1 h before or 3, 6, 10, and 24 h after CCN1 treatment. All experiments were done in triplicates and data presented as means ± S.D.