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. Author manuscript; available in PMC: 2011 Jan 1.
Published in final edited form as: Nat Cell Biol. 2010 Jun 6;12(7):676–685. doi: 10.1038/ncb2070

Figure 6. NOX1-RAC1 complex mediates CCN1-induced senescence.

Figure 6

(a) BJ cells were preincubated for 1 h with inhibitors of 5-LOX (MK886, 5 μM), NADPH oxidase (apocynin, 10 μM) or vehicle (0.1% DMSO), followed by CCN1 treatment for 3 days and SA-β-gal expression was determined. Chemical inhibitors were replenished daily. (b) Cells were infected with lentiviruses encoding shRNAs against 5-LOX (sh5LOX #1 and #2). Knockdown was assessed by immunoblotting (left) and cell proliferation after CCN1 treatment was monitored by counting cell numbers (right). (c) Cells treated with CCN1 for various times were assayed for expression of NOX1, NOX4, catalase and β-actin by RT-PCR. (d) Lentiviral shRNA-mediated knockdown of NADPH oxidase 1 (shNOX1 #1 and #2) and 4 (shNOX4) was accomplished as above and confirmed by RT-PCR (top). Proliferation of infected cells after CCN1 treatment was determined by counting cell numbers (bottom). (e) Lentiviral shRNA knockdown of RAC1 (shRAC1) was confirmed by immunoblotting (top), and proliferation of knockdown cells after CCN1 treatment was assessed (bottom). All experiments were done in triplicates and data presented as means ± S.D.