Figure 7.

Requirement of reactive oxygen species (ROS) production by p66Shc in dihydrotestosterone (DHT)-induced LNCaP cell proliferation. (a) LNCaP cells were transfected with wild-type p66Shc cDNA (WT) or W134F mutant or empty vector alone (pcDNA). Cells were steroid starved for 48 h and treated with 10 nM DHT as described above. Cells were trypsinized and counted after 48 h of treatment (*P<0.05). (b) Immunoblot analysis was performed for Cyclin D1 level in LNCaP cells transiently overexpressing WT or W134F mutant in the presence or absence of DHT, as mentioned above. (c) MDA PCa2b cells were plated at 4 × 10⁴ cells per cm² and transiently transfected WT and W134F mutant of p66Shc and treated with DHT, as mentioned earlier. Seventy-two hours after DHT treatment cells were trypsinized and counted (*P<0.05). (d) Small interfering RNA (siRNA) fragments specific to p66Shc cloned in pSUPER vector (pSUP-p66) was transiently transfected into LNCaP cells as per the conditions mentioned for cDNA transfection. Cells were treated with DHT for 48 h and cell growth was measured (*P<0.01). (e) Immunoblot analysis was performed for Cyclin D1 levels in LNCaP cells transiently transfected with p66Shc-specific siRNA (pSUP-p66).