C. Cycling PC12

1. C. Cycling PC12 B. Neuronal PC12 eGFP eGFP Si(p130) Si(p130)+p130(h) Si(p130)+p130(h) Si(p130) Percent survival Percent survival Days after 0 1 2 3 4 5 Days after 0 1 2 3 4 5 A. Efficacy of siRNA constructs Hs-Rb Hs-Rb(r) si-Rb Hs-p130 Hs-p130(r) si-p130 - + - - - - - + + - - - - - - - + - - - - - + + + - - - - - + - + - - - - - - + - - - - - + - + Fig S2 Efficacy of si-Rb and si-p130 constructs in down-regulation of their perspective targets and loss of p130 evokes death of neuronal PC12 cells but not cycling PC12 cells. (A) si-Rb and si-p130 against rat Rb and p130 had no effect on expression of human Rb and p130 but down-regulated those when the target regions of the human genes were replaced with the target sequences of the rat genes. eGFP and indicated constructs were co-transfected into CHO cells. When siRNA was absent, an equal amount of empty vector (pSuppressor) was employed. Three days later, cells were fixed and eGFP-expressing cells were assessed for co-expression of tagged Rb or p130. (B) siRNA-mediated loss of p130 causes death of neuronal PC12 cells. Experiments were carried out as in Fig 3C except that neuronal PC12 cells were used and counts were obtained out to 5 days. Data are given as means ± SEM from 3 independent experiments. (C) siRNA-mediated loss of p130 expression has little effect on survival of cycling PC12 cells. Experimental details are as in A except cycling, NGF-untreated PC12 cells were used.