Figure 2: Myc inhibition reduces proliferation and viability of V¹²Ha-Ras neuroprogenitors.

(a) Neuroprogenitor cells freshly isolated from the subventricular zone (SVZ) of adult (3 months old) asymptomatic triple transgenic TRE-Omomyc;CMVrtTA;GFAP-V¹²Ha-Ras control mice were grown as neurospheres in culture in serum-free medium supplemented with growth factors (EGF and FGF). Immunostaining for GFAP (green) and Ki67 (red) are shown. Nuclei were counterstained with Hoechst (blue). (b,c) NPG growth curves. Neuroprogenitors were plated as single cells in 12-well plates and treated with doxycycline (+Dox) or untreated (–Dox) for 3 days. Triple transgenic TRE-Omomyc;CMVrtTA;GFAP-V¹²Ha-Ras cells expressing Omomyc show a significant reduction in the total number of cells after 3 days of treatment, compared with the untreated cultures. (P-value=0.0168, calculated by t-test; b). Notably, 3 days of Omomyc expression in control TRE-Omomyc;CMVrtTA does not affect significantly their growth rate (c). Data are shown as means±s.e. of triplicates (n=2). (d,e) Cell death was assessed by Trypan blue staining. Triple transgenic TRE-Omomyc;CMVrtTA;GFAP-V¹²Ha-Ras NPGs have elevated numbers of dead cells after Omomyc expression for 3 days (d), whereas there is no difference in double transgenic TRE-Omomyc;CMVrtTA NPGs (e). Data are shown as mean+s.e. of triplicates. P-values were calculated by t-test. (f) BrdU staining of triple transgenic TRE-Omomyc;CMVrtTA;GFAP-V¹²Ha-Ras and control TRE-Omomyc;CMVrtTA cells treated with doxycycline for 3 days or untreated. Data represent the mean of triplicates+s.e. (n=3). P-values were calculated by t-test. (g) Omomyc expression impairs the self-renewal ability of triple transgenic TRE-Omomyc;CMVrtTA;GFAP-V¹²Ha-Ras NPGs. Single neuroprogenitor cells were plated at a clonal density (500 per well) in a 12-well plate and treated or not with doxycycline. After 21 days, the number of spheres was evaluated. Data are shown as means+s.e. of triplicates (n=2). P-values were calculated by t-test.