Fig. 3. Perturbation of Abcg2 expression affects SP cell phenotype of late retinal progenitors. (A-C) E18 retinal progenitors, enriched as neurospheres, were transduced with ABCG2 retrovirus (G1-ABCG2) or empty retrovirus (G1-Control), followed by Hoechst dye efflux assay. There was a significant increase in the proportion of SP cells in G1-ABCG2 retrovirus-transduced neurospheres, compared with controls. (D-F) Neurospheres were transduced with retrovirus to express ABCG2 siRNA (pSuper-ABCG2_siRNA) or missense siRNA (pSuper-ABCG2_missense) as controls, followed by Hoechst dye efflux assay. There was a significant decrease in the proportion of SP cells in pSuper-ABCG2_siRNA retrovirus-transduced neurospheres, compared with controls. (G-L) The specificity of siRNA-mediated attenuation of Abcg2 expression is demonstrated by a decrease in levels of Abcg2 transcript (G) by RT-PCR analysis and ABCG2 protein by western blot analysis (H) and by immunocytochemical analysis (I-L) in pSuper-ABCG2_siRNA-transduced neurospheres, compared with controls. Data are expressed as the mean ± s.e.m. from triplicate cultures of two different experiments. ^***P<0.005 compared with levels in the control. Magnification, 200x.