Figure 5.

Validation of iR3 and aR3 in a Primary Cell Culture Model

(A) Cross used to generate embryos to test aptamers on cells with or without FGFR1, FGFR2, and FGFR3. The dorsal neocortex tissue (boxed region) on embryonic day 14.5 was dissected and dissociated to obtain a single-cell suspension of neocortical precursor cells. (B) Western blot analysis using cells from hGFAP:Cre;Fgfr1^Fx/Fx;Fgfr2^Fx/Fx;Fgfr3^Fx/+ embryos treated with FGF2 alone or FGF2 and iR3 (or the control Ctrl.36 aptamer) at a concentration of 1 μmol/L. (C) Quantitation of western blots. There was a significant reduction in pERK when cortical cells were incubated with iR3 (n = 3 embryos/condition; FG2+Ctrl.36 versus FGF2+iR3, p = 0.0046; FGF2+iR3 versus FGF2, p = 0.0008). (D) Western blot of cells as in (C) but without FGF2. (E) aR3 demonstrated a modest but significant increase in pERK. N = 7 embryos; aR3 versus cells only, p = 0.0027; aR3 versus FGF2, p = 0.001; aR3 versus ctrl.36, p = 0.0007.