Inhibition of PI3K activity has no effect on BMP7-induced Lhx2/9 expression. (A) [i] explants treated with BMP7 or BMP6 (20 ng/ml) with or without 2.5 μM LY and labeled with α-pSmad1/5/8 (upper panels) and α-Lhx2/9 (lower panels). Scale = 20 μm. (B) Quantification of Lhx2/9+ cells/100 μm 2 in [i] explants: BMP7 - LY = 7.7 ± 1.0 cells; BMP7 + LY = 7.7 ± 1.9 cells; BMP6 - LY = 8.6 ± 1.6 cells; BMP6 + LY = 6.8 ± 1.7 cells; (n = 2) (mean ± SEM for each condition). LY had no effect on Lhx2/9 expression induced by either BMP7 or BMP6 (BMP7 - LY versus BMP7 + LY, P = 0.9997; BMP6 - LY versus BMP6 + LY, P = 0.4942 (Student's t-test)). (C) Lhx2/9-labeled (red) [d] explants co-cultured with control- or BMP-7-expressing COS-1 cells with or without 2.5 μM LY. Double-headed arrows mark the width of the control and induced Lhx2/9-expression regions. Scale = 50 μm. (D) Quantification of Lhx2/9 induction in [d] explants, measured as in Figure 3C: pMT23 - LY = 4.48 ± 0.44 (n = 8); pMT23 + LY = 4.85 ± 0.55 (n = 8); BMP7 - LY = 14.02 ± 1.0 (n = 17); BMP7 + LY = 12.42 ± 0.78 (n = 18). Mean × 105 ± SEM for each condition. Student's t-tests: there was no difference in the expression levels of Lhx2/9 with or without LY in either control [d] explant co-cultures (pMT23 - LY versus pMT23 + LY, P = 0.8002) or BMP7-induced [d] explants (BMP7 - LY versus BMP7 + LY, P = 0.5978).