Figure 3.

Inhibited expression of the RB tumor suppressor gene augments induction of PNECs by DAPT. (A) Schematic of PNEC production from hESC cells carrying a tetracycline-inducible transgene that produces shRNA targeting RB1 (shRB). The format is similar to Fig. 1 A, except that mixture VI is supplemented with DAPT (5 µM) with or without DOX. Internal colored circles at day 55 indicate PNECs induced by DAPT (red) or by DAPT and DOX (blue). AFE, anterior foregut endoderm. (B–D) Increased numbers of putative PNECs detected by costaining for CGRP and NKX2.1 (B) or by FACS sorting with anti-human CGRP antibody (C and D) as described in the legend to Fig. 1 and the Materials and methods section. **, P < 0.01 by one-way ANOVA test. In D, horizontal red lines denote average values; biological repeats (n) = 20. Scale bars, 100 µm (left) and 20 µm (right). (E) Expression of shRNA-RB increases the percentages of ASCL1⁺, CHGA⁺, or NCAM⁺ cells. The indicated markers were detected by immunostaining as in B. Scale bars, 100 µm and 20 µm (in small window).