Single-cell RNA profiling of RUES2-derived LCs in which NOTCH signaling was blocked by DAPT and RB and P53 protein levels were reduced by shRNA. (A) Schematic of PNEC production from RUES2 cells carrying DOX-inducible transgenes encoding shRNAs against RB and TP53 after exposure to DAPT and DOX (as in Fig. 2 A). AFE, anterior foregut endoderm. (B) Single-cell RNA profiling of day 55 LCs treated with DAPT (5 µM) and DOX from day 25 to 55. Scaled expression of the most differentially expressed genes (horizontal rows) specific to different clusters of cells (vertical columns). (C) PNEC-like cells are principally in cluster 4, but additional cells expressing some PNEC markers are found in other clusters. tSNE map colored by cluster assignment as in Fig. 2 D. (D) Individual cells positive for RNA encoding the indicated PNEC markers and for other proteins associated with neuroendocrine differentiation are denoted with red dots as in Fig. 2 E. (E) Left: A tSNE map of reclustering of the PNEC-enriched cluster only (cluster 4 in C); Right: A scaled expression of the most differentially expressed key genes (horizontal rows) specific to cells in three PNEC subclusters called 0, 1, and 2 on the left (vertical columns). (F) Individual cells positive for PNEC markers and other genes associated with neuroendocrine differentiation are denoted by red dots. (G) Individual cells positive for cell cycle–related genes are denoted by red dots in PNECs in which RB levels were reduced (top row) or both RB and TP53 levels were reduced (bottom row). Biological repeats (n) = 2.