FIGURE 8.

Validation of Bmyc regulon. (A), Gene set activity plot displays Bmyc regulon expression in scRNA-Seq dataset. The more red a dot is, the greater the regulon activity in that given cell. (B) Bmyc binding motifs for Bmyc downstream targets (Cd44, Met, Pax3). (C) Feature plots demonstrating expression of Bmyc and Pax3 in the intermediate cell cluster of the scRNA-Seq dataset. Maximum expression (normalized counts) is noted in the black outlined gray box for each gene in the lower left corner of each feature plot. Histogram is depicted from 0 to maximum (Max) in terms of normalized counts. Feature plots for Cd44 and Met have been shown previously in Figure 2. (D) smFISH demonstrates co-expression of Cd44, Met, and Pax3 transcripts in red with Bmyc transcripts in green in intermediate cells on representative cross-sections of the P30 mouse SV. Panels to left depict Bmyc transcripts (green) and downstream target transcripts (red) with DAPI. Panels to the right depict Bmyc transcripts (green) and downstream target transcripts (red) without DAPI. DAPI (4’,6-diamidino-2-phenylindole). White arrows point to examples of intermediate cells that exhibit both Bmyc and downstream target transcript expression. In the panels without DAPI, examples of cells that exhibit co-expression of Bmyc (green) and downstream target transcripts (red) are encircled with dashed yellow lines. All scale bars 20 μm. (E) Enlarged images of example cells from (D) are shown. White arrows in (E) point to encircled cells from (D).