Fig. 5.

Genes and miRNAs regulated by p73 and mTOR are associated with MSC differentiation. (A) Analysis of p73 binding level to select target genes by ChIP-qPCR, performed in human MSCs. Error bars indicate SD from triplicate analysis. (B) p73/mTOR-regulated genes, p73-bound genes (ChIP-on-Chip), and genes significantly altered during 5AZA-induced myogenic differentiation of MSCs (multiple testing, corrected P value < 0.05) were compared. MSC microarray data were publicly available (17). The Venn diagram shows the number of Affymetrix probes in each category. (C) Treatment of MSCs with 5AZA results in decreased mTOR activity as shown by Western blot analysis of pS6, total S6, and actin levels. (D) This core set of nine direct p73 target genes from those regulated by 5AZA was sufficient to segregate ARMS and ERMS. Fold-changes are indicated in two bar charts for: 5AZA-treated MSCs relative to control (Left), and ARMS relative to ERMS (Right). (E) Quantitative RT-PCR was performed in MSCs treated with 5AZA, after knock-down of p73 (shp73) or control (shGFP) for the indicated genes, normalized to GAPDH levels and control. (F) Quantitative RT-PCR analysis of mature miR-133b levels was performed in Rh30 cells treated with control RNAi (shGFP), or three different RNAi constructs that target p73 (shp73-1, -2, and -3), normalized to RNU19 levels. Error bars represent SDs from three independent experiments.