Figure 4

PRMT5 inhibition induces alternative splicing of MDM4 and p53 pathway activation. (A) MDM4 splicing time course evaluating relative abundances of MDM4-FL and MDM4-S isoforms in Z-138 following 1, 2, or 3 days of GSK3203591 treatment at concentrations of 200 nM or 1 μM using ethidium bromide gel electrophoresis. (B) MDM4 splicing dose response in Z-138 following 3 days of GSK3203591 treatment using ethidium bromide gel electrophoresis. (C) Western blot dose response for p53 and p21 protein levels in Z-138 following 3 days of GSK3326595 treatment. (D) Gene expression EC₅₀ values in Z-138 cells treated with GSK3326595 for 2 or 4 days. Gene panel was selected based on results of RNA-sequencing. Representative dose-response curves for CDKN1A (days 2 and 4, left panel) and gene panel EC₅₀ summary table (day 4, right panel) are shown. (E) MDM4 splicing analysis (via ethidium bromide gel electrophoresis) and p53/p21 induction analysis (via Western blot) in a panel of p53 wild-type (black text) and mutant (red text) breast and lymphoma cell lines treated with DMSO (−) or 200 nM GSK3326595 (+) for 3 or 5 days arranged in order of increasing gIC₅₀ value in a 6-day proliferation assay with GSK3326595. MDM4-FL, full-length isoform of MDM4; MDM4-S, short isoform of MDM4 with skipped exon 6.