Fig. 5. MSI2 mediates PRMT5 resistance through control of c-MYC translation.

A mRNA expression of c-MYC and MSI2 from RNA-Seq experiment Fig. 4E, n = 3. p-value 0.020 determined by two-sided Student’s t test. B Representative immunoblots of c-MYC and MSI2 upon treatment with GSK-591 and Ro at the indicated times in Z-138 cells. Similar results were observed in <4 different experiments. C Representative immunoblots of c-MYC and MSI2 upon MSI2 knockdown. Similar results were observed in two independent experiments. D Effect of GSK-591 and Ro treatment on cell viability upon c-MYC depletion (c-MYC OFF) for 72 h in P-4936 cells. Data represent mean ± SEM of three different experiments, n = 3 replicates. E Representative immunoblots showing decreased MSI2 protein abundance, in P-4936 cells after 72 h of MYC depletion (MYC OFF). Similar results were observed in two independent experiments. F Effect of GSK-591 and Ro treatment on Z-138 cells overexpressing empty vector or c-MYC after 96 h. Values are the mean ± SEM of three separate experiments. G qRT-PCR of recovered RNA from MSI2 RNA-IP from Z-138 cells treated with 5 μM GSK-591 and/or Ro for 24 h. c-MYC mRNA enrichment is shown as the percentage (IP/input) and normalized to DMSO  ±  SEM of three independent experiments. IgG served as a non-specific binding control. p-value determined by two-sided Student’s t test. H Luciferase reporter assay using MYC 3′UTR in 293T cells. 293T cells were treated with GSK-591 and/or Ro for 24 h. Luciferase activity was normalized using firefly values. n = 3 independent experiments. I Luciferase reporter assay using MYC 3′UTR in 293T cells. 293T cells were transfected with control or MSI2 and PRMT5 shRNA constructs. Luciferase activity was measured 48 h post-transfection and was normalized using firefly values. n = 3 independent experiments.