Fig. 4

MSI1 positively regulates the expression of YTHDF1 through stabilization of YTHDF1 mRNA in GBM cell line. a Western blot analysis of the expression of different YTH domain family proteins in DBTRG-05MG cells transfected with FLAG-tagged MSI1 (left panel) or MSI1-targeting siRNA (right panel). GAPDH used as a loading control. b qRT-PCR analysis of MSI1 and YTHDF1 mRNA levels in DBTRG-05MG cells transfected with FLAG-tagged MSI1. Data are expressed relative to cells transfected with empty vector (Flag). Means from three separate experiments are shown with SD error bars. **P < 0.01, ***P < 0.005 (Student’s t-test). c qRT-PCR analysis of MSI1 and YTHDF1 mRNA levels in DBTRG-05MG cells transfected with MSI1-targeting siRNA. Data are expressed relative to cells transfected with scrambled sirNA control (siCtl). Means from three separate experiments are shown with SD error bars. **P < 0.01, ***P < 0.005 (Student’s t-test). d Assay showing stability of YTHDF1 mRNA in DBTRG-05MG cells transfected MSI1-targeting siRNA (siMSI1) as compared to scrambled siRNA control (siCtl). Transcription was blocked by treatment of cells with actinomycin D and YTHDF1 mRNA levels were assessed by qRT-PCT at the indicated time points. Data are expressed relative to the siCtl control. Means from three separate experiments are shown with SD error bars. **P < 0.01, ***P < 0.005 (Student’s t-test). e The knockdown efficiency of MSI1 mRNA assessed by qRT-PCR. Quantitative data are presented as means from three independent experiments with SD error bars. **P < 0.01 (Student’s t-test)