Figure 3
TEC-1 does not strongly impact GALC cryptic splicing. (a) qPCR analysis of GALC transcripts in GM03813 cells treated with each compound for 24 h. The amounts of GALC mRNA were normalized to those of GAPDH. (b) Schematic representation of exon construction and cryptic exon inclusion of GALC mRNA splicing. (c) RT-PCR analysis of GALC transcripts in GM03813 cells treated for 6 h with both compounds and CHX. RT-PCR products were separated by agarose gel electrophoresis and visualized with ethidium bromide. Standard GALC fragments (Oligo STD) with or without the cryptic exon are shown. Molecular weight markers are shown on the right (bp). The full-length gel is presented in Supplementary Fig. 2c. (d) qPCR analysis of the GALC cryptic exon in GM03813 cells treated for 6 h with both compounds and CHX. Aberrant GALC mRNA amounts with cryptic exons were quantified and normalized to those of GAPDH. (e) GALC enzymatic assay from human oligodendroglioma (Hs683) cells treated with each compound for 3 days. The amounts of standardized product (fluorogenic HMU-β-D galactopyranoside) were normalized to those of total protein. (f) Ratio of psychosine and sphingosine in Hs683 cells treated with each compound for 12 days. The amounts of psychosine were determined by LC/MS/MS whose signal was normalized by sphingosine signals. Data are from two biologically independent samples in (a). Data in (d), (e), (f) represent means ± standard error of the mean (SEM) of three independent assessments per concentration.
