Fig. 3: Knockdown of SON increases exon 10 inclusion in PTBP2 transcript via PTBP1-independent mechanisms.

a RT-PCR analyses of PTBP2 exon 10 inclusion upon SON knockdown in the U87MG cell line and two patient-derived GSC lines (GSC #83 and GSC #84). PTBP2 exon 10 skipping cases frameshifting, resulting in a premature termination codon (PTC; indicated with a red circle). Data are representative of n = 3 independent experiments. b SON, PTBP1, and PTBP2 mRNA expressions at the indicated time points after SON siRNA transfection were analyzed by RT-qPCR. GAPDH was used for normalization (n = 3). c SON, PTBP1, and PTBP2 expression after SON siRNA transfection in U87MG cells were analyzed by Western blotting at the indicated time points. GAPDH was used as a loading control. The inclusion of PTBP2 exon 10 was analyzed by RT-PCR upon SON knockdown. Data are representative of n = 3 independent experiments. d A schematic illustrating the qPCR strategy used to measure PTBP2 exon 10 inclusion and skipping. A specific reverse primer detecting exon 9-11 junction (top; for exon 10 skipping) and exon 9–10 junction (bottom; for exon 10 inclusion) were used for qPCR. e The ratio of PTBP2 exon 10 inclusion/exon 10 skipping determined by qPCR analysis using the primer sets shown in panel (d) (n = 3). f, g RT-qPCR (f, n = 3) and Western blot (g) assays demonstrate that SON knockdown-mediated-PTBP2 upregulation is through a PTBP1-independent mechanism. U87MG cells were transfected with SON siRNA and/or Myc-PTBP1 constructs as indicated and harvested in 48 h. WB Data are representative of n = 3 independent experiments. h RT-PCR analysis of PTBP2 exon 10, PBX1 exon 7, and RTN4 exon 3 usages (skipping or inclusion) under the condition described for (f) and (g). Data are representative of n = 3 independent experiments. Error bars in all graphs represent the standard deviation (SD) of tests. NS; not significant, *p < 0.05, **p < 0.01. Statistical significance was determined by an unpaired two-tailed t-test. The number under each band in the Western blots indicates relative band intensity. Source data are provided in the Source data file.