Fig 2: PTBP1 represses inclusion of BIM exon 3 independently of the 2,903-nt polymorphic fragment.(A) Schematic of the deletions made on the ?10E minigene to remove the putative PTBP1 binding sites. +2,582 to +2,662 of the polymorphic fragment has been expanded to show the nucleotide sequence. The predicted PTBP1 binding sites on ?10E are boxed, whereas the deletions are indicated by dashes. (B) Real-time RT-PCR analysis of RNA from K562 cells nucleofected with the minigene constructs described in (A) to assess the ratio of exon 3- to exon 4-containing minigene products. The ?11 minigene serves as a positive control for enhanced exon 3 inclusion. Results are presented as an average of triplicates and the relative minigene E3: E4 ratio was determined by normalizing to the E3: E4 ratio of K562 cells nucleofected with the ?10 minigene. Error bars represent ± SEM. (C) K562 cells were either nucleofected with control or PTBP1-specific siRNA duplexes. 24 hours later, these cells were nucleofected with either the WT, ?10, ?10E or ?11 minigene. Real-time RT-PCR analysis of RNA from these cells was performed after another 24 hours to determine the ratio of exon 3- to exon 4-containing minigene products. Results are presented as an average of triplicates and the relative minigene E3: E4 ratio was determined by normalizing to the E3: E4 ratio of K562 cells nucleofected with the same minigene, but without any siRNA. Error bars represent ± SEM.

Fig 3: SiRNA-mediated knockdown of PTBP1, but not hnRNP C, inhibits imatinib-induced apoptosis in CML cells.(A) K562 cells were nucleofected with either control or PTBP1-specific siRNA duplexes. 24 hours later, these cells were treated with increasing doses of imatinib (0, 0.25, 0.5 µM). The cells were harvested after another 24 hours and western blot was performed to determine the protein levels of PTBP1, PARP, cleaved caspase 3 and BIM. Equal loading in each lane was determined by blotting of GAPDH. (B) The same experiment was performed as described in (A). The only difference was that siRNA duplexes against hnRNP C were used instead of PTBP1-specific siRNA duplexes.