Impact of pharmacologic inhibition of the NMD pathway on F11 splicing and quantification of the alternative transcript lacking exons 6+7. (A) The NMD pathway was blocked in HepG2 cells using puromycin (“NMD analysis”). Variations in the expression levels of splicing isoforms (compared with nontreated samples, set as 1) were quantified using the ΔΔCT method; in all experiments, connexin 32 was used as reference. Bars represent mean ± SEM of 3 independent experiments, each performed in triplicate on different days in different cell batches. The results were analyzed by unpaired t test. **P < .01; ***P < .001; ns indicates not significant. (B) Standard curves for both PCR assays (ie, for the amplification of the wild-type transcript and of the one lacking exons 6+7) are generated from dilution series constructed from the fusion plasmid pcDNA3/FXI-wt+Δ6/7. The quantification of wild-type and Δ6/7 FXI transcripts in liver (C) and platelets (D) was performed by real-time RT-PCR. Amplification curves and schematic visualization of the abundance of the 2 transcripts in liver and platelets are represented. The absolute quantities of the mRNA containing exon 6+7 (wild-type) was set as 100%. Bars represent mean ± SD of 3 independent experiments, each performed in triplicate on different cDNA preparations.