(A) For mRNAs from 495 genes reproducibly down-regulated by PTBP1 depletion in RNAseq of biological replicates, fold change comparing siPTBP1/siUPF1 to siPTBP1 alone was plotted vs. fold change comparing siPTBP1 to siNT. Mean fold change among all analyzed genes (dashed line; p<0.0001 in two-tailed Student’s t-test of null hypothesis of zero fold change), best fit determined by the least squares method (solid line) and correlation coefficient (Spearman’s ρ) are indicated. See also Figure 6—source data 1. (B) Mean fold change in transcript abundance upon PTBP1 depletion versus siNT treatment was plotted against the mean fold change in abundance between siUPF1 and siNT conditions for the set of mRNAs as described in A. (C) Table of Spearman’s correlation coefficients for all mRNAs reproducibly downregulated by siPTBP1, mRNAs containing one or more putative PTBP1 hexamer binding sites within 400 nt of the termination codon, and mRNAs lacking putative PTBP1 hexamers in that region. (D) mRNAs protected from NMD by PTBP1 have long 3’UTRs. Continuous distribution function (CDF) plot of annotated 3’UTR lengths among all expressed exemplar mRNAs (blue), mRNAs down-regulated by PTBP1 depletion (red), and mRNAs rescued by co-depletion of PTBP1 and UPF1 in RNA-seq analyses (green; see Materials and methods for details). Statistical significance was evaluated by two-tailed K-S test, comparing the indicated mRNA sets to the distribution of 3’UTR lengths among all exemplar mRNAs. (E) qRT-PCR analysis of mRNAs protected from NMD by PTBP1. Graph of average abundance of mRNAs normalized to housekeeping UBC control mRNAs (n=3, error bars indicate SD). Statistical significance was determined by two-tailed Student’s t-test, comparing siPTBP1 to siNT and siPTBP1/siUPF1 to siPTBP1. See also Figure 6—figure supplements 1, 2 and Figure 6—source data 1.