Figure 1: The tricistronic construct efficiently silences endogenous normal and expanded PABPN1 without affecting optPABPN1 expression in vitro.

(a) Three constructs were cloned into pAAV vectors and used in this study: a tricistronic shRNA construct including hairpins sh-1, sh-2 and sh-3 driven each by a different polymerase III promoter (U61, U69 and H1, respectively), the sequence-optimized PABPN1 (optPABPN1) driven by the SPc5-12 promoter and tagged with a MYC-tag, and the human expanded PABPN1 (expPABPN1) driven by the SPc5-12 promoter and tagged with a FLAG-tag. (b) HEK293T cells were transfected with 4 μg per well of pAAV-shRNA3X in triplicate with or without AAV plasmids expressing expPABPN1 or optPABPN1. Untransfected cells and cells transfected with AAV plasmid expressing shRNA for HBVpol were used as a control. Seventy-two hours post transfection, samples were collected and PABPN1 was detected by western blot and quantified by densitometric analysis using ImageJ software. Transfection was performed twice. PABPN1 expression in each condition was normalized by GAPDH expression level and then by the value of untransfected cells. Transfection with a plasmid expressing shRNA3X induced efficient PABPN1 knockdown even when expPABPN1 was co-expressed compared to untransfected cells. The co-expression with optPABPN1 restored PABPN1 expression to the normal level. (c) Representative image of a western blot used for these analyses. (d) FLAG is not detected when samples are transfected with shRNA3X. However, in samples prepared from cells transfected with pAAV-opPABPN1, MYC-Tag is detected by western blot. Detection of MYC-Tag shows that optPABPN1 is resistant to degradation by shRNA3X in vitro. Data are presented as mean±s.e.m., n=4 (shRNA3X+optPABPN1) or n=6 (all the other groups). One-way ANOVA test with Bonferroni post-hoc test *P<0.05, **P<0.01, NS, not significant.