Figure 1.

Knockdown of PC1 induces polyploidy in vitro. (A) Scheme of the self-inactivating VIRHD/P lentiviral vector and its derivatives VIRHD/P/siLuc and VIRHD/P/siPKD1, expressing the control anti-luciferase siRNA and anti-Pkd1 siRNA, respectively. CMV, cytomegalovirus early promoter; RRE, rev responsive element; cPPT, central polypurine tract; H1, human H1 promoter; SV40 ori, SV40 early promoter; Puro, puromycin acetyltransferase; WPRE, Woodchuck Hepatitis Virus post-transcriptional regulatory element; Δ deletion in 3′-LTR. (B) Expression of PC1 in parental IMCD3 cells untransduced or transduced with the VIRHD/P/siLuc (IMCD/P/siLuc) or VIRHD/P/siPKD1₃₂₉₇ (IMCD/P/siPKD1₃₂₉₇) lentivectors at the indicated days post-transduction (Days pt). The faint band below the main one varies in parallel to the PC1 signal and likely represents a form of partially processed PC1. Actin expression was determined as control of sample loading. Molecular weight standards are indicated (kDa). The numbers under each lane indicate the PC1:Actin expression ratio reported as percentage of the control parental IMCD3 cells. (C) Cells cycle profiles of IMCD3 at different time post-transduction with VIRHD/P/siPKD1₃₂₉₇ or VIRHD/P/siLuc. The ploidy of the subpopulations and the values of the subG₀/G₁ cellular fractions are indicated. (D) Lack of homology between the murine specific PKD1₁₂₆₃₂ siRNA target sequence (boxed) and human PKD1 gene. (E) VIRHD/P/siPKD1₁₂₆₃₂ suppresses the expression of the endogenous PC1 but not human flagged PC1 3 days following transduction (+) of IMCD3 untransfected or transfected with the control vector pCI or with the pCI/hPKD1-Flag plasmids. Control VIRHD/P/siLuc vector had no effect. Tubulin was used as sample loading control. The numbers under each lane indicate the PC1:Tubulin expression ratio reported as percentage of the control cells (untransfected and untransduced cells). (F) Parallel cell cycle analysis showed that following endogenous PC1 knockdown by VIRHD/P/siPKD1₁₂₆₃₂, abnormal ploidy (3n or >4n) was induced in untransfected or control pCI-transfected cells but not in cells expressing the exogenous human PC1. Transduction with control lentivector VIRHD/P/siLuc had no effect at any time.