Knockdown of I-2 yields multinucleated cells. (A) ARPE-19 cells were transfected with siRNA against I-2, or luciferase, as control. Cells were collected 72 h after transfection, and extracts were prepared and immunoblotted for I-2, with actin as a loading control, and anti-pan-PP1 as an additional control. (B) Control and I-2 knockdown cells were fixed and stained to quantitate the percentage of cells with multiple nuclei (500 cells in each group). Data are plotted as mean ± SD of results from three independent experiments. (C) Cells were fixed 72 h after siRNA transfection and stained with anti-α-tubulin (green) and Hoechst 33342 (blue) to show multiple nuclei. Bar, 25 μm. (D) ARPE-19 cells were nucleofected with pSUPER empty vector, pSUPER-I-2 shRNA, and pSUPER-I-2 shRNA + pK7-HA-I-2. The shRNA targeted a different sequence in I-2 than that used for siRNA. The pK7-HA-I-2 encodes I-2 with silent mutations to preserve the amino acid sequence but changes the nucleotide sequence to avoid knockdown by RNAi. Cell extracts were analyzed by immunoblotting with affinity-purified sheep anti-I-2, mouse anti-actin, and chicken anti-PP1. The migration of endogenous I-2 and ectopic HA-tagged I-2 are indicated with arrows. (E) Cells with multiple nuclei were scored (500 cells/group), and data are plotted as mean ± SD of values from three independent experiments.