Fig 2: PPP1R2 targets PP1 to the midbody. a-f ARPE-19 cells were transfected, fixed, and labeled for PP1 (green) to determine the localization of endogenous PP1 along the central spindle (red) following overexpression of PPP1R2 mutants: a phosphonull Thr73 mutant PPP1R2A (R2A), a phosphomimetic Thr73 mutant PPP1R2E (R2E), a C-terminal truncation PPP1R2?C (R2?C), and an N-terminal truncation PPP1R2?N (R2?N). Average intensity from the green channel was measured along the central spindle in equal sized squares (n = 30 dividing cells) and is plotted in (g). The area under the curve was calculated for total PP1 localization intensity throughout the midbody’s structure (h). Error bars represent standard error of the mean. Statistically significant differences (p = 0.05) between experimental groups and empty vector control are indicated by differing letters above the bars and asterisks indicate *p = 0.01 and **p = 0.001

Fig 3: PPP1R2 mutant overexpression increased midbody length. a ARPE-19 cells were transfected with FLAG, PPP1R2, and PPP1R2 mutants (R2A, R2E, R2?C, R2?N), fixed, and labeled with DAPI (blue) as well as a-tubulin (red) to stain the central spindle. Size bar equals 5 µm. b Midbody length was quantified using Metamorph software and averages were calculated within three biological replicates (n = 30 dividing cells). Statistically significant differences (p = 0.05) between experimental groups and empty vector control are indicated by differing letters above the bars and asterisks indicate *p = 0.01 and **p = 0.001

Fig 4: The effect of PPP1R2 on PP1 and AURKA activity at the centrosome was dependent on its C-terminus. a A graphical representation of the PP1 and AURKA activity of cells expressing each construct is shown. b Protein expression and phosphorylation levels of proteins indicated by the table below the graph were measured using ELISA. Cells were transfected with the indicated constructs, fixed, and labeled for ?-tubulin (red) and either (c) phosphorylated PP1 (pPP1, green) or (d) phosphorylated AURKA (pAURKA, green) antibodies. pPP1 and pAURKA levels at the centrosome were quantified using Metamorph software and are displayed as graphs in C and D. Statistically significant differences (p = 0.05) between experimental groups and empty vector control are indicated by differing letters above the bars and asterisks indicate *p = 0.01 and **p = 0.001

Fig 5: PPP1R2 overexpression increased cell ploidy and accumulation of cells with enlarged nuclei. a Cells were transfected with the indicated FLAG-tagged fusion proteins, fixed and stained for a-tubulin to detect microtubules (red), and DAPI to stain the nucleus (blue). Size bars equal 10 µm. b Quantitation of DAPI intensity after transfection with the indicated constructs and normalized to the control. c Quantitation of cells with increased ploidy after transfection with PPP1R2. Statistically significant differences (p = 0.05) between experimental groups and empty vector control are indicated by differing letters above the bars and asterisks indicate *p = 0.01 and **p = 0.001