Fig 2: Knockdown of PCTAIRE1 causes mitotic arrest and apoptosis. (A) Immunofluorescence of HeLa cells transfected with esiRNA for PCTAIRE1 and stained 72 h post-transfection showed aberrant mitosis. Many cells exhibited abnormal metaphase, with lagging chromosomes at anaphase and misoriented midbodies during cytokinesis. (B) Western blot analysis confirming knockdown of PCTAIRE1 in U2OS cells by shRNA (after 72 h of shRNA induction using doxycycline). (C) DAPI and tubulin staining in control U2OS cells to detect the spindle and nuclear morphologies during different stages of mitosis. (D) Effect of shRNA for PCTAIRE1 on mitosis in U2OS cells (after 72 h of shRNA induction using doxycycline): immunofluorescence of PCTAIRE1 knockdown in U2OS cells predominantly led to aberrant mitosis with multipolar, misaligned or distorted spindles, in addition to presence of multiple centrosomes, lagging chromosomes and abnormal midbody, as was observed by tubulin staining. (E) Quantitative analysis of aberrant mitosis in sh-PCTAIRE1 cell lines. Data shown are the mean±s.d. of n=3 independent experiments; *P<0.05 (two-tailed unpaired Student's t-test). All images were taken at 63× magnification.

Fig 3: Overexpression of PCTAIRE1 resists PolST-mediated mitotic arrest and cell death. (A) Crystal Violet staining showing PCTAIRE1-overexpressing U2OS cells exhibiting higher survival in the presence of PolST expression. Cells were stained after 7 days of doxycycline addition. (B) PCTAIRE1-overexpressing U2OS (PolST-PCTAIRE1) cells were sensitive to PolST-mediated cell death in the presence of PCTAIRE1 inhibitor, indirubin-E804 (500 nM). Cells were stained with Crystal Violet after 7 days. (C) Timeline showing the experimental strategy for live-cell time-lapse imaging for cells in which PolST was induced for 72 h (PolST was induced for 56 h before imaging and the cells were then monitored for 16 h). (D) Examples of time-lapse sequences illustrating the fate of the nucleus of a single representative cell (stained by siR-Hoechst) in the presence of PolST expression (arrows indicate a reference cell during mitosis). (E) Examples of time-lapse sequences illustrating the fate of the nucleus of a single cell (stained by siR-Hoechst) in the presence of both PolST and PCTAIRE1 expression. Numbers indicate minutes with relation to nuclear envelope breakdown (NEBD), which is denoted as 0 min (arrows indicate a reference cell during mitosis). The images in D and E depict areas of 152.18 × 133.16 µm. (F) Representative still images from live-cell time-lapse imaging of PolST cells in U2OS background (72 h induction), showing many mitotically arrested and apoptotic cells in the controls, whereas in the presence of PCTAIRE1 overexpression, there are many cells exhibiting normal cell division (cell structures and the nuclei can be seen when monitored under both differential interference contrast and fluorescence channels; arrows indicate reference cells). (G) Live-cell imaging analysis of PolST cells in U2OS background (in the presence and absence of doxycycline for 72 h), showing PCTAIRE1 overexpression leading to a significant percentage of cells exhibiting normal mitotic progression and decreased apoptosis (the percentage of cells not undergoing mitosis is not shown). Cells in prometaphase-like stage include the cells that were delayed in prometaphase for at least 800 min until the end of the experiment. (Time taken from NEBD to anaphase onset was considered to calculate the duration of mitosis. NEBD was observed by chromosomal condensation and appearance of irregular nuclei, whereas anaphase onset was observed by separation of the two chromosome masses.) (H) Dot plot analysis after live-cell imaging of PolST cells in U2OS background (in the presence and absence of doxycycline for 72 h), showing the time taken for cells in mitosis. Forty mitotic cells were included from each group for the analysis, and the cells undergoing death are not represented in the analysis. The cells that were delayed in a prometaphase-like stage for a minimum of 800 min until the end of the experiment have been categorized separately. (I) FACS analysis of PolST cells in U2OS background (in the presence and absence of PolST for 48 h), showing a decreased population of cells in the sub-G1 and G2/M phases in the presence of PCTAIRE1 overexpression, with a concomitant increase in the G0/G1 population. (J) Graphical representation of the FACS data obtained as in F. Data are presented as mean±s.d.; *P<0.05, **P<0.01 (two-tailed unpaired Student's t-test).

Fig 4: Localization of PCTAIRE1 in different phases of cell cycle. (A) Immunofluorescence studies of U2OS cells using actin binding dye, phalloidin and anti-PCTAIRE1 antibody. During prometaphase and metaphase, PCTAIRE1 can be seen on the mitotic spindle, including the spindle poles. At anaphase and cytokinesis, it is also seen at the midplate and midbody, respectively. DAPI was used for chromosomal staining to identify a particular phase of mitosis. (B) Staining of U2OS cells by anti-?-tubulin and anti-PCTAIRE1 antibodies showing that PCTAIRE1 colocalizes with ?-tubulin in the centrosomes and at spindle poles throughout mitosis (63× magnification). (C) PCTAIRE1 and PLK1 localizations during mitosis in U2OS cells: PCTAIRE1 colocalizes with PLK1 at centrosomes (prophase), spindle poles (prometaphase) and then later at the midbody (telophase, cytokinesis) (63× magnification), but not at metaphase and anaphase stages. (D) PCTAIRE1 localizes just adjacent to PLK1 at the midbody (63× magnification).

Fig 5: PCTAIRE1 protein levels change during cell cycle progression. (A,B) Western blots showing increased PCTAIRE1 protein amounts and phosphorylation levels, as indicated by reduced band mobilities in U2OS (A) and HeLa cells (B), as the cells progressed towards mitosis after release from thymidine block. Mitotic stage was apparent at ~6–7 h (as shown by the red arrows) after thymidine release, as was confirmed by enhanced phospho-CDK substrate and cyclin B signals. (C) Western blot showing increase in endogenous PCTAIRE1 levels in U2OS cells after the addition of nocodazole (50 ng/ml) and paclitaxel (430 ng/ml) for 24 h. (D) Endogenous PCTAIRE1 levels increased in PolST stable cell lines with increasing time of PolST expression by doxycycline induction up to 24 h. Thereafter, PCTAIRE1 levels decreased sharply. PolST and PCTAIRE1 were detected by anti-HA and anti-PCTAIRE1 antibodies, respectively. (E) Endogenous PCTAIRE1 was pulled down in 293T cells by anti-PCTAIRE1 antibody. A and C subunits of PP2A were co-immunoprecipitated along with PCTAIRE1, as detected by anti-PP2A-A and anti-PP2A-C subunit antibodies, respectively. SIRT1 and DBC1 were used as negative controls for interaction with PCTAIRE1.