Figure 1. Borealin structure-function analysis.

A.) Immunofluorescence using antibodies to FLAG and Hec1 in HeLa M cells transfected with FLAG-tagged Borealin and then blocked in taxol. Whole cell maximum projections (scale bar = 10μm), individual sister kinetochores (scale bar = 0.5μm) and line scans of Hec1 and Flag intensity in kinetochore images are shown. B.) Maximum projections from cells treated as in A and stained with antibodies against FLAG and INCENP (scale bar = 10μm). C.) Intensity of INCENP in the DNA area of greater than 30 cells. Pixel intensities in INCENP images are relative to the average level of INCENP in untransfected samples from the same experiment. Bars represent standard deviation. D.) Representative still images of GFP foci from FRAP experiments in nocodazole-arrested HeLa M cells after transient transfection with GFP-tagged Borealin truncations (scale bar = 1μm). E.) Post-bleach recovery of GFP-Borealin measured by FRAP. Averages and standard deviations of more than eight centromeres per condition are shown. F.) Average time-constant values of FRAP curves from E (see methods). G.) Borealin knockdown in HeLa M cells after transient transfection with pBabePURO, and either empty pSUPER (Control) or pSUPER-Borealin shRNA. Cell lysates obtained after puromycin selection to remove untransfected cells were analyzed by western blotting with antibodies against Borealin and Actin. H.) Expression of FLAG-tagged Borealin truncations. HeLa M cells were transfected with pSUPER-Borealin shRNA, pBABEpuro along with FLAG-tagged forms of Borealin. FLAG and Actin were detected by western analysis. I.) Percentage of cells with greater than 4N DNA content (Polyploid). HeLa M cells were transfected with pSUPER-Borealin shRNA, pBABEpuro and FLAG-tagged Borealin, exposed to puromycin and analyzed by flow cytometry for DNA content 4 days post-transfection. J.) Frequency of mitotic phases in Borealin-knockdown cells expressing truncated forms of Borealin. HeLa M cells were transfected with pSUPER-Borealin shRNA, a plasmid encoding H2BGFP and Borealin truncations. Three days post-transfection, mitotic phases of GFP-positive cells were determined. Scoring was carried out in a blinded manner (n≥100 for each condition). Bars from I and J represent standard error from three independent transfections. In sections C, F, I and J the asterisk (*) indicates p<0.05 using a student’s t-test.