Immunostaining and Multinucleated Cells Analysis

SW480, SW620, T98G, HeLa cells were seeded in 6 cell plates to 30% density. 24 hours later cells were fixed with cold methanol for 15 min at -20°C. The cells were washed with PBS 3 times and peameablized with 1%Trixon-X for 20 min. The cells were blocked with 2.5% BSA (in PBS) for 30min at room temperature. The cells were incubated with γ-tubulin or HA-flurescent primary antibody with appropriate dilutions overnight at room tempature. The next day, the cells were washed with PBS three times and secondary Goat anti-Rabbit antibody was incubated with cells for additional two hours. For multinucleated cell analysis, DAPI (1mg/ml) was applied to stain transfected cells and examined with Confocal microscope. 150 cells were counted from each slide to analyze the percentage of multinucleated cells.

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Fig 4.1 HA-PKM2 Expression Induces the Formation of Multinuclei in SW480 Cells (A) Fluroscent immunostaining of exogenous expressed PKM2 using the antibody

PabHA in SW480. The green signal represents staining of exogenous PKM2 in the cells (a). (b) is the overlay of HA antibody staining and DAPI staining. The blue signal represents DAPI staining of DNA. (B) shows the percentage of the number of multinuclei

cells in the whole populations of expressing HA-PKM2. (a) is the population of two nuclei in the whole populations of expressing HA-PKM2. (b) is the percentage of triple nuclei population in the whole populations expressing HA-PKM2. (c) is the percentage of single nucleus population in the whole population expressing HA-PKM2. Error bars in (B) are standard deviations of three measurements

Figure 4.2 Cdc14A Expression is Up-regulated by PKM2 and p68 RNA Helicase

(A) Cellular levels of Cdc14A in SW480 cells 48 hours after transfection of the cells with

plasmids vectors that carry PKM2 cDNA expression vector (HAPK), p68 RNA helicase (WTp68), Y593F mutant of p68 RNA helicase (593F) or empty vector (Vec) were analyzed by immunoblot with anti-Cdc14A (IB: Cdc14A). The exogenously expressed HA-PKM2 or p68 was examined by immunoblot using anti-HA antibody. Immunoblot of

β-actin (IB:β-actin) were loading control. (B) is the centrosome staining in multi nuclei

cell induced by PKM2 overexpression. (a) The red signal represents staining of γ-tubulin. (b) The blue signal represents DAPI staining of DNA. (c) The green signal represents staining of exogenous expression of PKM2 in in the cells. (d) is the overlay of HA antibody, γ-tubulin and DAPI staining.