Pmcherry c2

NIH 3T3 cells were plated at 1×10⁶ per 100 mm-diameter dish the day before transfection. The cells were transfected by Lipofectamin 2000 (Invitrogen) with total 24 μg of DNA containing either 12 μg each of GFP-HMGN5,¹⁴ CFP-NLS, or 12 μg each of GFP-HMGN5-SE¹⁴ and mCherry-NLS, as recommended by the manufacturer. Expression vector for mCherry-NLS was constructed by inserting nuclear localization signal encoding oligonucleotide between NheI and AgeI site of pmCherry-C2 (Clontech). CFP-NLS was constructed by adding NLS between NheI and XhoI site of pECFP-N1 (Clontech). After 5 hours of transfection the cultures were fed with fresh medium. After 48 hours of transfection, cells were harvested by trypsin treatment and dissolved in phosphate buffered saline (PBS). Aliquots containing 1 × 10⁶ of each transfected cells type were mixed in a 15 ml Falcon tube, sedimented at 300xg and re-suspended in 1 mL of 0.1 × PBS and transferred into 1.5 mL centrifuge tube. Using 1 mL syringe the cell were repeatedly passed through a 26 G needle, at a rate of 5 passages in 1 minutes. After each 10 passages, 50 μL of cell suspension was collected, fixed by 3.7% formalin, and the number of red and blue nuclei remaining determined with a Nikon Eclipse E800 microscope using the NIS Elements AR3.10 software.