Overexpression of p27KIP1 induced cell cycle arrest in G1 phase and subsequent apoptosis in HCC-9204 cell line

Figure 1 RT-PCR results of human p27^KIP1 RNA in Bak transfected HCC-9204 cell line. Predicted PCR product size is about 600 bp. lane 1: Molecular we ight Marker (Takara, DL 2000); lane 2: Bak (0 h); lane 3: Bak (24 h); lane 4: Bak (48 h)

Figure 2 The nucleotide sequences of p27^KIP1. Only one nucleotide is changed from c→t at site No 524, while the sequences of amino acids are the same

Figure 3 Cell slides under Nikon Eclipse TE200 fluorescence microscope using excitation light at either 488 nm to detect GFP (emission filter 522 nm). Distribution of GFP p27^KIP1 fusion protein expressed in living HCC-9204 cells 8 after 100 μmol/L ZnSO₄ treatment, indicated by an arrow. × 200

Figure 4 Transient transfected HCC-9204 cells exhibiting typical active cell death such as cell blebbing, condensed and rounded cells as seen under, Nikon Eclipse TE200 phase contrast microscope indicated by an arrow. × 200

Figure 5 The results of Western blots of lysates from transfected HCC-9204 cells, probed with antibodies against human p27^KIP1. Excellent p27^KIP1 expression was observed in HCC-9204 cells even 4 h after induction with zinc.

Figure 6 p27^KIP1 detected by DAKO LABC kit in transfected HCC-9204 cells after 24 h of addition of ZnSO₄. p27^KIP1 dark-brown granules are present in the nuclei of p27^KIP1 transfected HCC-9204 cells, as in dicated by an arrow. × 200

Figure 7 Time course of growth of cells transfected with pMD-KIP1 expressing p27^KIP1. HCC-9204 cells were induced by 100 μmol/L ZnSO₄. HCC-9204 cells viability was determined by microscopic examination of cells stained by 1 g/L trypan blue, counting cells on a hemocytometer at the time point indicated. Each treatment was triplicate.

Figure 8 Time course effect of p27^KIP1-on HCC-9204 cells growth. Cells were seeded at 10\+5 per dish. HCC-9204 cells were induced by 100 μmol/L ZnSO₄ at the time indicated. Cells morphology was observed under Nikon Eclipse TE 200 phase-contrast microscopic at 0, 24 and 48 h post addition of zinc. “Uppe r-row” represents pMD-neo transfected cells; “Lower-row” represents pMD- Bak transfected cells. × 200

Figure 9 Result of flow cytometric analysis. Cells were stably transfectd with p27^KIP1 and induced by 100 μmol/L ZnSO₄. The percentage of each phase for each group (before or 24 h after addition of zinc) is indicated in each panel.

Figure 10 TUNEL assay demonstrating obvious apoptotic changes in HCC-9204/p27^KIP1 24 h after addition of zinc under laser confocal microscope as in dicated by an arrow. (Bar represents 10 μm)

Figure 11 Analysis of DNA ladder formation at 0 (lane 1), 4 h (lane2), 24 h (lane 3), 48 h (lane 4), 72 h (lane 5) post addition of zinc in HCC-9204 cell line. Eac h lane was loaded with 10 μg DNA.

Figure 12 Comparative study of whether apoptotic cells can still express GFP. A GFP positive clone was chosen and counterstained with PI. Apoptotic cells were stained with PI and no longer expressed GFP. HCC-9204 cells were observed under Nikon Eclipse TE200 fluorescent phase-contrast microscopic. × 200

Figure 13 In situ fluorescence hybridization with FITC labelled p27^KIP1 probe. Two signals were observed in the chromosome under laser confocal micro scopy. (Bar represents 5 μm)