Expression of PKCθ-EGFP fusion protein in HeLa cells and its effect on the cell morphology

Aim: To investigate the expression and subcellular localization of protein kinase Cθ(PKCθ) fused with enhanced green fluorescent protein (EGFP) (PKCθ-EGFP) in HeLa cells and to analyze its effect on the cell morphology. Methods: HeLa cells were transfected with expression vectors pPKCθ-EGFP and pEGFP-N1 using LipofectAMINE 2000, respectively. Their expression in the cells was analyzed by flow cytometry and subcellular localization by confocal microscopy. The effect of phorbol 12,13-dibutyrate (PDB) and berberine on cell morphology was analyzed by confocal microscopy too. Results: The expression rates of green fluorescence in HeLa cells transfected with pEGFPN1 and pPKCθ-EGFP plasmids were (70.9±4.4)% and (45.8±2.3)% respectively at 48 h after transfection (no statistics significance). PKCθ-EGFP fusion protein was evenly distributed in the whole cells, which was similar to the distribution pattern of EGFP protein in HeLa cells. After 30 min stimulation of PDB, the shape of the HeLa cells expressing PKCθ-EGFP was dramatically changed. From spindle to small round, and the green fluorescence intensity increased and concentrated in the central of the cells. In contrast, the cells expressing EGFP had no significant change both in the green fluorescence distribution and in cell morphology after PDB stimulation. Besides, berberine exerted no obvious effect on the PDB-induced changes in HeLa cells expressing PKCθ-EGFP. Conclusion: The HeLa cells that expressed PKCθ-EGFP had significant morphological change under PDB stimulation.