PKN2 is involved in aggregation and spheroid formation of fibroblasts in suspension culture by regulating cell motility and expression.

The role of Protein Kinase N2 (PKN2, also known as PRK2/PKNγ) in cell aggregate/spheroid formation in suspension culture was investigated using immortalized fibroblasts established from mouse embryos. cells formed cell aggregates in flat bottom low attachment well plates, such as 2% agar and poly-2-hydroxyethymethacrylate coated plates, however, Cre; cells in which PKN2 was depleted by the introduction of Cre-recombinase rarely formed aggregates. Time-lapse analysis revealed that the velocity of Cre; cell motility was significantly lower than that of in a low attachment flat-bottom plate, which likely resulted in a lower cell-cell contact frequency among Cre; cells. Conversely, Cre; cells could form initial cell aggregates in U-bottom low attachment well plates, however, the succeeding compaction process was delayed in Cre; cells with decreased roundness, although cells underwent compaction in a round shape spheroid within 24 h. Immunoblot analysis revealed that the preparation of the cell suspension from adherent conditions using trypsin/EDTA treatment significantly decreased the expression of N-cadherin in both and Cre; cells. The N-cadherin expression level recovered time-dependently; however, the recovery of N-cadherin was significantly delayed in Cre; cells compared to cells. Reverse transcription quantitative PCR revealed that mRNA in Cre; cells was significantly lower than that of cells. These results suggest that PKN2 controls the velocity of cell motility and the transcription of in fibroblasts, leading to cell aggregation and compaction for spheroid formation in suspension culture.