Figure 5

Cyclin D1b- and cyclin K-expressing LP-1 cells display opposite clonogenic and migration properties. a) Exponentially growing cells were observed with an inverted phase contrast microscope and photographed. b) Clonogenic assay of LP-1-derived clones. Cells were prepared at a density of 3 × 10³ cells/ml in MethoCult^® containing PHA-LCM as source of growth factor (StemCell Technologies). Cells were dispensed in triplicate in Petri dishes, incubated in humidified atmosphere at 37°C for 10 days. Colonies containing more than 50 cells were counted using inverted microscope and gridded scoring dish. Each experiment was done in triplicate and repeated thrice. Results are expressed as mean ± SD. * p < 0.05 with the Student's t test. c) Western blot analysis of LP-1-derived clones. Either cytoplasmic or nuclear extracts were prepared, separated by 12% SDS-PAGE. Blots were then sequentially incubated with anti-p27^Kip1 (sc-528), anti-phospho-p27^Kip1 (sc-9104 from Santa Cruz Biotech.) Abs and anti-β-tubulin Ab to control gel loading and transfer. In the cytosolic and nuclear extracts from LP-1K cells, the anti-phospho-p27^Kip1 Ab reveals a band which is not at the expected size and likely represents a non specific binding (black dot). d) Migration assay of LP-1-derived clones. SVF (1%) was placed in the lower chamber of a Matrigel-coated transwell, LP-1 cells were plated (2 × 10⁴ cells) in the upper chamber, incubated 4 h at 37°C. After incubation, invading cells were fixed, stained and counted. Each experiment was done in triplicate and repeated thrice. Results are expressed as mean ± SD. * p < 0.05 with the Student's t test.