Figure 1.

The level of calponin 2 changes during cell cycle. A, top: newly divided HEK293 cells were collected from shake-off culture and reseeded for adherent preconfluent culture of 72 h. The levels of calponin 2 were examined at 0, 6, 24, 48, and 72 h. The sodium dodecyl sulfate (SDS)-gel and Western blot showed a low level of calponin 2 in rounded-up dividing cells, which was subsequently increased during culture. Middle: densitometry quantification of the Western blot normalized to actin in SDS-gel confirmed the low level of calponin 2 in newly divided cells which increased during culture. There was a trend of a second dip of calponin 2 after 30 h of culture, corresponding roughly to the completion of the first round of cell cycle indicated by the cell growth curve in the bottom that shows the number of cells per unit area doubling at 24, 48, and 72 h. B: NIH3T3 cells were treated with nocodazole for 28 h and released for synchronized growth. Western blot and densitometry analysis showed that nocodazole blocked cells expressed slightly higher level of calponin 2 as compared with untreated cells. From 0 to 20 min after releasing from nocodazole block, the level of calponin 2 decreased significantly, demonstrating a rapid degradation in dividing cells indicated by the high level of Ser¹⁰-phosphorrylated histone H3. C: SDS-gel and Western blot further showed that HEK293 cells blocked at metaphase with colchicine also maintained a high level of calponin 2. Data were summarized from 3 or more parallel cell cultures. Values are presented as means ± SD. *P value: 0.001 in Student’s t test.