Figure 3. Reduced spreading defect of keratinocytes from old hpm mice.

(A) Freshly isolated keratinocytes from 2.5-month-old mice seeded on a mixture of Col1 and FN. In contrast to control cells, hpm cells neither spread nor grow ex vivo. Time points after seeding are indicated. (B) Proliferation rate of keratinocytes from 3-week-old mice cultured for 2 days on Col1/FN and pulse-labelled with BrdU for 10 or 24 hours. Compared to control cells hpm keratinocytes have significantly reduced proliferation rate. Bars represent data from keratinocytes of 3 mice per genotype. Error bars indicate s.d. (C) Cell adhesion of hpm keratinocytes from 2.5-month-old mice plated on Col1/FN or on LM-332 is similar to control cells. 5 and 4 independent experiments were performed on Col1/FN or LM-332, respectively. Error bars indicate s.d. (D) Keratinocytes from 2.5-month-old control and hpm mice were cultured for 2 days on Col1/FN and immunostained for β1 integrin, paxillin and vinculin (red), and F-actin (phalloidin; green). Nuclei were stained with DAPI (blue). hpm cells failed to develop focal adhesions and to organize the actin cytoskeleton into stress fibers. (E) Freshly isolated keratinocytes from 7.5-month-old mice plated on Col1/FN. Both control and hpm keratinocytes were well spread and showed a similar proliferation rate. (F) Control and mutant keratinocytes isolated from 6.5-month-old mice plated on Col1/FN and cultured for 2 days were pulse-labelled with BrdU and harvested at times indicated. No significant difference in BrdU incorporation was observed between control and hpm keratinocytes. Bars represent data from at least 3 mice per genotype. Error bars indicate s.d. (G) Keratinocytes isolated from 7.5-month-old control and hpm mice were cultured for 2 days on Col1/FN and immunostained for β1 integrin, paxillin and vinculin (red) and F-actin (phalloidin; green). Nuclei were stained with DAPI (blue). Size and number of FAs and actin stress fibers is similar in hpm and control keratinocytes.