Figure 1.

The proteasome inhibitor MG132 decreases HR in HeLa cells. (a) HR substrate in HeLaG1 cells. HeLaG1 cells contain an intrachromosomal recombination substrate consisting of two hygromycin resistance (hyg^R) alleles inactivated by the insertion of 10 bp and separated by the Neo^R that confers resistance to G418. One hyg^R allele is inactivated at the PvuI site, whereas the other one is inactivated at the SacII site. An intrachromosomal recombination event leads to the reversion to hygromycin resistance phenotype (HYG^R) by gene conversion or reciprocal exchange. (b) MG132 reduces HR in HeLa cells. Cells were seeded in a 6-well plate at the concentration of 2 × 10⁵ per well. After 24 h cells were incubated for 5 hours with 0.3% DMSO as control or with 3 μM, 10 μM, and 30 μM MG132. Next, cells were trypsinized, counted, and seeded as described in Material and Methods. After 24 hours, hygromycin (200 μg/mL) was added. After 10 days, when the clones were visible, plates were stained with crystal violet and hygromycin-resistant clones were counted. The frequency of HR was calculated by dividing the total number of HYG^R colonies by the number of viable cells. Data are reported as mean of five independent experiments ± standard deviation. Results are statistically analyzed using Student's t-test. *P < .05.