Figure 2

Alterations in the cell cycle and apoptosis gene expression in α3 KO DF. A, PCR-based genotyping of α3+/+ and α3−/− mice. Genomic DNA extracted from neonatal mice was used in the RT-PCR assay, as detailed in “Materials and Methods,” with the following primers: 5′-GTGGATCCCTCCGGCCATCTTTAAGAG-3′ (wild-type forward), 5′-GACTGTGATGACAATGGACAAGGTGAC-3′ (wild-type reverse), and 5′-TGGCGCGAAGGGACCACCAAAGAACGG-3′ (mutant reverse). After 30 cycles, the PCR analysis identified the α3 homozygous null (−/−), heterozygous (+/−), and wild-type (+/+) phenotypes. The gel shows representative PCR profiles of homozygous mice from a progeny of a heterozygous α3+/− mouse. The homozygous null (−/−) mouse showed a single lower band, the wild-type (+/+) showed a single upper band, whereas the heterozygous (+/−) showed both the upper and the lower band (not shown). B, Analysis of the expression of cell cycle and apoptosis genes in α3 KO DF by RT-PCR. Total RNA was isolated from the second passage, ~75% confluent monolayers of DF grown from neonatal α3 homozygous null (−/−) and wild-type (+/+) mice and used in the RT-PCR assays described in “Materials and Methods.” Each primer set listed in Table 3 yielded a PCR product of the expected size: 233 bp for p21, 499 bp for Ki-67, 482 bp for cyclin D1, 307 bp for PCNA, 389 bp for p53, 470 bp for Bax, 474 bp for Bcl-2, and 494 bp for caspase 3. A gene expression ratio of 1 was given to the wild-type (+/+) animals. The α3 null mutation was associated with decreased transcription of the genes coding for p21, cyclin D1, Ki-67, PCNA, and Bcl-2 in a range from 1.4- to 50-fold and increased transcription of the genes encoding p53 (3.2-fold), Bax (1.8-fold), and caspase 3 (1.5-fold). The ratio data are the means ± sd of the values obtained in at least three independent experiments. The images represent typical appearance of the bands in gels. The negative control experiments did not yield any PCR products (not shown). Asterisks indicate significant (p < 0.05) differences from control. C, Analysis of the expression of cell cycle and apoptosis genes in α3 KO DF by WB. Total protein was isolated from the same cells as in B and used in the WB assay described in “Materials and Methods,” with the Abs listed in Table 2. The molecular weight of each protein is shown in kDa to the right of the gels. Changes in the gene expression of each of the cell cycle and apoptosis markers detectable by WB were consistent with those determined by RT-PCR. In α3−/− DF, the relative amounts of p21 decreased 2.7-fold, Ki-67 1.2-fold, PCNA 4.2-fold, and Bcl-2 1.2-fold; the relative amounts of p53, Bax, and caspase 3 increased 2.3-, 2.8-, and 1.8-fold, respectively; and the relative amount of cyclin D1 did not change compared with α3+/+ DF. The staining of these proteins was absent in the negative control experiments described in the legend to Figure 1B. Asterisks indicate significant (p < 0.05) differences from control.