Figure 2
Nf1−/− MEFs are more resistant than Nf1+/− or Nf1+/+ MEFs to cytotoxic drugs. SV40 (a–d) and primary (a) Nf1−/−, Nf1+/−, and Nf1+/+ MEFs were treated without or with staurosporine (Staur) (0.5 μM) or vincristine (Vinc) (0.07 μM) for the indicated times, or were UV-irradiated at the indicated intensities or not irradiated, and then grown for 15 h after which cell survival or death was determined. (a) Determination of cell viability in the different Nf1-genotype SV40 and primary MEFs by the MTT assay, performed as described in Materials and Methods. Survival was defined as the MTT value after each apoptotic treatment in each of the three Nf1 SV40 MEF genotypes, expressed as a percentage of the MTT values obtained in the absence of apoptotic treatment at the corresponding time points or UV irradiation intensities. The values shown are means±S.D. (bars) (n=3). *P<0.05, **P<0.005, significantly different from the corresponding treatment in Nf1+/+ MEFs. (b) Determination of cell death and viability in the three Nf1 SV40 MEF genotypes by the Trypan blue assay. After treatment with the apoptotic stimuli, blue (dead) and white (live) cells were counted under the microscope. Cell death was defined as the number of blue cells, expressed as a percentage of the total number of cells (blue and white) in each treatment. The values shown are means±S.D. (bars) (n=3). *P<0.05, **P<0.005, significantly different from the corresponding treatments in Nf1+/+ SV40 MEFs. (c) DEVDase activity in SV40 MEFs. The three Nf1 SV40 MEF genotypes were left untreated or were treated with Staur (0.5 μM) or Vinc (0.07 μM) for the indicated times, or were grown for 15 h with or without prior UV irradiation. DEVDase activity was assayed as described in Materials and Methods. The results are expressed as the ratio of DEVDase activity measured after each type of apoptotic treatment in each Nf1 genotype to the corresponding activity measured in the untreated cells. The values shown are from a representative experiment (n=3). (d) Determination of cell viability by colony formation. The three Nf1 SV40 MEF genotypes were treated with Staur (0.5 μM) for 6.5 h, and surviving cells were then allowed to form colonies as described in Materials and Methods. Colony formation for each Nf1 genotype was defined as the number of clones obtained after treatment with Staur, expressed as a percentage of the number obtained in untreated cultures. The values shown are means±S.D. (n=3). *P<0.05, **P<0.005, significantly different from Nf1+/+ SV40 MEFs. (e, f) Nf1−/−, Nf1+/−, and Nf1+/+ SV40 MEFs express similar amounts of apoptotic proteins. Nf1−/−, Nf1+/−, and Nf1+/+ SV40 MEFs were grown in medium supplemented with 10% serum. (e) Protein extracts prepared from these cells were subjected to immunoblot analysis for Bcl-xL, Mcl-1, Bax, Bak, caspase-8, -9, -3, and XIAP expression, as described in Materials and Methods. Membranes of immunoblots were reprobed with β-tubulin to assess the uniformity of sample loading. The results shown are from a representative experiment (one of two or three independent experiments). (f) Quantitative analysis of Bcl-xL, Mcl-1, Bax, Bak, caspase-8, -9, -3, and XIAP expression. Immunoblots from each of the different independent experiments were scanned and the intensity of each of the apoptotic bands was quantified by densitometry and normalized relative to the expression of their corresponding β-tubulin proteins. The normalized apoptotic proteins value for each Nf1 genotype is expressed as the fold increase of the corresponding normalized value in Nf1+/+ SV40 MEFs. Values shown are means±S.D. (bars) (n=3) or means±range (bars) (n=2)
