FIG. 1.

c-Myc-induced apoptosis in fibroblasts requires Bax but not Bid or Bak. (A) MEFs of the indicated genotypes were grown in 20% heat-inactivated FCS and were microinjected with either empty pcDNA3 or pcDNA3(Myc) (100 ng/ml) mixed in RNase-free water with pEGFP-N1 (20 ng/ml) indicator as described in Materials and Methods. Eighteen hours after microinjection, the number of GFP-expressing cells was evaluated by fluorescence microscopy; cells were then washed twice in PBS and cultured in the absence of FCS for an additional 24 h at 37°. The number of GFP-expressing cells still viable was then determined. Cell loss or cell gain was expressed as a percentage of the initial number of GFP-positive cells prior to serum deprivation. Data presented are mean values ± standard errors of the means (SEM) from the indicated number of independent experiments. wt, wild type. Insert: MEFs with genes knocked out were validated for loss of relevant BH3 protein by immunoblot analysis. Lysates of wild-type MEFs and bid^−/−, bax^−/−, or bak^−/− MEFs were fractionated by SDS-PAGE (10⁵ cells per lane) and immunoblotted with anti-Bid, anti-Bax, or anti-Bak antibodies as indicated. (B) bax^−/− MEFs were infected with either pBp(Bax) or retrovirus vector alone, and their sensitivities to c-Myc-induced apoptosis were assayed by microinjection with either empty pcDNA3 or pcDNA3(Myc) mixed with pEGFP-N1 indicator as described above. Data presented are mean values ± SEM from the indicated number of independent experiments. For each type of infected cell, each experiment comprised two paired observations, with either pcDNA3 or pcDNA3(Myc) plasmid DNA being injected within distinct areas of the same cell population seeded on glass coverslips the previous day. For each type of infected cell and each type of microinjected plasmid, the average number of GFP-positive cells prior to serum deprivation is indicated in brackets.