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To be able to compare the impact of a given gene into the BIK-mediated DDR, it is necessary to identify the upstream stimuli that induce BIK. For example, if BIK is activated by the effect of ATM activation upon DNA damage, then it is expected that the DDR induced by BIK activation should be similar to the one induced by ATM activation. To this end, we treated U2OS cells for 2 hours with the DNA-damaging agent bleomycin and then tested the response of BIK-transfected cells to a variety of stressors (Figure 3a). Bleomycin induced phosphorylation of ATM, CHK2, and H2AX in a manner similar to that observed with the ATM activator 3MB-PP1 (Figure 3b, right panel). However, bleomycin was clearly less effective than 3MB-PP1 in activating BIK (Figure 3c, middle panel). This suggests that BIK requires more than ATM activation to be fully activated. Further, the BIK-mediated DDR was not identical to that induced by bleomycin because BIK-transfected cells were less resistant to bleomycin than ATM- and BRCA1-deficient cells, which in turn were more sensitive than ATM- and BRCA1-deficient cells that were transfected with BIK (Figure 3d). Thus, although BIK can substitute for ATM and BRCA1 in the induction of the DDR, the BIK-mediated DDR is distinct from ATM and BRCA1 activation. As these three molecules play central roles in the DDR, BIK clearly can substitute for ATM and BRCA1, but not all of their biological functions.
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One of the drawbacks to this approach was that we needed to recompile every time we made even a small change to the code. This slowed everything else we were doing down and because we couldn't always have someone waiting for the application to finish building. 827ec27edc