It needed to be determined if F45D3.4 is being activated as a general response to stress or a specific response to hypoxic conditons (cobalt chloride and hypoxic conditons). Therefore, other stressors that were tested, including the following: hypertonic stress, heat shock, calcium chelation, reactive oxygen species, heavy metal exposure, and starvation. The results of this experiment can be seen in table 1 of the paper (as displayed).

Through testing to see if the F45D3.4 gene and the NHR-57 gene (an HIF-dependent reporter, and a positive control in this experiment) it was found that none of the stress treatments were able to induce the F45D3.4 gene, except for the 12-hour starvation. The NHR-57 gene was shown to also be unresponsive to the treatments, except when it was treated with ROS. Cobalt chloride, anoxia, and hypoxia were also treatments that were tested. It was found that NHR-57 is not triggered by cobalt chloride. It simply showing that the mimetic does not have 100% coverage (this is why it is only a primary screening method).

The oter stress factors, as listed above, did not activate F45D3.4 (and NHR-57). As a result, the conclusion of this experiment is that the expression of F45D3.4 (and NHR-57) is not simply a generalized response to stress. The activation of this pathway is a very specific response. Actuvatuib if the genes through GFP was viewed under the microscope (methods for this can be seen in the support pages).

Table 1: This table is taken directly from Padmenhaba et al (2015). It displays whether or not there was induction of GFP for the genes F45D3.4 and NHR-57 when the round worms were exposed to the various stress treatments.