The previous experimetns confirmed that there were conditons or factors triggering the F45D3.4 gene to be expressed. Therefore, it is of interest to narrow down and determine what exactly is causing the response of this gene. This was one of the key experiments in this paper. In order to figure this out, 387 transcription factors in the round worms were targeted through a procedure known as RNAi (more information on this can be found in the support pages). Through the use of RNAi to knock down the transcription factors, microscopy and the GFP construct (as explained in the support pages), eight targets were identified (found in table 2 of the paper) as being able to turn on the GFP after being treated with cobalt chloride. This means that when every transcription factor was knocked down in the entire worm, there were eight transcription factors remaining that had no response to cobalt chloride, and did not light up, thus showing they are necessary triggers to activate the F45D3.4 gene.

After passing this primary cobalt chloride test, further analysis was done to the eight targets by exposing them to real hypoxic conditions. Only three of the eight transcription factors were actually responsive to hypoxic conditions (tbx-38, lin-40 and blmp-1). The table that summarizes these results in the paper can be seen on the right.

Table 2: This table is taken directly from Padmenhaba et al (2015). It displays the transcription factors that are responsive to hypoxic conditions.