Pickett et al. identified genes that they proposed are unique to the various brands by using a twofold threshold and a Venn diagram approach.
The logic of using a twofold threshold is as follows; a confident dataset of 154 SUMOylated proteins in Drosophila early development has been published (Nie et al. 2009).
We labeled each wave by the timestamp at which the mean gene expression level of the corresponding transcriptional module(s) crossed the twofold threshold relative to the pre-starvation condition.
As expected, we found HSP70A among the genes induced by RB treatment of Chlamydomonas (Table 3) however in sak1 it was not significantly induced above the twofold threshold, suggesting that SAK1 might function in the same signaling pathway as MBS.
Interestingly, the number of PN-specific genes activated above the twofold threshold in iPou3f1/iHoxa5 ESC-MNs (22 genes) is higher than the sum of the two target gene sets in single transgenic MNs (12+3 genes), which suggests that several PN target genes, such as Pcdh10, require the combined activity of both determinants (supplementary material Table S6).
This can be shown specifically by analysing the differences in gene induction; for example hac1 and der1 are above the twofold change threshold in the secretion strain at 2.23 and 2.02, but below the threshold in the intracellular strain at 1.45- and 1.73-fold.
Cut-off for up- and downregulation of gene expression was set to twofold change threshold in all the analyses performed.
In the 48 68 min time frame, three other TFs crossed the twofold upregulation threshold: Cre12.g523000 (a C3H Zinc finger TF), RWP4 and GSM1.
SEPs were recorded at Erb, C6 and C4' (N20, P25, N35, P45, N50) following electrical stimulation of the median nerve (3 Hz, intensity of twofold motor threshold, 200/average, bandwidth 0.02 2 kHz).
Further analysis of the secretion vs. intracellular strain at these relevant time points, and comparison of the genes with significant changes, demonstrated that similar changes had occurred for both forms of production, although the relative level of fold change in regulation was lower for the intracellular production and often did not exceed the twofold change threshold.
