The proteins of GmGBP1 and GmGBP1c were both capable of inducing LacZ expression in yeast cells and could grow in the medium lacking His, indicating that GmGBP1 protein had transcriptional activity in its C-terminal domain.
All three cell lines were induced to undergo ER stress by the addition of 300 nM thapsigargin (Tg) (Alexis Biochemicals/Enzo Life Sciences, Plymouth, PA), 5 μg/mL of tunicamycin (Tun) (Sigma), or culture in medium lacking glucose, for indicated time points.
3-amino-1,2,4-triazole (3-AT; Sigma) was added to SC medium lacking leucine and histidine at the indicated concentrations.
Medium lacking serum was termed 'basal medium' (BM), while medium containing 10% FBS was termed as "full medium" (FM).
YEpOLEX- Lca-QPAQ transformants were able to grow on medium lacking supplemental unsaturated fatty-acids (UFAs), indicating that the Lca-QPAQ cDNA was encoding a desaturase that complemented the UFA auxotrophic ole1 strain.
For imaging, embryoid bodies (EBs) were generated by placing 300 cells, after infection with shRNA-expressing lentiviruses as indicated, in 30 μl of medium lacking LIF and incubating in hanging-drop cultures.
Further observations (data not shown) indicated that the embryos appeared very rapidly, (after 1-2 days) following transfer to medium lacking β-Glc Y.
In contrast, cells passaged in the absence of 4OHT showed no increase in the GFP+ fraction over this one-month period, indicating that the NEIE cell line is tightly regulated when cultivated in medium lacking estrogenic compounds.
As shown in Figure 3B, H4-R36A cells had modest growth on medium lacking histidine and containing glucose and much stronger growth on medium lacking histidine and containing galactose, indicating that cryptic initiation occurs in H4-R36A cells and that this defect is much more pronounced in the context of elevated levels of transcription.
RT-PCR experiments indicate that both genes were separately transcribed for growth on chitin as well as on tryptone-containing medium lacking chitin [ 8].
