Figure 1
Defining applicable ER stress stimulation conditions. ER stress was induced by incubating N. crassa in the presence of concentration gradients of dithiothreitol (DTT) or tunicamycin (TM), and several criteria were used to define ER stress levels, including (A) sensitivity of growth to the drugs. WT conidia were inoculated onto solid GFS agar in a multi-well plate containing the indicated concentrations of DTT or TM and incubated at 25°C for 3 days. (B) Monitoring hac-1 non-canonical splicing using semi-quantitative RT-PCR. WT conidia were inoculated into Vogel’s medium with Avicel as carbon source for 36 h, then either DTT or TM was added at different concentrations as indicated for an extra 1 h before harvesting. The product sizes representing hac-1 spliced and un-spliced forms were 201 bp and 224 bp, respectively. The gene actin (NCU04173) was used as a loading control. (C) Monitoring UPR and RESS activation by qPCR. Culture conditions were as in (B). The data are normalized to expression on mock = 1 and re-calculated to log2-fold (standard error of the mean (SEM), n = 3). The asterisk indicates a significant difference from mock (**P < 0.01, ***P < 0.001) via one-way ANOVA.