Fig. 5
The XBP1s mRNA-based determination of C-1305 impact on IRE1α endoribonuclease activity in ER stressed HeLa cells. HeLa cells were treated with ER stressors (Tm, 0.5 µg/ml and Tg, 25 nM), for 6 h in a presence of specified concentrations of C-1305 and 4µ8C. Following the treatments, the XBP1s mRNA levels were accessed with qPCR and expressed as a change versus ER stressed cells treated with vehicle. The results from 3 independent experiments (n = 9) are plotted normalized to GAPDH and TBP mRNA. Error bars represent standard deviations. Concentration response curves and IC50 values of a C-1305 and b 4µ8C in Tm treated cells were determined using Sigma Plot 1.1 software. Similar approach was used to determine Concentration response curves and IC50 values of c C-1305 and d 4µ8C in Tg treated cells. The IRE1α kinase assays were performed in the presence of serial dilution and were measured with ADP-Glo kinase assay. Concentration response curve and IC50 values of e C-1305, f 4µ8C and g staurosporine (used as a control of kinase activity) were determined using Origin Pro software. The results were expressed as remaining activity, normalized to the uninhibited control for 100% activity and to the fully inhibited one for 0% activity as described in “Materials and methods”. Data are plotted as means ± SD from triplicate measurements of two independent measurements (n = 6)