X-Box Binding Protein 1 (XBP1s) Is a Critical Determinant of Pseudomonas aeruginosa Homoserine Lactone-Mediated Apoptosis
Figure 2
Absence of the XBP1s transcription factor is responsible for reduced C12 cytotoxicity.
A. Fluorescence images of calcein AM stained wt MEFs treated with inhibitors of IRE1α RNase activity (STF-080310) and JNK (SP600125) in control conditions (top) and with C12 (25 µM, 4 hours, bottom). B. Normalized caspase 3/7 activation in wt MEFs in control conditions (black) and after treatment with STF-083010 (grey). C. Assessment of IRE1α activation in C12-treated cells. (left) Schematic of luciferase-based IRE1α activity reporter. Luciferase (luc) expression is prevented under control conditions by an ‘in-frame’ stop codon (top); however, IRE1α activation results in non-conventional splicing and removal of 26 nucleotides (26 nt) from the reporter pre-mRNA which shunts the stop codon ‘out-of-frame’ and the luciferase ‘in-frame’ (bottom). (right) IRE1α activation in wt MEFs in control conditions (black) and after 2 hours treatment with 25 µM C12 (grey) or 250 nM thapsigargin (blue). Statistical analysis was by ANOVA with Dunnett post hoc test; ** p<0.0001 versus control cells. D. Deletion of Xbp1 prevents C12-cytotoxicity assessed by calcein AM labelling (left) and caspase 3/7 activation (right). (bottom) Analysis of Xbp1−/− MEF by RT-PCR and western blot. E. Analysis of time- (left) and dose-dependent (right) C12-mediated normalized caspase 3/7 activation in wt (black) and Xbp1−/− (grey) MEFs. Cells were treated with 25 µM C12 over 0–8 hours and for 4 hours with 0–100 µM C12. Scale bar in panel A refers to all images.