Leucine zipper and transcriptional activation domains of XBP1s are sufficient for C12-mediated caspase activation.

<p>A. Acute inhibition of RNA or protein synthesis with actinomycin (actin.) or cycloheximide (cyclo.) does not inhibit C12-induced caspase activation in wt MEFs. B. Domain structure of XBP1s and XBP1s truncations. XBP1s contains a polybasic domain (<i>grey</i>), leucine zipper domain (<i>black</i>) and transcriptional activation domain (<i>blue</i>). The location of the splice site that converts XBP1u pre-mRNA to XBP1s mRNA is also shown (<i>red</i>). The FLAG-tag on all constructs is located at the amino-terminus. C. Analysis of cellular localization of XBP1s and XBP1s truncations by immunocytochemistry. Panels are shown for anti-FLAG immunostaining (<i>top</i>), DAPI staining (<i>middle</i>), and merged images (<i>bottom</i>). D. Expression of XBP1s truncations that contain the leucine zipper and transcriptional activation domains (XBP1_Δ1 and XBP1_Δ2) is sufficient to restore C12-mediated caspase activation in <i>Ire1α</i>^−/− (<i>top panels</i>) and <i>Xbp1^−/−</i> (<i>bottom panels</i>) MEFs. Expression of XBP1_N failed to restore C12-mediated caspase activation. Images are shown for DAPI staining (magenta) and caspase immunostaining (red) in the absence and presence of C12. E. XBP1s truncations that restore C12-mediated caspase activation (XBP1_Δ1 and XBP1_Δ2) are transcriptionally inactive. Normalized XBP1s transcriptional activity was measured in wt MEFs using an XBP1s-responsive luciferase-based reporter and co-expression of control plasmid, XBP1s, or XBP1 truncations. Individual scale bars are shown for panels C and D.</p

XBP1s mediates cellular responses to the C14 homoserine lactone and in different cell types.

<p>A. Structure of <i>N</i>-(3-oxo-tetradecanoyl) homoserine lactone (C14). B. <i>Ire1α</i>^−/− MEFs are protected from C14-mediate cell death as assessed by calcein AM staining. C. Normalized caspase 3/7 activity in wt (<i>black</i>) and <i>Ire1α^−/−</i> (<i>grey</i>) MEFs in control conditions and after treatment with C12 or C14 (25 µM, 4 h). D. Caspase activation in C14-treated MEFs assessed by cleaved caspase 3 immunocytochemistry. Images are shown in the absence (<i>top</i>) and presence (<i>bottom</i>) of C14 (25 µM, 2 hours) with DAPI (magenta) and cleaved caspase 3 staining (red). Wildtype MEFs (<i>first panels</i>) and <i>Ire1α^−/−</i> MEFs that are either untransfected (<i>second panels</i>) or transfected to express XBP1s (<i>third panels</i>) or XBP1_Δ2 (<i>fourth panels</i>) are shown. E. Over-expression of XBP1_Δ2 enhances executioner caspase activation when heterologously expressed in FRT cells. (<i>left</i>) Fluorescence images of cell enriched for expression of XBP1_Δ2 (FRT-XBP1_Δ2), anti-FLAG immunostaining (<i>top</i>), DAPI fluorescence (<i>middle</i>) and a merged image (<i>bottom</i>) are shown. (<i>right</i>) Caspase 3/7 activation is enhanced in FRT-XBP1_Δ2 cells (<i>grey bars</i>) relative to control cells (<i>black bars</i>), by C12 (25 µM) at 2 and 4 hours and by C14 (25 µM) at 4 hours. F. Over-expression of XBP1_Δ2 enhances C12- and C14- (25 µM) mediated caspase 3/7 activation when heterologously expressed in Hela cells. Statistical analysis was by t-test and for pairwise comparison of control and XBP1_Δ2 overexpressing cells, * p<0.001 and ** p<0.0001.</p

Correction: X-Box Binding Protein 1 (XBP1s) Is a Critical Determinant of <i>Pseudomonas aeruginosa</i> Homoserine Lactone-Mediated Apoptosis

Correction: X-Box Binding Protein 1 (XBP1s) Is a Critical Determinant of <i>Pseudomonas aeruginosa</i> Homoserine Lactone-Mediated Apoptosi

Role of XBP1s in C12-mediated cellular responses.

<p>Activation of caspases is dependent upon XBP1s, albeit by a mechanism that does not involve transcription. Phosphorylation of p38 MAPK and eIF2α is XBP1s-independent. Stimulation of cells with C12 does not activate IRE1α and presumably does not activate other ER stress response pathways. No inferences about c-Jun synthesis can be drawn from the current study.</p

Deletion of <i>Ire1α</i> prevents C12 cytotoxicity.

<p>A. Structure of the <i>Pseudomonas aeruginosa N</i>-(3-oxo-dodecanoyl) homoserine lactone (C12). B. (<i>left</i>) Cell density of wt (wild-type; <i>first panels</i>), <i>Perk^−/−</i> (<i>third panels</i>) and <i>Ire1α^−/−</i> (<i>fourth panels</i>) MEFs under control conditions (<i>top</i>) and after C12 challenge (25 µM, 4 hours, <i>bottom</i>) assessed by calcein AM labelling. Caspase inhibition prevents C12 cytotoxicity as assessed by calcein AM staining of z-VAD-fmk (z-VAD) treated wt MEFs cells (<i>second panels</i>). (<i>right</i>) RT-PCR (<i>top</i>) and western blot (<i>bottom</i>) analysis of <i>Perk^−/−</i> and <i>Ire1α^−/−</i> MEFs. C. Quantitative analysis of change in MEF cell density after C12 challenge in wt, <i>Perk^−/−</i> and <i>Ire1α^−/−</i> MEFs and in z-VAD-fmk-treated wt MEFs (<i>grey bar</i>). D. Normalized caspase 3/7 activity in wt, <i>Perk^−/−</i> and <i>Ire1α^−/−</i> MEFs in control conditions (−) and after C12 treatment (+; 25 µM, 4 hours). E. Dose response of normalized caspase 3/7 activity in wt MEFs treated with C12 for 4 hours. Statistical analysis was by t-test with reference to data from wt MEFs (C) or control experiments (E); * p<0.005, ** p<0.0001.</p

Absence of the XBP1s transcription factor is responsible for reduced C12 cytotoxicity.

<p>A. Fluorescence images of calcein AM stained wt MEFs treated with inhibitors of IRE1α RNase activity (STF-080310) and JNK (SP600125) in control conditions (<i>top</i>) and with C12 (25 µM, 4 hours, <i>bottom</i>). B. Normalized caspase 3/7 activation in wt MEFs in control conditions (<i>black</i>) and after treatment with STF-083010 (<i>grey</i>). C. Assessment of IRE1α activation in C12-treated cells. (<i>left</i>) Schematic of luciferase-based IRE1α activity reporter. Luciferase (<i>luc</i>) expression is prevented under control conditions by an ‘<i>in-frame</i>’ stop codon (<i>top</i>); 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 ‘<i>out-of-frame</i>’ and the luciferase ‘<i>in-frame</i>’ (<i>bottom</i>). (<i>right</i>) IRE1α activation in wt MEFs in control conditions (<i>black</i>) and after 2 hours treatment with 25 µM C12 (<i>grey</i>) or 250 nM thapsigargin (<i>blue</i>). Statistical analysis was by ANOVA with Dunnett post hoc test; ** p<0.0001 versus control cells. D. Deletion of <i>Xbp1</i> prevents C12-cytotoxicity assessed by calcein AM labelling (<i>left</i>) and caspase 3/7 activation (<i>right</i>). (<i>bottom</i>) Analysis of <i>Xbp1^−/−</i> MEF by RT-PCR and western blot. E. Analysis of time- (<i>left</i>) and dose-dependent (<i>right</i>) C12-mediated normalized caspase 3/7 activation in wt (<i>black</i>) and <i>Xbp1^−/−</i> (<i>grey</i>) 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.</p

XBP1-independent cellular responses to C12.

<p>A. Immunoblot analysis of phosphorylated eIF2α (p-eIF2α, <i>left</i>) and phosphorylated p38 (p-p38, <i>right</i>) levels in wt, <i>Ire1α</i>^−/− and <i>Xbp1^−/−</i> MEFs. Cells were treated with 25 µM C12 for 45 minutes. B. (<i>top</i>) C12 treatment (25 µM, 45 min) of <i>Perk^−/−</i> MEFs results in phosphorylation of eIF2α (<i>bottom</i>) Control experiments confirm that ER stress (brefeldin A (BFA), 2 hours) in wt MEFs, but not <i>Perk^−/−</i> MEFs, produces p-eIF2α. C. C12 treatment increases c-Jun levels in wt MEFs but not in <i>Ire1α</i>^−/−, and <i>Xbp1^−/−</i> MEFs. Cells were treated with 25 µM C12 for 90 minutes (<i>bottom</i>) and c-Jun levels were characterized by image analysis of immunostained cells (green). Images of DAPI staining are also shown (magenta). D. Quantitative analysis of normalized c-Jun levels in wt, <i>Ire1α</i>^−/−, and <i>Xbp1^−/−</i> MEFs. Scale bar in panel C refers to all images.</p