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. 2019 Aug 27;8:e47084. doi: 10.7554/eLife.47084

Figure 3. Ectopic expression of IRE1 LDTM attenuates apoptotic caspase activation independent of full-length IRE1.

(A) KMS11 parental cells or cells stably expressing a cDNA plasmid encoding LDTM (1-470) driven by the CMV promoter were treated with DMSO or 100 nM Tg for 24 hr. Cell viability was measured using CellTiter-Glo normalized by the number of cells at seeding (middle panel). The percentage of viable cells is graphed as an average of three biological replicates. Equal amounts of protein from cell lysates were analyzed by WB (top panel) or Caspase-Glo 3/7 assay (bottom panel). WBs are representative of two or more experiments and the graph depicts mean ± SD of three technical replicates. (B) KMS11 cells as in A were treated with different concentrations of Tg for 24 hr and analyzed for viability. The percentage of viable cells is graphed as an average of three biological replicates ± SD. (C) KMS11 parental cells, LDTM expressing KMS11, or single cell clones derived from the LDTM transfected KMS11 pool (C7 or C13) were treated with 0.3 μg/ml SubAB for 3 hr. Equal amounts of protein from cell lysates were analyzed using the Caspase-Glo 3/7 assay. The graph depicts mean luminescence signal normalized to the control ± SD of three technical replicates. (D) KMS11 cells were stably transfected with a DOX-inducible shRNA plasmid targeting IRE1 (Parental). The cells were then stably transfected with a cDNA plasmid encoding LDTM (1-470) driven by the CMV promoter as in A. Parental and ectopic LDTM-expressing cells were treated for 3 days in the absence or presence of 1 μg/ml DOX to induce shRNA-mediated depletion of endogenous IRE1. Cells were then treated with DMSO or 100 nM Tg for 24 hr to induce ER stress and analyzed by WB (top panel) or CellTiterGlo assay (bottom panel). The percentage of viable cells is graphed as an average of three biological replicates ± SD. (E) KMS11 cells expressing DOX-inducible IRE1 shRNA were treated in the absence or presence of 1 μg/ml DOX and then subjected to ER-stress induction with 100 nM Tg or 5 μg/ml Tm for 24 hr. Cells were analyzed by WB (top panel) or Caspase-Glo 3/7 assay (bottom panel). The graph depicts mean luminescence signal normalized to DMSO ± SD of three technical replicates.

Figure 3.

Figure 3—figure supplement 1. Ectopic expression of IRE1 LDTM attenuates apoptotic caspase activation independent of full-length IRE1.

Figure 3—figure supplement 1.

(A) KMS11 overexpressing an untagged version of LDTM (amino acids 1–470) or parental cells were treated with DMSO or 100 nM Tg for 24 hr. Equal amounts of protein from cell lysates were analyzed by Caspase-Glo 3/7 assay. Graphs depict mean luminescence signal ± SD as a ratio to the DMSO control from three technical replicates. (B) Two independent clones expressing ectopic LDTM (1-470) were isolated (C7 and C13). Cells were treated with different concentrations of Tg for 72 hr and viability was measured by CellTiter-Glo assay. The percentage of viable cells is graphed as an average of three biological replicates ± SD. (C) Parental KMS11 cells or cells stably transfected with a plasmid encoding a CMV-driven LDTM protein, representing a precise cleavage fragment of IRE1 with a Flag tag (1–507F), were treated with DMSO or 0.3 μg/ml SubAB for 3 hr and analyzed by Caspase-Glo 3/7 assay. The graph depicts mean luminescence signal ± SD as a ratio to the DMSO control from three technical replicates. (D) Parental JJN3 cells or JJN3 cells transfected as in C were treated with 100 nM Tg, or 5 μg/ml Tm, or 0.5 μg/ml BfeA for 24 hr, or 0.3 μg/ml SubAB for 3 hr, and analyzed by Caspase-Glo 3/7 assay. The graph depicts mean luminescence signal ± SD as a ratio to the DMSO control from three technical replicates. (E) Fixed MDA-MB-231 cells expressing the flag-tagged LDTM construct were stained with anti-flag (red), anti-calnexin (green), and DAPI. Cells were imaged using a confocal microscope and a z-stack image was acquired to determine localization of LDTM in the ER. An orthogonal projection is shown. (F, G) KMS11 cells expressing DOX-inducible IRE1 shRNA (Parental) were stably transfected with a plasmid encoding a CMV-driven LDTM protein without (1-507) or with a Flag tag (1–507F). The cells were incubated in the absence or presence of 1 μg/ml DOX for 3 days to induce IRE1 shRNA expression. Cells were then treated with DMSO, 100 nM Tg, or 5 μg/ml Tm for 6 hr and equal amounts of protein lysates were analyzed by Caspase-Glo 3/7 assay (F) or WB (G). Data represent at least three independent experiments; graphs depict mean ± SD of three technical replicates.