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. 2020 Jan 6;9:e52291. doi: 10.7554/eLife.52291

Figure 1. Misfolded proteins induce DR5-dependent apoptosis and assemble DR5-caspase 8 signaling complexes.

(A) Confocal images of epithelial cells HCT116 fixed 24 hr post-transfection with 0.25–1.0 μg of a plasmid containing myelin protein zero (MPZ) tagged with a C-terminal monomeric EGFP or 1.0 μg of the empty vector showing MPZ-GFP fluorescence (green) and immunofluorescence with an antibody against DR5 (red) (scale bar = 5 μm). (B) Western blot of HCT116 cell lysates harvested 24 hr post-transfection with a titration of MPZ-GFP plasmid or the empty vector (C8 = caspase 8, cC3 = cleaved caspase 3). p55 represents full-length, inactive C8; p43 indicates a C8 intermediate after release of the active p10 subunit, and p29 corresponds to the released p18 and p10 subunits. (C) Western blot of HCT116 cells transfected with siRNA against a non-targeting (Nt) control or DR5 (48 hr) followed by the empty vector -/+ 100 nM thapsigargin (Tg), 1.0 μg MPZ-GFP, or cytosolic GFP (24 hr; * denotes degradation products; L and S denote the long and short isoforms of DR5, respectively; FL and C denote full-length and cleaved PARP, respectively). (D) Average percent of annexin V staining for HCT116 cells transfected as described in C) from n = 3 biological replicates (error bars = SEM; * indicates p<0.05; ns indicates p=0.46 as analyzed by unpaired t-test with equal SD). See Figure 1—figure supplement 4D for gating. (E) Top: Caspase 8 activity in size exclusion chromatography fractions from lysates of HCT116 cells transfected with MPZ-GFP or cytosolic GFP (24 hr). Bottom: Size exclusion fractions were pooled according to dotted grid lines and immunoblotted for DR5 and GFP (* denotes degradation products). (F) Immunoprecipitation of GFP-tagged proteins from lysates of HCT116 transfected with MPZ-GFP, cytosolic GFP, or the empty vector (L and S denote the long and short isoforms of DR5, respectively). The percent of total DR5 recovered has been quantified in Figure 1—figure supplement 5C. (G) Fold change in caspase 8 activity relative to the empty vector control for beads with immunoprecipitated contents shown in Figure 1F (error bars = SEM for n = 3 biological replicates; * indicates p=0.023 and ns indicates p=0.83 as calculated by unpaired t-tests with equal SD).

Figure 1—source data 1. Caspase glo 8 measurements for IP of MPZ-GFP vs GFP.
This zip archive contains the measured luminescent units for caspase glo 8 activity shown in Figure 1G (IP beads) and Figure 1—figure supplement 3C (input lysates). Coomassie gels used to normalize lysate concentration are included as. tif files.
Figure 1—source data 2. Westerns and quantification of DR5 recovered on IPs.
This zip archive contains images of the Western blots and measurements used to quantify the amount of DR5 in the IP samples relative to the input lysate.
Figure 1—source data 3. FCS files and quantification of annexin V staining for MPZ-GFP.
This zip archive contains FCS files from n = 3 biological replicates of HCT116 transfected with the conditions outlined in Figure 1D. The excel file contains the quantification of annexin V staining exported frow FlowJo.
Figure 1—source data 4. qPCR analysis of MPZ-GFP titration.
This zip archive contains the compiled excel file for qPCR data shown in Figure 1—figure supplement 1A along with the Prism 6 file used to perform multiple t-tests with Holm-Sidak correction for multiple comparisons.
Figure 1—source data 5. Caspase glo 8 measurements for time course of MPZ-GFP transfection.
This zip archive contains the measured luminescent units for caspase glo 8 activity shown in Figure 1—figure supplement 1E and the tif file of the Coomassie blue-stained gel used to normalize lysate concentrations.
Figure 1—source data 6. qPCR and cell death measurement for CHOP expression.
This zip archive contains the qPCR analysis from CHOP expression in Figure 1—figure supplement 2B, and brightfield images of Trypan Blue staining measured on the Countess II for n = 3 biological replicates, summarized in Figure 1—figure supplement 2D.
Figure 1—source data 7. qPCR analysis of INS and RHO-GFP expression.
This zip archive contains the compiled excel file for qPCR data shown in Figure 1—figure supplement 4A along with the Prism 6 file used to perform multiple t-tests with Holm-Sidak correction for multiple comparisons.
Figure 1—source data 8. FCS files and quantification of annexin V staining for INS and RHO.
This zip archive contains FCS files from n = 3 biological replicates of HCT116 transfected with the conditions outlined in Figure 1—figure supplement 4E. The excel file contains the quantification of annexin V staining exported frow FlowJo.
Figure 1—source data 9. Caspase glo 8 measurements for IP of INS and RHO-GFP.
This zip archive contains the measured luminescent units for caspase glo 8 activity shown in Figures 1S5B (input lysates and IP beads). Coomassie gels used to normalize lysate concentration are included as. tif files.

Figure 1.

Figure 1—figure supplement 1. Sustained MPZ-GFP expression invokes a terminal, pro-apoptotic UPR at late time points.

Figure 1—figure supplement 1.

(A) qPCR for reverse-transcribed transcripts harvested from HCT116 cells transfected with 0.12–1.0 μg of a plasmid containing myelin protein zero (MPZ) tagged with a C-terminal monomeric EGFP or 1.0 μg of the empty vector for 24 hr (n = 3 technical replicates, error bars = SD; * denotes p<0.05 as analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons). (B) Quantification of the mean intensity for DR5 versus the mean intensity of intracellular MPZ-GFP per cell for HCT116 transfected with 0.25 μg (left) and 1.0 μg (right) of MPZ-GFP plasmid to show the correlation between DR5 and MPZ-GFP expression levels per cell. Intensity values given by CellProfiler algorithms were normalized to 0.02 for DR5 and 0.06 for MPZ-GFP to assign arbitrary values. P values were calculated from unpaired two-tailed t-tests. (C) RT-PCR for unspliced and spliced forms of Xbp1 mRNA isolated from HCT116 cells transfected for 24 hr with the empty vector or for various time points with 1 μg MPZ-GFP, followed by cells treated with 100 nM Tg for 2 hr and 24 hr. (D) Western blot of HCT116 cell lysates harvested 24 hr post-transfection with the empty vector, or 3–24 hr post-transfection with 1 μg MPZ-GFP. (E) Fold change in caspase 8 activity, as measured by a luminescent caspase glo 8 substrate, of lysates from HCT116 harvested 3–24 hr post-transfection with 1 μg MPZ-GFP relative to cells transfected with the empty vector control (error bars represent SD of n = 3 technical replicates; *** denotes p<0.005, and ns indicates p=0.15 by unpaired t-test with equal SD).
Figure 1—figure supplement 2. Upregulating DR5 levels in the absence of ER stress through ectopic expression of CHOP is not sufficient to induce apoptosis.

Figure 1—figure supplement 2.

(A) Western blot of HCT116 cell lysates harvested 24 hr post-transfection with a titration of 0.03–0.50 μg of a CHOP expression vector, 1 μg MPZ-GFP plasmid, or the empty vector (FL = full length, C = cleaved). (B) qPCR for reverse-transcribed transcripts harvested from HCT116 cells transfected with 0.03–0.50 μg of a CHOP expression vector, 1.0 μg of MPZ-GFP, or 1.0 μg of the empty vector for 24 hr (n = 3 technical replicates, error bars = SD, * denotes p<0.05). (C) Representative images of automated counting for Trypan blue-stained cells, where green outlines denote non-stained (live) cells and red outlines denote stained cells (Trypan blue+, dead). (D) Average percentage of cells transfected as described in (S3A) stained with Trypan blue as quantified by automated cell counting from n = 3 biological replicates (error bars = SEM; ** denotes p=0.008 and ns = non significant for unpaired t-test with equal SD; ns1 refers to p=0.19 from unpaired t-test with Welch’s correction for variance).
Figure 1—figure supplement 3. DR5 immunoprecipitates with FADD and MPZ-GFP.

Figure 1—figure supplement 3.

(A) Immunoprecipitation of DR5 from HCT116 transfected with MPZ-GFP or the empty vector and blotted for DR5, MPZ-GFP, and FADD. (B) Inputs for GFP pulldown performed in Figure 1F. (C) Caspase 8 activity of inputs relative to the empty vector control for the GFP pulldown performed in Figure 1F (n = 3 biological replicates, error bars = SEM, ** indicates p=0.0046, and * indicates p<0.05 from unpaired t-tests with equal SD). Source data can be found in Figure 1—source data 1.
Figure 1—figure supplement 4. Sustained overexpression of other ER-trafficked proteins induce UPR-mediated apoptosis in a DR5-dependent manner.

Figure 1—figure supplement 4.

(A) qPCR for reverse-transcribed transcripts harvested from HCT116 cells transfected with 1.0 μg of GFP-tagged rhodopsin (RHO), proinsulin (INS), or 1.0 μg of the empty vector for 24 hr (n = 2 biological replicates, each with three technical replicates; error bars = SD; * denotes p<0.05). (B) RT-PCR for unspliced and spliced forms of Xbp1 mRNA isolated from HCT116 cells transfected for 24 hr with 1 μg of empty vector -/+ 100 nM Tg for 2 hr, MPZ-GFP, INS-GFP, or RHO-GFP. (C) Western blot of HCT116 cells transfected with siRNA against a non-targeting (Nt) control or DR5 (48 hr) followed by 1.0 μg RHO-GFP or INS-GFP (24 hr). (D) Representative flow cytometry histograms of HCT116 cells transfected with the listed siRNA and vector and stained with annexin V-AlexaFluor647. Y-axis has been scaled so that the mode = 100%. Dotted lines represent gating to distinguish staining-positive cells. Left: Histograms of fluorescence at 647 nm to measure annexin V staining. Right: Histograms of fluorescence at 488 nm to compare level and distribution of GFP-tagged protein expression. To note, GFP expression profiles for the same construct are similar between different siRNA transfected samples. (E) Average percent of annexin V-positive cells for HCT116 cells transfected with siRNA and GFP-tagged rhodopsin/proinsulin (n = 3 biological replicates, error bars = SEM, * indicates p=0.011, ** indicates p=0.005 from unpaired t-test with equal SD). Gating for annexin V-positive staining is shown in Figure 1—figure supplement 4D.
Figure 1—figure supplement 5. DR5 engages a selective subset of ER-trafficked client proteins upon prolonged ER stress.

Figure 1—figure supplement 5.

(A) Pulldown of GFP-tagged proteins from HCT116 transfected with INS, RHO, or cytosolic GFP. Inputs (left) and immunoprecipitated samples (right) were immunoblotted for GFP and DR5 (L and S indicate long and short isoforms, respectively). (B) Fold change in caspase 8 activity relative to cytosolic GFP for beads with immunoprecipitated contents described in Figure 1—figure supplement 5A as measured by caspase glo 8 luminescence (n = 2 biological replicates, error bars = SEM, * indicates p<0.05, ** indicates p=0.0013, **** indicates p<0.005 from unpaired t-tests with equal SD). (C) Quantification of the percent of total DR5 recovered in the IPs of GFP-tagged proteins, shown in Figure 1G and Figure 1—figure supplement 5A. (n = 3 biological replicates for GFP and MPZ, while n = 2 biological replicates for INS and RHO. * denotes p=0.016, ** denotes p=0.0035, and ns denotes p=0.39 from unpaired t-test with equal SD) The DR5 signal of the input and IP lanes were quantified from the same exposure and then normalized to the amount loaded on the gel. Source data of blots and quantification are provided in Figure 1—source data 2.