Fig 2: IRE1 does not oppose Tg-induced upregulation of DR5 mRNA. a-d LNCaP (a and b) or HCT116 (c and d) cells were transfected for 2 d with the indicated siRNAs (siCtrl = non-targeting control siRNA), employing two different siRNA oligoes for each target (designated by - 1 and - 2). Subsequently, cells were treated with 100 nM Tg or 0.01% DMSO (also transfected with siCtrl) for 6 h, and subjected to real-time RT-PCR to quantify DR5 mRNA levels (a and c) or western blotting to validate efficient knockdown of IRE1 and XBP1s protein levels (b and d). In (a and c), relative mRNA levels are shown normalized to the DMSO control-treated condition (set to 1 and indicated by the dotted line in the graphs), i.e. the conditions shown are all with Tg treatment. Mean ± SD of triplicate measurements from one representative experiment out of two. Of note, the reduction observed with siXBP1–1 in (a) was not reproducible. In (b and d), one representative blot out of 2 independent experiments is shown

Fig 3: CQ induces DR5 up-regulation through sustained protein stability in Caki cells.(a) Caki cells were treated 30 μM CQ for 12 h, then changed with fresh medium and pre-treated 20 μg/ml cycloheximide (CHX) for 30 min, and treated with or without 30 μM CQ for the indicated time periods. The protein expression of DR5 was determined by Western blotting (left panel). The band intensity of DR5 protein was measured using the public domain JAVA image-processing program ImageJ (http://rsb.info.nih.gov/ij) (right panel). (b) Caki cells were treated with 1 μM MG132 (as a positive control) and indicated concentrations CQ for 24 h. The cells were lysed, and the proteasome activity was measured as described in the Materials and Methods. (c) Caki/ZsGreen cells were treated with 1 μM MG132 (as a positive control) and indicated concentrations of CQ for 24 h. Proteasome activity was analyzed by using FACS analysis. (d) Caki cells were treated with indicated concentrations of CQ for 24 h. The protein levels of PSMD4/S5a and PSMA5 were determined by Western blotting. (e) Caki cells were treated with 30 μM CQ for the indicated time periods. The protein levels of Cbl was determined by Western blotting. (f) Caki cells were transfected with Cbl siRNA or control siRNA. Twenty-four hours after transfection, cells were treated with 30 μM CQ for an additional 24 h. The protein expression of DR5 and Cbl were determined by Western blotting. The level of actin was used as a loading control. The values in panel (b,c) represent the mean ± SD from three independent samples. *p < 0.05 compared to the control.

Fig 4: Involvement of RNF183 in the localization and degradation of DR5. (a) Effect of RNF183 overexpression on DR5 subcellular localization. HeLa cells transfected with EmGFP-tagged RNF183 or EmGFP alone were subjected to immunofluorescence staining with DR5 and LAMP1 antibodies (grey, EmGFP; green, DR5; red, LAMP1). (b) Effect of RNF183 knockdown on DR5 protein levels. RNF183-siRNA or non-target control (NC)-siRNA were transfected into HEK293 cells stably expressing V5-tagged RNF183. Cell lysates were subjected to western blotting with DR5 and V5 antibodies. The full-length blots are presented in Supplementary Fig. S10. (c) Quantitative graph of data from (b). Asterisks represent significant differences (n = 3; paired t-test, ***p < 0.001). (d) Cell surface expression level of DR5. HEK293 cells expressing V5-tagged RNF183 were treated with 100 µM CQ for 12 h. Then, cells were labeled with PE-conjugated anti-DR5 antibody. (e) DR5 protein levels in colonic epithelial cells from DW- and DSS-treated mice at 5 days. Cell lysates were subjected to western blotting with anti-DR5 and anti-ß-actin antibodies. The full-length blots are presented in Supplementary Fig. S11. (f) Quantitative graph of data from (e). Asterisks represent significant differences (n = 6; paired t-test, **p < 0.01).

Fig 5: TY (5 μg/mL) induced the expression of the death factors/receptors Fas L/Fas and TRAIL/DR5. (A) The expression levels of FAS mRNA at 48 h after TY treatment estimated by qRT-PCR (left figure). Time-dependent protein expression of p-FADD and death factors/receptors Fas-L/Fas and TRAIL/DR5 were examined by Western blot analysis. (B) Time-dependent expression of MIR34a after treatment with TY estimated by qRT-PCR. Expression levels of MIR34a are shown as the relative ratios with respect to the RNU6B expression levels. (C) Ectopic expression of MIR34a inhibited cell growth dose-dependently and induced apoptosis according to Hoechst 33348 staining at 72 h after transfection. The apoptotic cells are indicated by white arrows. (D) Expression of MIR34a-targeting E2F3, SIRT1, and CyclinD1 was downregulated by the TY. (E) Anti-MIR34a (Anti; 10 and 20 nM) partly canceled the activation of caspase-8 and PARP cleavage elicited by co-transfection with MIR34a. (F) Silencing of E2F3 by siR-E2F3 or MIR32a increased in the expression levels of DR5, TRAIL, and Fas.