Fig 2: DR4 is critical for Andro-mediated sensitization of 786-0 cells to TRAIL. (A and B) DR4 and DR5 protein levels in 786-0 cells treated with TRAIL (50 ng/ml) and/or Andro (5 µM) for 24 h. (C) DR4 and DR5 protein levels in siRNA-targeted DR4 or DR5-transfected (siDR5 and siDR4) cells. (D) Cell viability of 786-0 cells transfected with siDR4 and/or siDR5 and treated with the combination of TRAIL (50 ng/ml) and Andro (5 µM) for 24 h (two-way ANOVA, Tukey). (E) DR4 and DR5 protein levels in siRNA-targeted DR4 or DR5-transfected cells and treated with Andro (5 µM) for 24 h. (F) Cell apoptosis was determined by Annexin V-FITC after transfection with siDR4 or siDR5 and treated with the combination of TRAIL (50 ng/ml) and Andro (5 µM) for 24 h. (G) Effects of TRAIL (50 ng/ml) and Andro (0.5 µM) on the clonogenic formation of 786-0 cells after transfection with siDR4 or siDR5. Data are shown as mean ± SD; n.s. (not significant), P>0.05, ***P<0.001, n=3). Andro, andrographolide; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; DR, death receptor; PI, propidium iodide.

Fig 3: Orthogonal genetic screens and surface marker analysis specify SLC39A7 loss-of-function phenotype on receptor trafficking. a Spider plot summarizing the results of 8 independent CRISPR/Cas9 screens in KBM7 wildtype, KBM7 FADD-, or HAP1 cells using an SLC-specific gRNA library. Each plot section represents one screen with the indicated stimuli. Screen analysis was performed by identifying differentially enriched sgRNAs using DESeq2 and then aggregating sgRNAs to genes using Gene Set Enrichment Analysis. Identified hits are ranked according to the adjusted p-value of enrichment (–log10(padj)), bubble size indicates the number of significantly enriched sgRNAs and color the average log2 fold-change (aLFC) of the enriched sgRNAs. Screens were performed in duplicate. SLC39A7 is highlighted in orange. No gene was identified in KBM7 wildtype cells upon TRAIL treatment. b MCA of Jurkat E6.1 SpCas9 cells transduced with a GFP marker (GFP+) and sgRNAs targeting either SLC39A7 or Renilla luciferase (sgRen) as control, against cells transduced with sgRen and an mCherry marker (mCherry+). The cell populations were mixed at 1:1 ratio, treated with TRAIL (20 ng/ml) or FASL (1 ng/ml), and analyzed after 14 days by flow cytometry. Data represent mean value ± s.d. of two independent experiments performed in duplicates, n.d. (not determined) indicates wells with no outgrowth. c Flow cytometry analysis for TRAILR1 (left) and TRAILR2 (right) surface expression in KBM7 FADD-, KBM7 FADD- sgRen or KBM7 FADD- SLC39A7- cells. Data shown are representative of two independent experiments

Fig 4: CtBP loss activates caspase 8 via death receptor 4.a, b KURAMOCHI cells were infected with indicated shRNAs. Cell death receptor expression was examined by western blotting (a), DR4 and DR5 mRNA levels were examined by qPCR (b), **p < 0.01 as compared with shCtrl group. Arrow in FAS blot points to unmodified FAS, and higher molecular weight forms are consistent with known glycosylation58. c–e KURAMOCHI cells were treated with indicated siRNAs along with shRNAs. Cleaved caspase 8, and the knockdown efficiency of DR4 and CtBP1/2 was determined by immunoblotting (c); caspase 8 activity was examined by enzymatic assay (d), **p < 0.01 as compared with shCtrl group; and viable cells were counted by Trypan blue assay at indicated time points (e). ***p < 0.001 as compared with shCtrl group.

Fig 5: CtBP represses DR4 and DR5 depending on cell context.a DR4 and DR5 protein levels were examined by western blotting in indicated HGSOC cell lines. b–d OVCA429, KURAMOCHI, and SKOV3 were infected with indicated shRNAs. DR4 protein was examined by western blotting (b, c), mRNA level of DR4 was analyzed by qPCR (d), **p < 0.01; ***p < 0.001 as compared with shCtrl group. e, f OVCA429 and KURAMOCHI cells were infected with indicated shRNAs, and DR5 protein level was examined by western blotting (e), DR5 mRNA level in OVCA429 cells was analyzed by qPCR (f), **p < 0.001 as compared with shCtrl group.