Lcl161

Induction of apoptosis was performed using a combination of 20 ng/ml recombinant mouse or human TNF (PeproTech), 10 μg/ml CHX (Selleckchem), and 5 μM LCL-161 (MedChemExpress) as reported^{23 (link)}. The concentrations of GSK’872 (Selleckchem) and Z-IETD-FMk (Selleckchem) used were 10 μM as reported^{23 (link)}. To induce necroptosis, the same concentrations were used, with the addition of 20 μM zVAD-fmk (Selleckchem) as reported^{23 (link)}. At the endpoint of the experiment, cells were stained with the FITC Annexin V Apoptosis Detection Kit (BD Biosciences) following the manufacturer’s instructions and analyzed using a BD LSRFortessa flow cytometer (BD Biosciences). To detect the cell-surface level of TNFR1, cells were stained with APC-CD120a (Biolegend). Flow cytometry data were analyzed by Flowjo_V10. For live imaging of organoid cell death, the organoids were incubated with CellEvent Caspase-3/7 Green Detection Reagent (Thermo Fisher Scientific) and PI (BD Biosciences) for 30 minutes at 37 °C. Images were acquired using the ZEISS Celldiscoverer 7 imaging system (ZEISS).

The compounds used in this study include LCL161 targeting XIAP, Fludarabine inhibiting DNA synthesis, OSI‐027 blocking mTOR, Rucaparib, and Niraparib targeting PARP1, which were chosen based on their selective inhibitory effect on MDA‐MB‐231 cells (Garnett et al,2012; Iorio et al,2016). Compounds LCL161, Fludarabine, OSI‐027, Niraparib, and Rucaparib were obtained from MedChemExpress and dissolved in DMSO. For scRNA‐seq experiment, 0.1 µM of LCL161, 0.15 µM of Fludarabine, 2.5 µM of OSI‐027, 15 µM of Rucaparib, and 12.5 µM of Niraparib were used to treat the cells for 4, 8 or 24 h.

LCL-161,
AZD5582, birinapant,
and BV6 were obtained from MedChemExpress. Human TNF-α was obtained
from Miltenyi Biotec. MG132, MLN4924, and MLN7243 were purchased from
SelleckChem.
Primary antibodies used for immunoblotting include
BIRC2 (BioRad; VMA00532; clone AB01/3B4), cIAP2 (Cell Signaling; 3130S;
clone 58C7), XIAP (Cell Signaling; 14334S; clone D2Z8W), VHL (Cell
Signaling; 68547S), CRBN (Sigma-Aldrich; SAB2106014), Ikaros (Cell
Signaling; 14859S; clone D6N9Y), Aiolos (Cell Signaling; 15103S; clone
D1C1E), HIF-1α (BD BioSciences, 610958; clone 54), α-tubulin
(Sigma-Aldrich; T5168; clone B512), and beta-actin (Sigma-Aldrich;
A1978). Secondary antibodies include anti-rabbit IgG HRP-linked antibody
(Cell Signaling; 7074) and anti-mouse IgG HRP-linked antibody (Cell
Signaling; 7076).

Birinapant and LCL-161 were purchased from MedChemExpress. zVAD-fmk was purchased from Bachem. Necrostatin-1 was obtained from Tocris Bioscience. MRT67307, TPCA-1 [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide and Protease Inhibitor Cocktail Set V were purchased from EMD Millipore.

Restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA); Reagents: ETV (Bristol-Myers Squibb, New York, NY, USA), Debio 1143 (Debiopharm, Lausanne. Switzerland), Birinapant (HY-16591, MedChemExpress, Monmouth Junction, NJ, USA), LCL-161 (HY-15518, MedChemExpress). Antibodies: HBsAg for WB (H166, Abbott, Chicago, IL, USA), HBcAg for WB (ID8, in house), cIAP1 for WB (ALX-803-335-C100, Enzo, New York, NY, USA), β-actin conjugated to HRP (5125S, Cell Signalling Technology, Danvers, MA, USA), HBcAg for liver IHC (B0586, Dako, Santa Clara, CA, USA), HBcAg for organoid IHC and IF (ab115992, Abcam, Cambridge, UK), cleaved caspase 3 for IF (C10423, Thermo Fisher Scientific, Waltham, MA, USA), DAPI for IF (D1306, Thermo Fisher Scientific).

All reagents and solvents were procured from Thermo Fisher or Sigma‐Aldrich and employed without further purification. Small‐molecules (Table S3, Supporting Information), namely 2‐Fucosyllactose, 2‐NP, Baicalein, Eganelisib, Entinostat, KIN‐1408, LCL161, MSA‐2, Picroside, Pidotimod, R848, Ruxolitinib, SR‐717, and Tilorone, were purchased from MedChemExpress; CRX527, MPLA, M‐TriDAP, and Murabutide were obtained from InvivoGen, while RBN2397 was acquired from AmBeed. The compounds were dissolved in dimethyl sulfoxide (DMSO) as appropriate and were utilized without further processing. MilliQ water was sourced from the Waters filtration system.