Anti mrp2

Cells were lysed by ice-cold cell lysis buffer (Cat# 17081, Intron Biotechnology, Seongnam, Korea) with the protease inhibitor cocktail. Protein concentrations were determined with a BCA assay kit (Cat# 23117, Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Protein was separated via 10% SDS-PAGE, transferred to a PVDF membrane, and blocked with 5% non-fat milk. Membranes were incubated with anti-HOXB9 (Cat# PA5-101087, Thermo Fisher Scientific, Waltham, MA, USA), anti-ERCC-1 (Cat# ab129267), anti-MRP-2 (Cat# ab172630), anti-Bcl-2 (Cat# ab182858, abCAM, Cambridge, UK), anti-XIAP (Cat# 14334), anti-MMP-2 (Cat# 40994), anti-Bax (Cat# 5023), anti-cleaved caspase-3 (Cat# 9661), anti-cleaved PARP (Cat# 5625, Cell Signaling Technology, Inc., Danvers, MA, USA), and alpha-tubulin (Sigma-Aldrich, St. Louis, MO, USA) antibodies. Next, the membranes were incubated with HRP-conjugated anti-secondary Mouse IgG antibody (Cat# 7076) or HRP-conjugated anti-secondary Rabbit IgG antibody (Cat# 7074, Cell Signaling Technology, Inc., Danvers, MA, USA), and the visualized using Immobilin Forte Western HRP Substrate (Cat# WBLUF0100, Merk Millipore, Burlington, MA, USA).

Immunofluorescence staining of organoids was performed essentially as described previously^{25 (link)}. Briefly, organoids were fixed with 4% PFA for 2 hours at RT and washed twice with PBS. Organoids were blocked and permeabilized with 5% BSA-PBS including 0.2% Triton-X for 1 hour at RT. Organoids were washed once with PBS and incubated with primary antibody diluted in 2.5% BSA-PBS O/N at 4 ºC. Primary antibodies included anti-AFP (Thermo Fisher, PA5-16658, 1:250); anti-ALB (Bethyl, A80-229A, 1:500); anti-Ki-67 (Thermo Fisher, 14-5698-82, 1:1000); anti-CK7 (Thermo Fisher, MA5-11986, 1:400); anti-CK19 (Cell Signaling Technology, 13092 S, 1:500); anti-MRP2 (Abcam, ab3373, 1:500); anti-BCAT (Santa Cruz, sc-7199, 1:500); anti-A1AT (Bethyl, A80-122A, 1:250); anti-HNF4A (Abcam, ab201460, 1:250); anti-CYP3A4 (Thermo Fisher, MA5-17064, 1:250); anti-ZO1 (Thermo Fisher, PA5-19090, 1:250). Organoids were washed with PBS three times and subsequently incubated with the appropriate secondary Alexa Fluor antibodies diluted in 2.5% BSA-PBS for 3 hours at RT. After one wash with PBS, organoids were incubated with 1 μg/ml DAPI (Thermo Fisher, 62248), and cell membranes were optionally stained with Phalloidin-Atto 647N (Sigma–Aldrich, 65906, 1:1000), both in PBS for 20 min at RT. After two washes with PBS, organoids were transferred to a 96-well Sensoplate and imaged on a Leica Sp8 microscope.

The mouse monoclonal antibodies used in this study were: anti-MUC1 (clone BSB-44, Bio SB, dilution 1:50), anti-CEA (clone BSB-31, Bio SB, prediluted), anti-CA19-9 (clone 121SLE, dilution 1:75), anti-MRP2 (clone M2III-6, dilution 1:100), anti-MRP3 (clone M3II-9, dilution 1:5), anti-Pg-P (clone JSB-1, dilution 1:5), anti-TIM3 (clone TIM3/3113, dilution 1:50), anti-PD-L1 (clone ABM4E54, dilution 1:500), anti-HLA-ABC (clone EMR8-5, dilution 1:50), anti-IL-8 (clone 807, dilution 1:50) (Abcam, Cambridge, UK), anti-survivin (clone 8E2, dilution 1:30), anti-TNF-a (clone F6C5, dilution 1:50), and anti-TGF-b (clone TB21, dilution 1:50) (ThermoFisher Scientific, Waltham, MA, USA). The rabbit polyclonal antibodies used were: anti-A2aR (PA-33323, ThermoFisher Scientific, dilution 1:200), anti-IFN-g (ab25101, dilution 1:500), anti-CXCR4 (p-S339) (ab74012, dilution 1:75), anti-CCR7 (ab140758, dilution 14 mg/mL), and anti-IL-6 (ab6672, dilution 1:600) (from Abcam).