Mrp 1

An antibody against PAR1 was purchased from BECKMAN COULTER. Anti-E-cadherin, fibronectin, YAP1, phospho-YAP1 (pYAP1), Lats1 and phospo-Lats1 (pLats1) were purchased from Abcam. Anti-ABCG2, -MRP1, and -P-glycoprotein (P-gp) were purchased from GeneTex. Anti- ribophorin II (RPN2) was purchased from OriGene. Anti-GAPDH was from IMGENEX. The selective PAR1 agonist TFLLR-NH₂ and PAR1 antagonist SCH79797 was purchased from Tocris Bioscience. We used TFLLR-NH₂ (EC50: 1.9 μM) at the 20 μM, and SCH79797 (IC50: 70 nM) at the 70 nM. C3 transferase and Y27932 were purchased from Cytoskeleton. We used C3 at the 2 μg/ml and Y27632 at 10 μM. Small interfering RNA (siRNA) directed against PAR1 (5′-AAGGCUACUAUGCCUACUACU-3′) was synthesized by TOYOBO, and siRNA directed against YAP1 (5′-GGUCAGAGAUACUUCUUA-3′) and Snail (5′- CCACAGAAAUGGCCAUGGGAAGGCCUC-3′) were synthesized by Sigma. Control siRNA is a scrambled sequence with no homology in the human genome (Qiagen) is listed as follows: Scrambled, UUCUCCGAACGUGUCACGUdTdT.

Cells were plated at 6×10⁵ cells/well in six-well plates and transfected as described above.
The proteins were extracted, and the total protein concentration was determined by Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA) following manufacturer’s manual. The absorbance was measured by a spectrophotometer (CT-5600; ChromTech, Inc, Apple Valley, MN, USA). Proteins were separated in 8%–13.5% polyacrylamide-SDS gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% skim milk in TBST (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20), the membranes were incubated with specific antibodies against Calnexin, HuR, HA, c-Myc, caspase-3, -9 (Cell Signaling Technology Inc., Beverly, MA, USA), galectin-3, P-gp, MRP1, Bcl-2 (GeneTex Inc., Hsinchu, Taiwan), and β-actin (Millipore, Billerica, MA, USA) overnight at 4°C. These membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Inc., PA, USA) for 1 h at room temperature. The detection of the signal was performed by incubating blotted membranes with enhanced chemiluminescence detection kit (PerkinElmer Life Sciences, Waltham, MA, USA).

Primary antibodies specific for HIF-1α, Nrf2, p-Nrf2, GCLM, MRP-1, GAPDH, and Histone H3 were purchased from Genetex. The NQO1-specific antibody was purchased from Epitomics. The GCLC antibody was purchased from BTLAB. The rabbit secondary antibody conjugated with HRP and the rabbit secondary antibody conjugated with dylight 488 were purchased from Jackson. The following chemicals, including Thiazolyl Blue Tetrazolium Bromide (MTT), dimethyl sulfoxide (DMSO), cisplatin (CDDP), trigonelline, buthionine sulfoximine (BSO), dicoumarol (DIC), dithiothreitol (DTT), and N-acetyl-L-cysteine (NAC), were purchased from Sigma Aldrich. Neomycin (G418) was purchased from Merck. The following siRNAs were purchased from Omic Bio: HIF-1α (CCA UAU AGA GAU ACU CAA ATT), Nrf2 (GCA CCU UAU AUC UCG AAG UTT), GCLC (GCA UAG ACA CCA UCA ATT), and NQO1 (GUG GCU UCC AAG UCU UAG ATT). The PCDNA3-Myc3-Nrf2 plasmid was purchased from Addgene (Plasmid #21555).