Vu0155069

Cells following 48 h of siRNA transfection or intact cells were seeded in a 96-well plate at 5000 cells per well. After the cells attached to the bottom, a series of doxorubicin concentrations was applied to the wells. For PLD1 inhibitor experiments, a series of doxorubicin concentrations combined with 10 μM of PLD1 selective inhibitor VU0155069 (Santa Cruz Biotechnology) was applied to the wells after 48 h of seeding intact cells. After 48 h or 72 h of treatment, WST-1 assay reagent (Roche Life Science, Indianapolis, IN) was added according to the manufacturer's protocol and incubated for 2 h, followed by detecting the absorption at 450 nm. We measured 3 technical replicates for each of 4 biological repeats for each experimental group.

Pharmacological inhibitors used in this study are summarized in Fig. S1 and Table S1. HLEKs or hTCEpi cells were plated in a six-well plate. After overnight incubation, the cells were exposed to ir-antago, antago-103, or antago-107 (or mixture of antago-103 and antago-107) with and without an inhibitor. For the sequential treatment schedule, cells were treated with ir-antago, antago-103, or antago-107 for 24 h for the generation of vacuoles. After 24 h, each inhibitor was applied in cells. Amiloride, EIPA, roscovitine, manumycin A, Ro-32-0432, FAK inhibitor 14, NSC23766, EUK134, 8-bromo-cAMP, VU0155069, Go6983, Rottlerin, IBMX, and propranolol hydrochloride were obtained from Santa Cruz Biotechnology, Inc.; O-tricyclo[5.2.1.0(2,6)]dec-9-yl dithiocarbonate potassium salt (D609), l-α-phosphatidic acid sodium salt, and chloroquine were obtained from Sigma-Aldrich. BafA1 was from Cayman. PP2 was from EMD Millipore. ZCL278 was from Tocris Bioscience. SB203580 was from Cell Signaling Technology. BI-D1870 was from Enzo Life Sciences. Z-VAD-FMK was from BD. For short-term treatment of BafA1, cells were pretreated with BafA1 for 1 h, then medium with BafA1 was removed and cells were incubated in fresh medium with antagomirs for 24 h.