Resazurin assay

The cytotoxicity assay was performed using the methods described previously [18 (link),19 (link)]. Huh7 cells were used in resazurin assay (Cayman Chemical) and treated with indicated concentrations (12.5, 50.0, and 200.0 µM) of compounds for 72 h. The DMSO control was assigned 100% of relative cell viability. The positive control, Sorafenib, inhibited the 52% growth of Huh7 cells at 12.5 µM.

The human hepatocellular carcinoma (HCC) Huh7 cells were used in the resazurin assay (Cayman Chemical) to evaluate the cytotoxicity of compounds (Supplementary Table S13). The method was performed as described previously [18 (link),19 (link)], and the detailed process of the cytotoxicity assay was the same as in our previous publications [12 (link),25 (link)]. Sorafenib, the positive control, inhibited 52% of the growth of Huh7 cells at 12.5 µM, and the DMSO controls were assigned 100% of relative cell-viability.

The human cancer
cell lines, lung adenocarcinoma A549, multiple myeloma RPMI8226, and
glioma U87 were obtained from the American Tissue Culture Collection
(ATCC, Manassas, VA, USA). Human breast adenocarcinoma MDA-MB-231,
human embryonic kidney cells HEK-293T, and human vascular endothelial
EA.hy926 cell lines were purchased from Bioresource Collection and
Research Center (BCRC, Hsinchu, Taiwan). Cells were maintained in
a humidified incubator at 37 °C in 5% CO₂, and cell
viability was determined with the resazurin assay (Cayman Chemical,
USA) after the incubation with compounds for indicated concentrations
and times or DMSO as control.^{72 (link)}