*4.6. Western Blot Analysis*

After stimulating with compounds, we washed the cells twice with phosphate-buffered saline (PBS) and lysed the cells with the RIPA buffer including 1% phosphatase and 1% protease inhibitors. Protein lysis was quantified by a BCA kit (Solarbio, Beijing, China) and separated by 10% SDS-PAGE. Later they were transferred onto nitrocellulose membranes (GE Healthcare) at 90v for 2 h and incubated with primary antibodies at 4 ◦C overnight. After that, they were incubated with anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h, detected with chemiluminescence HRP substrate (Millipore, Billerica, MA, USA) and photographed by Chemiluminescence Imaging System (Tanon 5200, Shanghai, China).

#### *4.7. Cell Viability and Antiproliferation Activity Assay*

Cell viability and antiproliferation activity was evaluated by the resazurin indicator [51]. Cells (3500 cells/well) were seeded into 96-well plates overnight and treated with the vehicle or indicated drug concentrations for 72 h. An amount of 10 μL of 1 mg/mL resazurin solution was added per well and, after incubating for 3 h, relative cell viability was detected using a SpectraMax Mode Plate Reader (Molecular Devices, Shanghai, China) at a 595-nm emission wavelength and a 549-nm excitation wavelength. The halfmaximal inhibitory concentration value (IC50) of **11c** was determined using GraphPad Prism 8 software.
