Annexin 5 fitc and propidium iodide kit

Apoptosis was detected from flow cytometry using an Annexin V‐FITC and propidium iodide (PI) kit (BD Biosciences, 556547) according to the manufacturer's instructions. Cells were collected by centrifugation at 3000 × g for 5 minutes, washed twice with cold PBS and gently resuspended in 1 × binding buffer at a concentration of 1 × 10⁶ cells/mL. Annexin V‐FITC (5 μL) and PI (5 μL) were introduced, gently mixed with cell suspension and incubated for 15 minutes at illuminated room temperature. The cells were immediately analysed using flow cytometry.

Licochalcone A (LicA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were used in this research: anti-Bcl-2, anti-p-JNK, anti-JNK, anti-p-ERK1/2, anti-ERK1/2, anti-p-p38, anti-p38, anti-p-Akt, anti-Akt, and anti-β-actin were purchased from Santa Cruz (CA, USA). siRNA-Beclin1, siRNA-Akt, siRNA-Atg12 were purchased from Santa Cruz (CA, USA). The anti-cleaved-caspase-3, anti-cleaved-caspase-9, and anti-cleaved-PARP, mTOR Pathway and Autophagy Antibody Sampler Kit were purchased from Cell Signal Technology. Acidic vesicular organelle was purchased from AAT Bioquest, Inc. Annexin V-FITC and propidiumiodide (PI) kit was purchased from BD Biosciences (San Diego, CA). Z-VAD-FMK was purchased from BioVision. Bafilomycin A1 was purchased from Enzo Life Sciences. LY294002 and Rapmycin were purchased from Calbiochem. pBABEpuro GFP-LC3 was a gift from Jayanta Debnath (Addgene plasmid # 22405).

Compounds 1, 2, and 3 were synthesized according to the procedure developed by our group [12 (link)]. They were dissolved in dimethyl sulfoxide (DMSO), the concentration of which never exceeded 0.1% (v/v); 50 mM of stock solution was stored at −20 °C. The Cell counting Kit-8 (CCK-8), Hoechst 33258, JC-1, and the ROS assay kit were purchased from Beyotime Institute of Biotechnology Company (Shanghai, China). The annexin V-FITC and propidium iodide (PI) kit was obtained from BD Pharmingen (BD, San Diego, CA, USA). The primary antibodies used are as follows: CDK4 (D9G3E, Cell Signaling Technology, Danvers, MA, USA), Cyclin D1 (E3P5S, Cell Signaling Technology), Caspase-3 and cleaved Caspase-3 (D3R6Y, Cell Signaling Technology), PARP (46D11, Cell Signaling Technology), Cytochrome c (AF0146, Affinity, Changzhou, China), Caspase-9 (AF6348, Affinity), cleaved Caspase-9 (D8I9E, Cell Signaling Technology), BAX (D2E11, Cell Signaling Technology), Bcl-2 (D17C4, Cell Signaling Technology), Bad (D24A9, Cell Signaling Technology), and β-actin (AF0003, Beyotime Biotechnology, Shanghai, China). All other chemicals used are commercially available and reagent grade.