Ham s f 12k media

Acetylshikonin was purchased from Yuanye Bio-technology Co., Ltd. (Shanghai, China). Emodin was purchased from National institutes for Food and Drug Control (Beijing, China). Dihydrotanshinone was purchased from Shanghai Shunbio Bioengineering Co., Ltd (Shanghai, China). Structures of dihydrotanshinone, emodin and acetylshikonin are shown in Fig. 1.
Hank's balanced salt solution (HBSS), HEPES, fetal bovine serum (FBS) were obtained from Gibco, McCoy's 5A and Ham's F12K media were obtained from Sigma (St Louis, MO, USA). Epic® 384-well biosensor cell culture compatible microplates were obtained from Corning Incorporated (Corning, NY, USA).
GW9508, TUG891, GSK137647A, MEDICA16, AH7614 and ALA were stocked at 100 mM and TAK875, GW1100 were stocked at 10 mM. The stock solution were stored at −20 °C. ALA was prepared in 95% ethanol, and others were dissolved in 100% dimethyl sulfoxide (DMSO). All compounds were freshly diluted with the assay buffer (1× Hank's balanced salt solution (HBSS) buffer, 10 mM HEPES, pH = 7.2) containing 0.1% bovine serum albumin (BSA).

A549 (human NSCLC cell) and its GEM-resistant cells (A549/GemR) were provided by Procell Life Science & Technology Co., Ltd. (Wuhan, China). The cells were maintained in Ham’s F-12K media (Sigma, USA) with 10% fetal bovine serum (Gibco, USA) in an incubator with 5% CO₂ at 37°C.
3-Methyladenine (3-MA), rapamycin (Rapa), and GEM were purchased from Selleck Chemicals (Shanghai, China). For drug handling, A549 cells were cultivated in 5 μM GEM, whereas A549/GemR cells were grown with 40 μM GEM. The concentration and processing time of 3-MA was 10 mM for 4 h, and Rapa was 10 μM for 4 h.