Bcl 2

For the luciferase reporter assay, the 3′UTR of Bcl-2 was amplified and inserted into the pmirGLO luciferase vector (GeneCreat, Wuhan, China). The Bcl-2 (Ribo Bio, Hangzhou, China) was transfected with miR-877-3p mimic or miR-877-3p inhibitor into 293T cells, respectively, using lipo2000 (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s procedures. Forty-eight hours after transfection, the cells were harvested, and luciferase activity was detected using the dual-luciferase reporter assay system. Renilla luciferase served as the internal control.

Monoclonal antibodies specific of Caspase-3 (cleaved Caspase-3), Caspase-9 (cleaved Caspase-9), Bcl-2, myeloid cell leukemia-1 (Mcl-1), Bax, Bim, Cytochrome C (Cyt-C), and STAT3 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Caspase-3 and Caspase-9 activity assay kits were ordered from Abcam (Cambridge, MA, USA). Lipofectamine 3000 reagent was purchased from Life Technologies (Carlsbad, CA, USA). SiSTAT3 (#1,2,3) and all qRT-PCR primers including Bcl-2, Mcl-1, Bax, Bim, STAT3 and GAPDH were purchased from Ribobio (Guangzhou, Guangdong, China). All primer sequences and siSTAT3 target sequences were shown in Additional file 1: Table S1. NSCLC cells A549 were obtained from the Cell Line Bank at the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China) and PC9 were obtained from the Chinese Academy of Sciences Cell Bank of Type Culture Collection (Shanghai, China). All cells were grown at 37 °C, in a humidified 5% CO₂ and 95% air and cultured in RPMI-1640 medium (Life Technologies, Carlsbad, CA, USA) containing 10% FBS (Gibco, USA) and 0.5% penicillin–streptomycin sulfate (Invitrogen, Carlsbad, CA, USA). Cells were counted using the automated cell counter star (Invitrogen, Carlsbad, CA, USA).

Normal human astrocyte (NHA) and GBM cell lines U251-MG (U251), U87-MG (U87), and LN229-MG (LN229) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA), glutamine, and antibiotics at 37°C with 5% CO₂. Cell transfection was performed using Lipofectamine 3000 reagent (Thermo Fisher Scientific). Cell lines were transfected with either GFP-tagged LC3 plasmid (GFP-LC3; Addgene plasmid # 11546), lentivirus containing SOCS5 shRNA or control shRNA, pcDNA plasmids expressing SOCS5 or control protein, or pmirGLO plasmids containing the 3′ untranslated regions (UTRs) of wild-type or mutated Bcl-2 with the predicted binding sites of SOCS5 (RiboBio, Guangzhou, China). Transfections were performed in 24-well or 96-well pretreated culture plates, according to the manufacturer’s recommendations. TMZ (catalog no. ab141055) was obtained from Abcam (Cambridge, UK), dissolved in dimethyl sulfoxide, and used at a final concentration of 40 μM as previously reported [27 ].