Bax d2e11

PPI was purchased from the Institute for Drug Control (Shanghai, China, #111590) and dissolved in dimethyl sulfoxide (DMSO). Doxorubicin was purchased from Sangon Biotech (Shanghai, China, #DB3456) and dissolved in ultra-purified water. FITC Annexin V and propidium iodide were obtained from BD Biosciences (Franklin Lakes, USA, #556420). The cell cycle detection kit was from Key GENBioTECH (Nanjing, China). Antibodies against PARP (#9542), BAX (D2E11, #5023), BCL-2 (#2876), Vimentin (5G3F10, #3390), C-Myc (D84C12, #5605), GSK-3β (27C10, #9315), phosphor-GSK-3β (#8213), active β-catenin (D13A1, #8814) and β-catenin siRNA II (#6238) were from Cell Signaling Technology (Danvers, USA). Trypsin-EDTA, Lipofectamine 2000 (#11668) and TRIzol reagent (#15596) were from Invitrogen (Rockville, MD, USA). CHIR99021 (#SML1046) and antibody against β-actin (#A2228) were from Sigma-Aldrich (Saint Louis, USA). Monoclonal antibody of rabbit anti-Ki-67 and RIPA Cell Lysis Buffer (#P0013B) were got from Beyotime Institute of Biotechnology (Shanghai, China). SuperSignal Chemiluminescent HRP Substrate (#34078) was from Thermo Fisher scientific Inc (Rockford, USA). Matrigel (#356234) was purchased from BD Biosciences (Bedford, USA). PrimeScript RT reagent kit (#RR037A) and SYBR Premix Ex Taq II (#RR420L) were bought from TaKaRa (Dalian, China).

Total proteins were extracted from human glioma cells using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) and heated for 5 min. 4–15% Mini-PROTEAN® TGX™ Precast Gels (BIO-RAD, California, USA) were used to separate the proteins which were then transferred onto PVDF membranes. The membranes were blocked for 20 min at room temperature using StartingBlock™ (PBS) Blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 4 °C overnight with primary antibodies including CD13 antibody (rabbit, Cat #ab108310, 1/500; Abcam, Cambridge, UK), BAX (D2E11) (rabbit, Cat#5023, 1/1000; Cell Signaling Technology, Danvers, MA, USA ), BCL-2 (mouse, Cat#15071, 1/1000; Cell Signaling Technology), NOXA (mouse, Cat#ab13654, 1/1000; Abcam), Caspase-3 (D3R6Y) (rabbit, Cat#14220, 1/500; Cell Signaling Technology) and GAPDH antibody (mouse, Cat #ab9484, 1/1500; Abcam). After washing several times with TBS-T (0.05% Tween20), the membranes were incubated with HRP-conjugated secondary antibodies (1/200; Dianova, Hamburg, Germany) for 2 h at room temperature. The blots were washed several times before the enhanced chemo-luminescence detection kit (ECL Advance) was applied and luminescence was measured.

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.