Propidium iodide staining solution

The cell death kinetics assay of GSCs was conducted using the Nikon laser confocal microscope AX/AX R imaging system. GSCs were seeded in 24-well culture plates at a well-established growth state of 10⁵ cells/well. The different research groups of GSCs were treated and incubated with propidium iodide (PI) staining solution (BD Bioscience, USA) following the provided instructions. Real-time fluorescence and differential interference contrast imaging of cells in the culture plates were performed every 2 h from 0 to 48 h and the cell death kinetic curve was calculated based on counting and analyzing the PI-positive dead cells.

Bortezomib (Velcade^®) and antibodies against Bax, tubulin, cytochrome c, GAPDH, caspase-3, caspase-9, and PARP were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against BID and cleaved caspase-3 were purchased from Cell Signaling Technologies (Beverly, MA, USA). XIAP, cIAP1, and caspase-8 antibodies were purchased from R and D (USA). BD Cytofix/Cytoperm plus fixation and permeabilization solution kit with BD GolgiPlug, propidium iodide (PI) staining solution, annexin V binding buffer, mitochondrial membrane potential detection (JC-1) kit, stain buffer (FBS), annexin V-FITC antibody, H2AX (pS139)-Alexa Fluor 647 antibody, rabbit anti-active caspase-3- Bv605 antibody and PARP cleaved form-AF700 antibody were obtained from BD Biosciences (NJ, USA). Apoptotic DNA-ladder kit was obtained from Roche (Penzberg, Germany).

To determine the apoptosis rate, cells were treated with HJC0152 (0, 1.25, 2.5 or 5 μmol/L) for 24 hours, washed with PBS and then incubated for 15 minutes in a binding buffer containing Annexin V‐fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining solution (BD Biosciences) before flow cytometric analysis. To determine intracellular reactive oxygen species (ROS) levels, A549 or H460 cells were treated with HJC0152 (0, 1.25, 2.5 or 5 μmol/L) for 24 hours and then preincubated with 10 μmol/L 2,7‐dichlorodihydrofluorescein diacetate (DCFH‐DA) for 30 minutes at 37°C. Images were acquired under a fluorescence microscope, and the mean fluorescence intensity of DCFH‐DA was measured using a flow cytometer (Accuri C6, BD Biosciences) as previously described.21

To analyse the effects of miR-134 on apoptosis induction, cells were transfected with miR-134-mimic or NC-mimic and, 24 h later, transfected cells were treated with 15 μM cisplatin for a further 24 h. CM was then collected and cells were trypsinised and pelleted. CM was used to neutralise trypsin and to collect any apoptotic cells which may be present in the CM. Cell pellets were re-suspended in 2 ml 1X binding buffer (BB) (0.1 M HEPES, 1.4 M NaCl, 25 mM CaCl₂, pH 7.4), centrifuged at 200 g and supernatant discarded and resuspended in 30 μl of BB solution. 20 μl of cell suspension, 5 μl of Annexin-V-allophycocyanin (APC) (BD Biosciences) and 5 μl of propidium iodide (PI) staining solution (BD Biosciences) were mixed. 70 μl of 1X BB was added and incubated at room temperature in the dark for 15 mins. 400 μl of 1X BB solution was subsequently added and mixed by pipetting. Levels of apoptosis was analysed on 2 × 10⁴ cells using a BD Accuri™-C6 flow cytometer.

Following indicated treatments, cells were trypsinized, centrifuged at 240g for 5 minutes, and washed in PBS. For apoptosis detection of nonfixed cells, Alexa Fluor 647–Annexin V (Invitrogen, A23204) and Propidium Iodide (PI) Staining Solution (BD Biosciences, 556463) were used following the manufacturer’s instructions. For dichlorodihydrofluorescein diacetate (DCFDA) staining, cells were stained with 5 μM H2-DCFDA (Thermo Fisher Scientific, D399) for 30 minutes at 37°C, followed by Annexin V/PI staining. For CD36 staining, cells were stained with Brilliant Violet 421 anti–human CD36 (clone 5-271, BioLegend, 336229) antibody diluted at 1:400 in PBS containing 2% FBS for 30 minutes on ice. Cells were then washed and sorted into CD36⁺ and CD36^– populations, or fixed for subsequent intracellular staining. For tumor samples, freshly resected tumors were minced and digested in 4 mg/mL collagenase A (Sigma-Aldrich) at 37°C for 1 hour to obtain single-cell suspensions and were stained with indicated antibodies as well as a live/dead discrimination dye (see Supplemental Figure 11 and Supplemental Table 3). For all other panels, representative images showing gating strategy and antibody information can be found in Supplemental Figure 11. All flow cytometry experiments were conducted on an LSRFortessa (BD Biosciences).