Low attachment 96 well plates

A549 cells were grown in 96‐well low attachment plates (Corning, New York, NY, USA) for 5 or 10 days with 0.015% (w/v) FP001 (initial cell number, 2000 cells/100 μL/well). Spheroids were stained by adding 100 μL phenol red‐free DMEM supplemented with 40 μg/mL Hoechst 33342 (Life Technologies, Carlsbad, CA, USA) per well. After incubation for 45 min at 37°C, the culture plates were centrifuged at 420 g for 10 min and supernatants (150 μL) were changed to fresh media. The culture plates were centrifuged at 420 g for 10 min, and then spheroid size was analyzed using Cellomics Arrayscan VTI platform (Thermo Fisher Scientific, Waltham, MA, USA).

Cells were transfected with miR-367 or non-specific control and plated 24 h later into 96-well low-attachment plates (Corning, New York, NY), at a density of 1 × 10³ cells/mL in DMEM/F12 medium containing 20 ng/mL EGF, 20 ng/mL bFGF, 1× B-27 and 1× N2 (Invitrogen). Well-developed neurospheres (D283-Med and Daoy: ≥30 μm in diameter; CHLA-01-Med: ≥50 μm in diameter; USP-13-Med: ≥100 μm in diameter) were measured at day 4. D283-Med, Daoy and USP-13-Med neurospheres were dissociated with 0.05% trypsin/1 mM EDTA (Tryple, Life Technologies) and CHLA-01-Med neurospheres were dissociated with Cell Strainer Cap (Corning) to reach single cell suspension. For CHLA-01-Med, Daoy and D283-Med cells, 1 × 10⁵ cells were labeled with CD133/1 PE antibody (AC133; Miltenyi Biotec, Cologne, Germany) for 40 min at 4°C. Cells were analyzed in the flow cytometer FACS Aria II with DIVA software (BD Biosciences, Franklin Lakes, NJ). USP-13-Med cells were labeled as described above, fixed (PFA 4% for 1 h) and permeabilized (0.1% Triton X-100 for 10 min) before analysis in the Guava easyCyte 5HT Flow Cytometer (Millipore). Cells displaying fluorescence higher than the basal fluorescence of unstained cells were deemed CD133-positive.