Dmi3000

Cells were fixed in 4% paraformaldehyde for 10 min at room temperature and were blocked with blocking buffer (5% semi-skimmed milk with 0.1% Triton in PBS) for 2 hr at room temperature. Primary antibodies were diluted in blocking buffer and incubated at 4°C overnight. Primary antibody was carefully washed away with 0.1% Triton in PBS three times with 10 min incubation between each wash. Secondary antibody diluted in blocking buffer (1:1000) was incubated at room temperature for 1 hr followed by 3 washes with 0.1% Triton in PBS. Nuclei were counterstained with DAPI. Primary antibodies used were Nanog (eBioscience, 14–5761, RRID:AB_763613, 1:200) and Lin28a (Cell signalling, 3978, RRID:AB_2297060, 1:800; 8706, RRID:AB_10896850, 1:200). Images from random fields were taken with Leica DMI3000 and the images from different fields at each time point were combined and analysed using CellProfiler software (Broad Institute, RRID:SCR_007358) to conduct nuclear and cytoplasmic compartmentalisation and total fluorescent intensity for each sub-cellular compartments as well as the whole cell for each cell was extracted for correlation analysis.

Acridine orange (AO) staining was used to visualize the volume of the cellular acidic compartments. AO is an acidotropic flourescent dye that stains DNA and cytoplasm components a bright green. Cells were treated with AO (1 μg/ml) in serum-free medium for 15 min at 37°C.
For AO staining analysis using flow cytometry, cells were washed and then illuminated with blue excitation light (488 nm). The mean fluorescence emission of green light (510–530 nm) and red light (650 nm) of 1 × 10⁴ cells were measured with a BD FACSCanto II Flow Cytometer (BD Biosciences, USA).
For AO staining analysis using fluorescence microscopy, an inverted fluorescence microscope was used (Leica DMI3000, Solms, Germany).

U87MG (Uppsala 87 Malignant Glioma) human glioma cells used in the experiments were acquired from the American Type Culture Collection (ATCC). Cells were certified mycoplasma free by regular testing (microbiome.nl). Cells were cultured at 37 °C and 5% CO₂ and maintained as monolayer in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum, 1% Penicillin/streptomycin and 2% hepes serum. Sterile phosphate buffered saline (PBS) and 0.25% Trypsin-Ethylenediaminetetraacetic acid (Trypsin–EDTA) were obtained from Gibco (Paisly, UK) dilutions. U87 spheroids were formed by plating 3000 cells/well in a transparent U-bottom 96 well cell-repellent surface plate (Cellstar®). Four days were allowed for spheroid formation before starting the treatment. Images were made with a microscope (Leica DMI3000) using the Universal Grab 6.3 software (DCILabs). The size of the spheroids was determined using the Scratch assay 6.2 software (DCILabs) and analyzed with GraphPad.

The Transwell chamber (8 μm, Corning #3422, ME, USA) was placed into a 24-well plate. The transfected cells were diluted with 200 μl 0.1% FBS medium to generate a cell suspension with a density of 1 × 10⁶ cells/mL and seeded in the upper chamber. Then, 600 μl RPMI-1640 containing 30% FBS was added to the basolateral chamber. Each group was tested in 3 replicates. After culture at 37°C with 5% CO₂ for 72 h, the Transwell chambers were removed, the cells were rinsed with PBS twice, fixed and stained with 0.3% crystal violet for 15 min, and rinsed in ddH₂O three times. Cells in the upper chamber were removed. The invasive cells were observed with an inverted microscope (DMI 3000, Leica, IL, USA) (×100), and 5 fields were randomly selected for cell counts.

The cultured cells were washed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. Following inhibition, the cells were initially incubated with primary antibody overnight at 4°C, and then with fluorescein isothiocyanate- or Cy3-conjugated goat anti-rabbit IgG antibody (1:200; Sigma-Aldrich) and 5 μg/ml DAPI (Sigma-Aldrich) at room temperature for 30 min. Then, the cells were thoroughly washed with TBST and viewed through a fluorescence microscope (DMI3000; Leica, Allendale, NJ, USA).

TUNEL assay was performed as previously described (27 (link),29 (link)). Briefly, each group of cells treated as indicated was fixed with 4% paraformaldehyde, rinsed with PBS, then permeabilized with 0.1% Triton X-100 for fluorescein isothiocyanate (FITC)-end-labeling the fragmented DNA of apoptotic SH-SY5Y cells using a TUNEL cell apoptosis detection kit (Beyotime Institute of Biotechnology, Shanghai, China). The FITC-labeled TUNEL-positive cells were imaged under a fluorescent microscope (DMI3000; Leica, Allendale, NJ, USA) using 488 nm excitation and 530 nm emission.