Ix 73 inverted microscopy

To examine whether engineered hNSCs can migrate to lymph node–derived metastatic colorectal adenocarcinoma, SW-620 cells (1×10⁵ cells per well) and human dermal fibroblast cells (a control, 1×10⁵ cells per well) were plated in a 24-well plate containing 1% CD-FBS phenol free DMEM medium with incubation for 24 hours. The bottom surface of transwell plates (0.4 μm, BD Biosciences, Dickinson, Franklin Lakes, NJ) coated the with fibronectin (250 μg/mL, Sigma-Aldrich) was placed in the 24-well plates and CM-Dil (Invitrogen Life Technologies) pre-stained engineered hNSCs were plated in the upper chambers of the transwell plates at a density of 1×10⁵ cells per well in the same condition medium and cultured for 24 hours at 37°C. After washing the lower chamber, the cells were fixed with 10% formalin solution (Sigma-Aldrich) for 10 minutes and permeabilized using 100% cold-methanol (Sigma-Aldrich) for 10 minutes. Then, DAPI (4',6-diamidino-2-phenylindole, Invitrogen Life Technologies) was added to the lower chamber at 300 nM and the plates were incubated for 10 minutes at 37°C followed by washing with PBS. Cells stained with CM-Dil and DAPI were examined by fluorescence microscopy (IX-73 Inverted Microscopy, Olympus, Tokyo, Japan) and counted by Cell Sense Dimension.

Intracellular ROS generation in VM7Luc4E2 cells was measured by 20,70-dichlorofluorescein diacetate (DCF-DA) assay. VM7Luc4E2 cells were seeded at a density of 1×10⁵ cells per well in a 6 well plate with 200 μl phenol-free DMEM media containing 5% CD-FBS. After 24 h, the medium was replaced with new medium containing E2 (0.001 μM), TCS (1 μM), BPA (1 μM), Kaem (30 μM), DIM (15 μM), a combination of E2 (0.001 μM), TCS (1 μM) or BPA (1 μM) and Kaem (30 μM), and a combination of E2 (0.001 μM), TCS (1 μM) or BPA (1 μM) and DIM (15 μM), after which cells were incubated for an additional 72 h. Moreover, H₂O₂ was used as a positive control to detect ROS formation in VM7Luc4E2 cells. Briefly, 3% H₂O₂ solution was added to the wells with 1×10⁵ cells of VM7Luc4E2 cells at 1% concentration and incubated for 30 min. Following incubation, the culture medium was removed, and each well was treated with 200 μl H2DCF-DA solution (10 mM in PBS) for 30 min. This plate was then placed in a dark room at room temperature for 30 min, after which the fluorescence intensity of DCF (an oxidized form of H2DCF) was measured and samples were photographed using a fluorescence microscope (IX-73 Inverted Microscopy, Olympus, Japan) to detect ROS production. The ROS production induced by each treatment was quantified using the Cell Sens Dimension software (Olympus).

A terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay was processed to detect the fluorescence of apoptotic cells using TdT enzyme with the fluorometric TUNEL assay kit (Promega, Madison, WI, USA). Briefly, VM7Luc4E2 cells were seeded at 1×10⁵ cells per well in a 6 well plate with DMEM media. After 24 h, the medium was replaced with new medium containing E2 (0.001 μM), TCS (1 μM), BPA (1 μM), Kaem (30 μM), DIM (15 μM), a combination of E2 (0.001 μM), TCS (1 μM) or BPA (1 μM) and Kaem (30 μM), and a combination of E2 (0.001 μM), TCS (1 μM) or BPA (1 μM) and DIM (15 μM). After 48 h, cells were fixed with 4% methanol-free formaldehyde (pH 7.4) for 1 h, permeabilized using lysis buffer (1% Triton X-100 in 1% sodium citrate) for 15 min, and then treated with 50 μL TdT enzyme buffer, which attached to DNA strand breaks. Finally, labeled strand breaks were visualized by the attachment of fluorescein isothiocyanate-5-dUTP. All slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen Life Technologies, Carlsbad, CA, USA), then viewed using a fluorescence microscope (IX-73 Inverted Microscopy, Olympus). Fluorescence by each treatment was quantified with the Cell Sens Dimension software (Olympus).

To observe the ability of hNSCs to migrate to melanoma and normal human lung cells, both A375SM and L132 (1×10⁵ cells/well) were plated in a 24-well plate containing 5% CD-FBS phenol free DMEM and incubated for 24 hours. The bottom surface of the transwell with 8.0-μm pores (BD Biosciences, Dickinson, Bedford, MA) was coated with fibronectin (25 μg/mL, Sigma-Aldrich) and placed in 24-well plates and engineered hNSCs (1×10⁵ cells/well) were seeded in the upper compartment of the transwell on the next day. After 24 hours of incubation, all lower chambers were washed with PBS, migrated hNSCs were fixed using 3.7% formaldehyde solution (Sigma-Aldrich) for 15 minutes, and the cell underwent permeabilization using 10% coldmethanol (Sigma-Aldrich) for 15 minutes. Crystal violet was used to stain the surface of the transwell for 15 minutes and all transwells were rinsed thoroughly with PBS. Crystal violet stained hNSCs were visualized by fluorescence microscopy (IX-73 Inverted Microscopy, Olympus, Tokyo, Japan) and were counted with Cell Sense Dimension.