Ix53 dp80

Intracellular ROS levels were measured using a ROS Assay Kit (S0033S, Beyotime, Shanghai, China) according to the manufacturer’s protocols. CaP cells treated with T-96 or the control were incubated with serum-free DMEM or F12K containing 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA, 10 µM) for 20 min. Subsequently, cell suspensions were centrifuged and washed three times with serum-free DMEM, and then visualized using a fluorescence microscope (IX53/DP80, Olympus Corporation, Japan).

Glioma tissue was isolated from each group of nude mice (treatment groups treated with OA^nano and control groups with DMSO) as described above and fixed by 4% PFA. Then the tissue was dehydrated, embedded in paraffin through standard process (70% ethanol 3 h, 85% ethanol 1 h, 95% ethanol 1 h or overnight, 100% ethanol 1 h x 2, 1/2 ethanol + 1/2 xylene 20 min, xylene 30 min x 2, paraffin I/II/III 1 h respectively) and cut into slices. Sections, which were 4μm thick, were stained with HE and IHC using antibodies against Vimentin. HE and IHC staining results were analyzed by Image proPlus6.0. Another part of Glioma tissue was washed with PBS three times and then fixed by 4% PFA for 0.5h, then permeabilized by 0.1% Triton X-100 for 15min at room temperature and blocked in 5% BSA (Sigma, USA) for 1h. The cells were immunostained with antibody to Caspase-3 (rabbit anti-human Caspase-3, 1:200) at 4°C overnight and then incubated with the secondary antibody (goat anti-rabbit, Boster, Wuhan, China) for 0.5h at room temperature, exposing to no light as possible. The nuclei were counterstained with DAPI. The photos were taken by using a fluorescence microscope (Olympus IX53/DP80, Japan).

The average levels of intracellular ROS in MCF7 and ZR75-1 cells were detected using 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) by a ROS Assay Kit from Beyotime. MCF-7 and ZR-75-1 breast cancer cells (1 × 10⁵ cells/well) were seeded into six-well plates and were cultured for 24 h. After exposure to the mentioned magnetic field for 3 h, with or without pre-treatment with NAC (5 mM) for 1h, the cells were stained with 10 uM DCFH-DA at 37 °C for 30 min in the dark and washed with PBS for three times. After that, the intracellular ROS levels of cells were captured by a fluorescence microscope (Olympus IX53/DP80, Tokyo, Japan) for fluorescent photo and were analyzed by a multi-mode microplate reader (Biotek, Synergy H1, Winooski, VT, USA) for the fluorescence intensity.

Single-cell suspensions of ovarian cancer cells (4 × 10² cells/mL) were plated on ultra-low attachment 24 wells plates (Corning, America) and cultured in phenol red-free DMEM/F12 (Gibco, America) containing B27 supplement (Gibico, America, #12587010) and 20 ng/mL epidermal growth factor (EGF, Solarbio, China, #P00033), 20 ng/mL basic fibroblast growth factor (bFGF, Solarbio, China, #P00032), and 5 μg/mL insulin (Solarbio, China, #I8040). Tumorsphere were visualized under a phase-contrast microscope (Olympus, America, IX53 + DP80).

Thaw the stock 10 mL Matrigel® (Corning, America, #356234) overnight at 4 °C by placing inside at cold room. Dilute Matrigel at a 1:8 ratio with chilled serum-free growth medium before coating. 100 μL the chilled diluted Matrigel was placed directly onto the center of the Transwell insert. Place the plates in a humidified incubator at 37 °C for 60 min to allow gelling. Meanwhile, a proper density of cancer cells in 200 ml serum-free growth medium were added to the upper chamber of replicate and 500 μL serum-containing medium was added to the lower chamber. The plate was incubated for 24–72 h at 37 °C. The inserts were stained with crystal violet and were observed under microscope (Olympus, America, IX53 + DP80) for identification.

1% noble agar and 0.6% noble agar were prepared. The bottom layer was obtained by mixing 1% noble agar with equal volume of 2X cell culture medium. Once the lower layer of agar solidified, the upper layer containing with cells were plated onto lower layer. The cells and agar mixture were placed into a 37 °C cell culture incubator for around 21 days. Formed colonies were stained with 0.01% crystal violet and photographs of wells were obtained by using microscope (Olympus, America, IX53 + DP80).

Tissue chips containing with 4 cases of low grade ovarian serous carcinoma and 54 cases of high grade ovarian serous carcinoma were obtained from Shanghai Outdo Biotech Company (#HOvaC070PT01). The slides were deparaffinized in xylene for 10 min and then rinsed with distilled water. After that, the slides were treated with citrate buffer and microwaved for 10 min. Once cooled, the slides were rinsed with TBST for 3 min each. Subsequently, the slides were exposed to 1% H₂O₂ for 10 min, followed by another rinse with distilled water. The slides were then blocked in 5% BSA for 1 h. The tissues chips were incubated with the primary antibody overnight at 4 °C, and then in the secondary antibody for 1 h, as listed in Supplementary Table 2. Finally, the indicated proteins were visualized using the microscope (Olympus, America, #IX53 + DP80). The glass slides were scanned using a panoramic scanning instrument, and the staining results were analyzed using CaseViewer software. The used of tissue microarray for research purposed was approved by the Ethics Committee of Shanghai Outdo Biotech Company (license no. YBM-05-02).