Trypsin

All chemical reagents in this study were purchased and used directly without further purification. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, ≥ 98.0%), TanI (97%), dimethyl sulfoxide (DMSO, ACS, ≥ 99.9%), and 1,3-diphenylisobenzofuran (DPBF, 98%) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Dulbecco’s modified Eagle’s medium (DMEM) without phenol red were purchased from Sangon Biotech (Shanghai) Co., Ltd. Fetal bovine serum (FBS) was purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. Trypsin and all the KITs used in this study were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.
Light lamp board (30 W, 14 cm × 20 cm) with wavelength centered at 460 nm (460 ± 10 nm) were purchased from Xuzhou Aijia Electronic Technology Co., Ltd (China). The distance between light source and 96-plates was set at 24 cm. The light radiation flux density is 10 ± 2 W/m² and the luminous flux is 20 ± 5 lm. According to the light radiation flux density, 30 min light irradiation could bring light doses of 1.8 J/cm².

Human normal lung epithelial cell line BEAS-2B and NSCLC cell lines H1299, H1975, A549, and H2228 were available from American Type Culture Collection (ATCC) (Rockville, MD, USA). All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) containing 10 % fetal bovine serum (FBS) (Biyuntian, Shanghai, China) at 37 °C with 5 % CO₂. 0.25 % trypsin (Biyuntian, Shanghai, China) was employed for subculture. The GAS5 overexpression plasmid, shRNA targeting GAS5, miR-221-3p mimics, and miR-221-3p inhibitors, and the corresponding controls were constructed by GenePharma (Shanghai, China). The above plasmids or oligonucleotides were transfected into H1299 or A549 cells using Lipofectamine®3000 (Invitrogen, Carlsbad, CA, USA).

HMEC-1 cells in the logarithmic growth phase were digested by trypsin (C0202, Biyuntian). The cell suspension (5 × 10⁴/mL) was prepared and added to a 96-well plate according to 100 μL/well. The cells were placed in an incubator at 37°C until the cells adhered to the wall and covered the bottom of the well. At the same time, 10 μl CCK8 solution was added to each hole. After mixing and incubating in the incubator for 2 hours, the absorption value was measured at 450 nm wavelength by an enzyme labeling instrument. The cell proliferation was calculated according to the formula.

The study was approved by the local ethics committee of Tongji Medical College, Huazhong University of Science and Technology. Written informed consent was obtained from each patient. As we described previously [17 (link)], human primary glioblastoma cells and gaMSCs were isolated and cultured. In brief, for gaMSCs, fresh glioma specimens were washed with phosphate-buffered saline (PBS, HyClone, USA) and then processed for mechanical cutting and trypsin (BIYUNTIAN, China) digestion. The mononuclear cells were collected by Ficoll (2:1 Genview, USA) density gradient centrifugation and cultured in DMEM (HyClone, USA) containing 20% foetal bovine serum (FBS, BI, Israel) and 1% penicillin and streptomycin in a humidified atmosphere at 37 °C containing 5% CO2. For primary glioblastoma cells, fresh glioma specimens were processed for washing and digestion. Then, the obtained cell suspension was lysed with erythrocyte lysate (BIYUNTIAN, China), seeded into a neuroblast medium (containing N2, B27, hEGF, FGF and heparin) and transferred to an extracellular matrix culture flask (Corning, USA). The cells were cultured in a humidified atmosphere at 37 °C containing 5% CO2. U87MG cells were purchased from the American Type Culture Collection (ATCC, Gaithersburg, MD, USA) and cultured in DMEM containing 10% foetal bovine serum.

DNA smear image cytometry was performed on 20 patients with epithelial ovarian cancer. Deep-frozen cancer tissues were first cut into 8 μm-thick frozen sections. Laser capture microdissection was performed to dissect the cancer islets, which were digested with 0.25% trypsin (Biyuntian, Beijing, China) and 0.2% collagenase IV (Sigma-Aldrich, St Louis, MO, USA). Samples were filtered through a 70 μm screen mesh, fixed for 1 h in Bohm-Sprenger liquid (Mike Audi Corporation, Xiamen, China) and smeared to prepare single-cell-layer slides. The slides were then stainedwith Feulgen DNA dye (Mike Audi Corporation, Xiamen, China) for 75 min. DNA images were captured with a microscope, and data were analyzed withMotiCytometer and MotiClassify software (Mike Audi Corporation, Xiamen, China).