Opti mem serum free medium

Sixty colorectal cancer samples and their paired normal tissues were collected in the department of pathology of the First Affiliated Hospital of Fujian Medical University between Jan 2019 and Dec 2019. The ethics committee of The First Affiliated Hospital of Fujian Medical University had reviewed and approved all experimental protocols. All patients had read and signed the informed consent. The detached tissues were quickly frozen with fluid nitrogen and stored at − 80 °C. FHC, HCT116, SW480, DLD1, and LOVO cells were purchased from ATCC (Virginia, USA). Cells were cultured with RPMI 1640 with 10% FBS (Invitrogen, Carlsbad, CA) in a humidified chamber at 5% CO₂, at 37 °C. SW480 cells were plated on six-well plates (5 × 10⁵ cells per well). OPTI-MEM serum-free medium (M5650, Sigma Aldrich) and Lipofectamine 2000 reagent (Thermo Fisher Scientific, USA) were used in transfection tests. The final concentration of 100 nM siRNA was introduced in this study. Meanwhile, pEZ-Lv201 Vector (Biovector, China) was employed to construct the USP18 overexpression system in the DLD1 cells. Lentiviral particles generated with a standardized protocol were used to produce the highly purified plasmids. Endo Fectin-Lenti™ and Titer Boost™ reagents (CWBio, China) were used to co-transfect DLD1 cells. The supernatant was collected after 48 h transfection and stored at − 80 °C.

100 TNBC samples and normal tissues were collected in the Fujian Medical University Union Hospital from Jan 2019 to Dec 2019. The research was approved by the ethics committee of Fujian Medical University Union Hospital. The informed consents have been signed by patients. BT-549, MDA-MB-468, MDA-MB-453, MDA-MB-231, and MCF-10A cell lines were obtained from ATCC (Virginia, USA). Cells were cultivated using RPMI 1640 containing 10% FBS (Invitrogen, CA) on the condition of 5% CO₂ and 37° C. Cells (5×10⁵ each well) were seeded on six-well plate. Lipofectamine 2000 reagent (Thermo Fisher, USA) and OPTI-MEM serum-free medium (M5650, Sigma) were used for cell transfection. 100 nM siRNA was used to construct knockdown model of miR-205. Meanwhile, MiR-205 overexpression model was estabilished using pEZ-Lv201 Vector (Biovector, China). Titer Boost^TM and Endo Fectin-Lenti^TM (CWBio, China) were applied to co-transfect MDA-MB-231 cells. After 48 h transfection, the supernatant was collected.

HEK293T cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Hyclone, Logan, UT, USA), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM-Glutamine. All cells were incubated in a 5% CO₂ incubator at 37 °C. HEK293T cells transfection was carried out according to the manufacturer’s instructions of TransIntroTM EL Transfection Reagent (Transgen, Biotech, Beijing, China). Briefly, HEK293T cells were seeded into a 6-cm petri dish 24 h before transfection. On the next day, DNA and transfection reagent was added to an appropriate volume of opti-MEM serum-free medium (Sigma-Aldrich, St. Louis, MO, USA) in a ratio of 1:3, incubated at room temperature for 15 min, and then transferred to the HEK293T cells.