Ishikawa

The endometrioid carcinoma cell line Ishikawa was obtained from Procell LifeScience & Technology Company (Wuhan, PR China) and authenticated by short tandem repeat profiling. To observe the effect of estrogen (β‐estradiol, E2) (Sigma‐Aldrich, Santa Clara, CA, USA) on PRMT5 expression, the Ishikawa cells were cultured in phenol‐free medium RPMI 1640 (Macgene, Beijing, PR China) containing 10% activated carbon‐filtered fetal bovine serum (Biological industries, Beit HaEmek, Israel) in 6‐well plates for 24 h. Six concentration gradients of E2 were prepared and added to the Ishikawa cells once a day. The final concentrations of E2 were 0, 10⁻⁷, 10⁻⁶, 10⁻⁵, 10⁻⁴, and 10⁻³ mm. Total proteins were collected after 4 days of E2 treatment, and western blotting was performed to detect the PRMT5 expression.

The human EC cell line Ishikawa (RRID: CVCL_2529) and human embryonic kidney 293T (HEK 293T, RRID: CVCL_0063) were obtained from Procell and maintained in DMEM containing 10% FBS and 1% penicillin and streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. The human EC cell line HEC‐1‐A (RRID: CVCL_0293) was obtained from the National Collection of Authenticated Cell Culture and maintained in McCoy's 5A medium containing 10% FBS and 1% penicillin and streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. All cell lines have been authenticated using short tandem repeat profiling within the last 3 years and tested for mycoplasma contamination by DAPI staining. All experiments were performed with mycoplasma‐free cells. The cells were transfected with a miR‐548ag mimic, miR‐548ag inhibitor, and MOB1B‐overexpression plasmid, using the Lipofectamine 2000 reagent according to the manufacturer's instructions. The miR‐548ag mimic and miR‐548ag inhibitor were constructed by Genepharm. The MOB1B‐overexpression plasmid was constructed by SyngenTech.