Si ythdf1

Human breast cancer cells MCF-7 (ATCC, USA) were grown in DMEM (Thermo, USA) containing 10% FBS in an incubator with 5% CO₂ at 37°C. MCF-7 cells were seeded in 6-well plates. The culture medium was changed 1 day before transfection, and when the cells reached 70–90% of the growth density, the cells were transfected with synthetic siRNAs (si-YTHDF1; GenePharma, Suzhou, China) through the Lipofectamine^™2000 Transfection Kit (Invitrogen, USA). Simultaneously, non-interfering siRNA was transfected as a negative control. The transfection process strictly followed the instructions of the kit, and the cells transfected for 48 h were collected for next research.

WPMY-1 human normal prostate stromal immutable cells and human PCa cells, PC3, LNCAP, 22RV1, and PC-3 M-IE8 were purchased from the ATCC. The culture conditions for WPMY-1 were DMEM, high glucose, and 5% fetal bovine serum (FBS). PC3 was cultured using F-12 K containing 10% FBS. The culture conditions for LNCAP, 22RV1, and PC-3 M-IE8 consisted of RPMI 1640 containing 10% FBS. To interfere with the expression of YTHDF1, si-YTHDF1 was transfected into the human PCa cell lines, PC3 and LNCAP, for 48 h, and then divided into PC3 + si-NC, PC3 + si-YTHDF1, LNCAP + si-NC, and LNCAP + si-YTHDF1 groups. To investigate whether YTHDF1 plays a role in PCa by regulating TRIM44, we conducted recovery experiments in PC3 cells, which were divided into PC3 + si-NC, PC3 + si-YTHDF1, and PC3 + si-YTHDF1 + TRIM44 groups. si-YTHDF1 (GCUGGCAAAUAUGAAGGUATT) and si-NC (UUCUCCGAACGUGUCACGUTT) constructs were designed and synthesized by GenePharma (Shanghai, China). To overexpress TRIM44, the TRIM44 sequence was cloned into the LV003 vector (Cookgen, Guangzhou, China). Cell transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.