Hieff trans liposomal transfection reagent kit

The interactions between LDLRAD3 and miR-20a-5p or miR-20a-5p and SLC7A5 were indicated by ENCORI. The oligo sequence of LDLRAD3 and 3′-UTR of SLC7A5 carrying the wild type or mutant binding sites of miR-20a-5p and were amplified using Q960 PCR instrument (Dinai, China) and inserted into pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, USA) to create the plasmids (LDLRAD3-WT and LDLRAD3-MT; SLC7A5-WT and SLC7A5-MT). The mimic-NC and miR-20a-5p mimic were cotransfected with the above vectors using Hieff Trans Liposomal Transfection Reagent kit (Yeasen, China), and the luciferase activity was measured with DLR Gene Assay Kit (Yeasen, China) and GloMax 20/20 Luminometer (Promega, USA). The luminescence activity of firefly luciferase substrate was normalized to Renilla luciferase substrate [25 (link)].

For transient transfection, small interfering RNAs (siRNAs) directed against SRSF9 (#SIGS0008682-4, RIBOBIO, Guangzhou, China), DSN1 (#SIGS0013609-1, RIBOBIO), METTL3 (#SIGS00056532-1, RIBOBIO) and negative control RNAs (si-ctrl) were synthesized by RIBOBIO Company (Guangzhou, China). Transient transfection was performed using a Hieff Trans™ Liposomal Transfection Reagent kit (#40802, Yeasen, Shanghai, China) in accordance with the standard protocol. Cells were collected after 24 h for qRT-PCR and after 48 h for Western blotting and functional studies.
For lentiviral transfection, Flag-SRSF9 (ov-SRSF9), empty vector (Vector), shSRSF9 (sh1, sh2), and shNC were purchased from GeneChem Company (Shanghai, China). Caco2 cells and HT29 cells were used to establish stable SRSF9 overexpression models, and HCT116 cells and LOVO cells were used in the stable SRSF9 knockdown experiments. According to the manufacturer’s instructions, 4 × 10⁴ cells per well were seeded and transfected with the indicated lentiviruses. The infected cells were screened using 5 μg/mL puromycin (Solarbio, Beijing, China) for 1 week or longer, and transfection efficiency was determined by qRT-PCR and Western blotting analysis.