Si socs2

To transiently knock down METTL3 expression, siRNA transfection was used. Two siRNAs targeting METTL3 (si-METTL3#1 and si-METTL3#2) were synthesized by GenePharma Corporation. The siRNA sequences were as follows: si-METTL3#1, 5′-GGUGACUGCUCUUUCCUUATT-3′ and 3′-UAAGGAAAGAGCAGUCACCTT-5′; si-METTL3#2, 5′-GCUACCUGGACGUCAGUAUTT-3′ and 5′-AUACUGACGUCCAGGUAGCTT-3′. The negative control siRNA sequences were as follows: si-Ctrl, 5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′. The siRNA targeting SOCS2 (si-SOCS2) was obtained from Santa Cruz Biotechnology, Inc. The pCMV6 plasmid containing the full-length SOCS2 sequence (FULL-SOCS2) was purchased from Origene (RC203163).
Cells were cultured in 6-well plates (1×10⁵ cells per well) overnight prior to the experiment. The following day, siRNA (80 µM) was transfected into cells with Lipofectamine RNAiMAX (5 µl per well) (Thermo Fisher Scientific, Inc.). For plasmid transfection, cells were seeded at a density of 1×10⁵ cells per well in 6-well plates. Twenty-four hours later, Lipofectamine 2000 (5 µl per well; Thermo Fisher Scientific, Inc.) was used to transfect cells with the plasmid (2.5 µg per well) following the manufacturer's instructions. Cells were harvested 48 h after transfection for RNA and protein detection.

The specific siRNA to SOCS2 (si-SOCS2) was obtained from Santa Cruz Biotech, Inc. (sc-40998). A non-specific siRNA of a scrambled sequence from Santa Cruz was used as the control. siRNA was mixed with Lipofectamine RNAiMax (Thermo Fisher Scientific) and transfected into cells, as we previously reported (28 (link)). The pCMV6 plasmid with full-length SOCS2 sequence (pCMV6-SOCS2) was purchased from Origene (RC203163). The pCMVv6 empty vector from Origene was used as the control. AGS cells were transfected with the plasmid via Lipofectamine 2000 (Thermo Fisher Scientific) (28 (link)). Knockdown or overexpression of SOCS2 in AGS cells was validated by real-time PCR at 48 h after transfection.