Ov nc

The short hairpin (sh)RNA targeting PTP1B (sh-PTP1B#1/2), KLF2 (sh-KLF2#1/2) and the corresponding sh-negative control (NC; sh-NC) were synthesized and inserted into the pRNA-U6.1 plasmid (GenScript) by GeneCopoeia, Inc. The sequences of shRNAs were as follows: sh-PTP1B#1, 5'-GCTACAGGTTCCTGTTCAA-3'; sh-PTP1B#2, 5'-GGTTCCTGTTCAACAGCAA-3'; sh-KLF2#1, 5'-GCACCGACGACGACCTCAA-3'; sh-KLF2#2, 5'-GAGTGGTAGCTTTCTACAA-3'; sh-NC, 5'-CCGGCAACAAGATGAAGAGCACCAACTC-3'.
A pcDNA3.1 overexpression vector (GenScript) encoding the full-length KLF2 (Ov-KLF2) and the corresponding NC (Ov-NC) were produced by Shanghai GenePharma Co., Ltd. In total, 100 nM recombinant vectors were transfected into HUVECs for 48 h using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol at 37˚C. The transfected HUVECs were then collected for subsequent experiments.

Plasmids carrying the RAGE gene [overexpression (Ov)-RAGE] and its corresponding negative control (Ov-NC) were constructed by GenePharma (Shanghai, China). The above-mentioned plasmids were transfected into primary hepatocytes using Lipofectamine 2000 (Invitrogen, CA, USA) for 24 h. After 24 h, the transfection efficacy was tested utilizing real-time quantitative polymerase chain reaction (RT-qPCR) and western blot.

Small interfering RNAs (siRNAs/si) targeting TRIM22 (si-TRIM22#1/2; cat. no. siG000010346A-1-5/siG1072101848-1-5) were constructed by Guangzhou RiboBio Co., Ltd., with an siRNA negative control (NC; si-NC; non-targeting sequence; cat. no. siB06525141922-1-5). The pcDNA3.1 overexpression vector containing the FOXC1 gene (Ov-FOXC1) and NC overexpression vector (Ov-NC) were purchased from Shanghai GenePharma Co., Ltd. For TRIM22 silencing, RA-FLSs were transfected with 50 nM si-TRIM22#1/2 and si-NC. For FOXC1 overexpression, RA-FLSs were transfected with 2 µg Ov-FOXC1 and Ov-NC. Untransfected cells were regarded as the Control group. Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect the plasmids into RA-FLSs, which were seeded in 6-well plates at a density of 1×10⁶ for 48 h at 37°C according to the manufacturer's instructions. The cells were collected for subsequent experiments 48 h following transfection.

SKOV3 cells were seeded into 6-well plates at a density of 2x10⁵ cells/well and cultured at 37˚C until they reached 80% confluence. pcDNA 3.1 containing FBXW7 (Ov-FBXW7) or VEGF (pc-VEGF), and the corresponding empty vectors used as negative controls (Ov-NC and pc-NC, respectively) were synthesized by Shanghai GenePharma Co., Ltd. SKOV3 cells were transfected with the respective plasmids (50 nM) using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C for 24 h, according to the manufacturer's instructions. Cells were collected and the transfection efficiency was assessed via reverse transcription-quantitative (RT-q) PCR and western blot analyses 24 h post-transfection.