Inhibitor nc

Mac-T cells (Laboratory of Animal Stress Regulation Mechanism of Heilongjiang Bayi Agricultural University) were stimulated with 1, 10, 20, and 40 μg LTA for 3, 6, 12, and 24 h to induce inflammation. The bta-miR-223 mimic (5′-UGUCAGUUUGUCAAAUACCCCA-3′), mimic negative control (NC; 5′-CAGUACUUUUGUGUAGUACAA-3′), inhibitor (5′-UGGGGUAUUUGACAAACUGACA-3′), and inhibitor NC (5′-CAGUACUUUUGUGUAGUACAA-3′) sequences were purchased from Shanghai Sangon Biotech, China. The cells were transfected with 30 pmol/ml of the above oligonucleotides using RFect transfection reagent (Changzhou Biogenerating Biotechnologies, China), and the fluorescence intensity was checked at 0, 12, 24, 36, and 48 h post-transfection. In addition, the Mac-T cells were also transfected with The 4 μg/mL CBLB overexpression and interference vectors (Hedgehogbio Biotechnology, China) using Lipofectamine 3000 (Thermo Fisher Scientific, USA), and the fluorescence intensity was checked after 0, 24, 48, and 72 h of transfection. The expression/silencing efficiency was assessed by RT-PCR and Western blotting.

MiR-17-5p mimic, mimic-NC, miR-17-5p inhibitor, and inhibitor-NC were designed and synthesized by Sangon Biotech (Shanghai, China) with following sequences: hsa-miR-17-5p mimic: F: 5′-CAAAGUGCUUACAGUGCAGGUAG-3′, R: 5′-CUACCUGCACUGUAAGCACUUUG-3′; mimic-NC: F: 5′-UUCUCCGAACGUGUCACGUTT-3′, R: 5′-ACGUGACACGUUCGGAGAATT-3′; hsa-miR-17-5p inhibitor: 5′-CUACCUGCACUGUAAGCACUUUG-3′; hsa-miR-17-5p inhibitor-NC: 5′-CAGUACUUUUGUGUAGUACAA-3′. hsa-miR-17-5p mimic (50 nmol/l) was transfected into HCC cells grown on a lower-stiffness substrate using Lipofectamine 2000 (Invitrogen, United States). Hsa-miR-17-5p mimic-NC was set as the control. Contrarily, hsa-miR-17-5p inhibitor (50 nmol/l) was transfected into HCC cells grown on a higher-stiffness substrate and hsa-miR-17-5p inhibitor-NC also acted as the control. Transient transfections were performed according to manufacturers’ instructions.

miRNAs, including miR-199a-5p mimics, miR-199a-5p inhibitor and the nontargeting control (mimics -NC and inhibitor-NC), were obtained from Sangon (Shanghai, China). For cell transfection, miR-199a-5p mimics, miR-199a-5p inhibitor transfected and their negative control (mimics -NC and inhibitor-NC) were transfected into FTC cells with Lipofectamine 3000 reagent (Invitrogen, USA) according to the manufacturer’s instructions. RT–qPCR was used to measure the efficiency of transfection. All nucleotide sequences are listed in Table S2.

Circ-0002570 shRNA (circ-0002570-sh-#1 and circ-0002570-sh-#2), VCAN pcDNA, miR-587 inhibitor, miR-587 mimics, shRNA NC (sh-NC), inhibitor NC, mimic NC, and NC vectors were purchased from Sangon Bioengineering (Shanghai, Pudong, China). AGS and SGC7901 cells were transfected with siRNAs, miRNA, or the plasmid vectors based on the Manufacturer Directory. RT-qPCR and Western blot assays were applied to confirm transfection efficiency

MiR-21-3p mimics (mimic-21-3p) and its negative control (mimic-NC), miR-21-3p inhibitor (inhibitor-21-3p), and the negative control (inhibitor-NC), small interfering RNA (siRNA) against FGF2 (si-FGF2) and its matched control (si-NC) were bought from Sangon Biotech (Shanghai, China). The fragment of FGF2 was inserted into pcDNA vector (GenePharma, Shanghai, China) to construct overexpression plasmid (FGF2). The Lip2000 Transfection Reagent (Solarbio) was utilized for transfection. For viral infection, after transfection for 24 h, the washed A549 cells in the 24-well plate were infected with H5N1 at 0.1 multiplicity of infection (MOI) for 1 h. Then, the cells were incubated with fresh infection medium in an incubator.

Mimic NC, microRNA-603 mimic, inhibitor NC, microRNA-603 inhibitor, si-NC, and si-TBX5 were all designed by Sangon Biotech (Shanghai, China). Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) was applied to transiently transfect the synthesized sequences or expression plasmids into human cutaneous melanoma cells Malme-3M and A375. Afterward, the cells were cultivated in a corresponding medium with 5% CO₂ at 37°C for future use. Before transfection, all cells should be maintained in the complete medium for at least 24 h and be washed with phosphate-buffered saline (PBS, pH 7.4).