C jun sirna

Knowkdown using siRNA in CD4⁺ T cells were performed as described by Ciofani et al.^{27 (link)}. CD4⁺ T cells from wild-type mice were cultured for 16 h under Th0 conditions, and 300 pmol of control or c-Jun siRNA (Invitrogen) was introduced using the Mouse T cell Nucleofector Kit (Lonza) according the manufacture’s instruction. After recovery by incubation in fully supplemented culture media (Lonza), the cells were cultured for 3 days under Th17 conditions. Sequences for c-Jun siRNAs are as follows: #1, 5′-UACUGUAGCCGUAGGCACCGCUCUC-3′; #2, 5′-AUGACUUUCUGCUUAAGCUGUGCCA-3′; #3, 5′-AAACGUUUGCAACUGCUGCGUUAGC-3′.

MiR-139 mimic (miR10000250), mimic-NC, inhibitor (miR20000250), inhibitor-NC, antagomiR-139 (miR30000250), and antagomiR-NC (NC: negative control) were synthesized by RiboBio (RiboBio, Guangzhou, China). C-jun siRNA were purchased from GenePharma (Shanghai, China), and c-jun plasmids were purchased from Vigene (Jinan, China). The final concentration of miR-139 mimics, miR-139 inhibitor and C-jun siRNA in the transfection system was 100nM, respectively. The concentration of c-jun plasmid in the transfection system was 1ug/ml. When the confluence of ECFCs was approximately 70% to 80%, the miR-139 mimic, miR-139 inhibitor, C-jun siRNA, and c-jun plasmids were transfected with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). After 72 hours, the cells were subjected to the process of induction of differentiation.

The plasmids of shRNA for SPANXA were supplied by National RNAi Core Facility Platform (Academia Sinica, Taipei, Taiwan). They provided only 5 sequences to users. The clone ID of the fourth shRNA (sh4) for SPANXA was TRCN0000203935, and the target sequence was 5′-CGCTACAGGAGGAACTTTAAA-3′. The CL1-0 and H1437 cells were seeded onto 2 × 10⁴ plates for 24 h before infection. Viral infection was performed according to the manufacturer protocol. SNAI2 and c-JUN siRNA were purchased from Invitrogen (Life technologies, Carlsbad, CA).