Mir 205 5p inhibitor

The mouse lung epithelial (MLE‐12) cell lines were purchased from the BeNa Culture Collection. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS), in a humidified atmosphere of 95% air‐ 5% CO₂ at 37°C. MLE‐12 cells were cultured in 6 well culture plate, and then the cells were transfected with miR‐205‐5p mimic, miR‐205‐5p inhibitor, miR‐431‐5p mimic, miR‐431‐5p inhibitor, NC or inhibitor negative control (INC), with lipofectamine 2000 for 48 h. miR‐205‐5p inhibitor, miR‐431‐5p inhibitor, INC were synthesized by Genepharma. These miRNA mimic (or respective inhibitor) transfected MLE‐12 cells were simultaneously infected with influenza A virus (MLE‐12 Infection model). The cells cultured in complete medium were considered as normal control. The culture plate was cultured in a 37°C, 5% CO₂ incubator. The cells were harvested 48 h after transfection, and Western blot was carried out using the primary antibodies against NP (Abcam) and GAPDH (Cell Signalling) as an internal control.²³

Mimic-negative control (NC), miR-205-5p mimic, inhibitor-NC, miR-205-5p inhibitor, small interfering RNA (si)-NC, si-PRKCE#1, and si-PRKCE#2 plasmids were bought from the GenePharma (Shanghai, China). Based on the manufacturer’s manuals, the Lipofectamine 2000 (Invitrogen, Carlsbad, CA, U.S.A.) was adopted to transfect CD44⁺ CD133⁺ GBC-SD cells.