Adenoviral Knockdown of FYN and FGF18 in NRCMs

The pSilencer 2.1-U6 expression vector was purchased from Ambion (Ambion, AM5762). The RNU6-1 RNA polymerase III promoter and the polylinker region were subcloned into the adenoviral shuttle vector pDC311 (Microbix, PD-01-25). The rat FYN shRNA targeting sequence was 5ʹ-AGGATAAAGAAGCAGCGAAACTGAC-3ʹ. The rat FGF18 shRNA targeting sequence was 5ʹ-CCTGCACTTGCCTGTGTTT-3ʹ. For Scrambled shRNA, an in-house-generated shRNA adenovirus that encodes a scrambled sequence was used as a control. Recombinant adenoviruses were generated by homologous recombination in HEK293T cells as described above. For adenovirus-mediated gene knockdown, these constructed adenovirus vectors were transfected into NRCMs at an MOI of 20× PFU/cell for 24 h. After 24 h, the knockdown efficiency was evaluated.