Fig 3. Co-culture of hMSCs transduced with ADAMTS5-targeting shRNA can effectively knockdown the expression of ADAMTS5 in SW982 cells.

(A), A western blot of whole cell extracts from SW982 cells or hMSCs in mono-culture probed with anti-ADAMTS5 or anti-GAPDH antibodies, as indicated, shows that ADAMTS5 is most abundantly expressed in SW982 cells, although a modest amount is also observed in hMSCs. A representative blot is shown. (B), A cartoon depiction of the proposed model in which hMSCs, which have been “pre-loaded” with ADAMTS5-targeting shRNA, are co-cultured with synovial cells. These cells are capable of forming Cx43- containing gap junctions. These gap junction channels permit the communication of the siRNA from the hMSCs to the synovial cells, resulting in an attenuation of the production of ADAMTS5 in the recipient synovial cell. (C) A representative western blot is shown for co-cultured hMSCs and SW982 synovial fibroblasts. The hMSCs have been previously transduced with non-targerting control shRNA (hMSCs-shRNA-control), ADAMTS5-targeting shRNA (hMSCs-shRNA-ADAMT5), or left untransduced (hMSCs). The cells are co-cultured at the indicated ratio (2x hMSCs to 1x SW982 cells). The blot was probed with anti-ADAMTS5 or anti-GAPDH antibodies, as indicated. The images are from non-contiguous lanes of the same blot and exposure. The graph to the right reveals the combined data from quantitation of band intensity from 4 independent replicates of the experiment. Histograms indicate means ± standard deviation. *, indicates a p-value <0.05 relative to control.