Figure 5.

A20 is essential for the adipogenic capacity of human MSCs. (a) A20 specific gene silencing by lentivirus-mediated shRNA. Four-days transduced MSCs were collected and GFP expression was analyzed by flow cytometry (gray). Negative control: non-infected MSCs (white). Western blot analysis of A20 expression after specific knock down (MSC-A20i). Protein expression levels were analyzed in total cell extracts. MSCs transduced with the empty vector were used to compare the efficiency from the specific shRNA (MSC-pLV). Sam68 was used as a loading control. (b) Quantification by real-time qPCR of IL-6 after 6 days of culture in MSCs transduced with the empty vector or with the specific A20 shRNA. (c) A20 knockdown induced an inhibition of adipogenesis in MSCs. MSCs showing ∼50% infection efficiency with either empty vector (MSC-pLV) or pLV-specific shRNA (MSC-A20i) containing vectors were induced to adipogenic differentiation. Percentages of adipocytes were determined by flow cytometry. Results show the means±S.D. of triplicate samples. ***P<0.001. (d) Analysis of apoptotis in cells where A20 expression was knocked down. The percentage of apoptotic cells (annexin V positive) was measured at 7-days post-infection by flow cytometry (GFP positive). Necrotic cells were discarded by specific TO-PRO-3 staining. Results show the means of triplicate samples