Inhibition of FXIII-A TG activity increases adipocyte differentiation and lipid accumulation. (A) Inhibition of TG activity with the irreversible TG inhibitor NC9 increases lipid accumulation in a concentration-dependent manner as assessed by quantification of Oil Red O staining of 3T3-L1 cultures on day 8 of differentiation. Images show the increased size of lipid droplets in Oil Red O-stained cells. (B) Inhibition of TG activity during different stages of 3T3-L1 culture shows that TG activity has its most prominent inhibitory effect on lipid accumulation when given during days 0 to 4. (C-D) WB analysis and quantification of PPARγ expression (normalized to actin) during adipocyte differentiation shows increased expression (and thus accelerated differentiation) of NC9-treated cells. (E-F) WB analysis and quantification of Akt phosphorylation at Ser473 show that inhibition of TG activity significantly increases Akt activation. (G) The PI3K pathway inhibitor LY294002 used from day 0 to 4 reversed the NC9-mediated increase in adipogenesis; the graph shows quantification of Oil Red staining of the cultures on day 8. (H) Inhibition of TG activity with NC9 between days 0 and 4 can function in a similar manner as insulin (INS) in DM to promote preadipocyte differentiation; the graph shows quantification of Oil Red O-stained cultures on day 8. All error bars represent SEM (n = 3). *P < .05; **P < .01; ***P < .001.