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Fig. 4. hDkk1 inhibits Wnt signalling and promotes differentiation of 3T3-L1 preadipocytes. (A) Western blot analysis of whole-cell lysates from 3T3-L1 preadipocytes expressing empty vector (EV) or human Dkk1 (hDkk1) and pooled stromal-vascular cells from four mice (mPreads). Human recombinant Dkk1 protein (rDkk1) was used as positive control, and loading efficiency was assessed using p85 PI 3-kinase. (B) Western blot analysis of cytosolic ß-catenin levels during differentiation of control and hDkk1-expressing 3T3-L1 preadipocytes. (C) Effect of hDkk1 on TOPflash reporter activity in 3T3-L1 cells expressing empty vector (EV) or hDkk1. Results are expressed as fold difference relative to EV control. All results are the mean ± s.e.m. of three independent experiments. (D) Oil-Red O staining of EV and hDkk1-expressing 3T3-L1 adipocytes. Cells were induced to differentiate for 8 days using either differentiation medium lacking IBMX (sub-differentiation) or the full differentiation cocktail. (E) Effect of hDkk1 on adipogenic gene expression. Control and hDkk1-expressing 3T3-L1 cells were differentiated sub-maximally using DI and total RNA extracted at the time points indicated. PPAR1, PPAR2 and aP2 mRNA levels were measured using real-time PCR. Results are expressed as fold difference relative to the basal (time 0) value for control. All results are the mean ± s.e.m. of three independent experiments. All comparisons were made against control using Student's t test. PC, pre-confluent; C, confluent; 0, onset of differentiation (2 days post-confluence); 2, 4, 8 indicate 2, 4 and 8 days post-induction of differentiation respectively; RU, relative units; 3T3, 3T3-L1 cells; hDkk1, human Dkk1. ^*P<0.05, ^***P<0.001.

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