FIG. 4.
Membrane-bound DLK1 restricts in vitro preadipocyte proliferation by repressing G1-to-S-phase transition. A, B: The number of 3T3-L1 preadipocytes in the S phase of the cell cycle was quantified by EdU incorporation 48 h after transfection with Dlk1Total, Dlk1PS, or scramble siRNA. Cell doublets and debris were excluded from the flow cytometric analysis, and EdU+ gating was established within each siRNA treatment from negative controls that did not receive any EdU. C–F: Proliferation capacity was examined in Dlk1Total, Dlk1PS, or scramble siRNA-treated cells using Coulter counting (C, D), MTT (E), and qRT-PCR (F). G, H: Dlk1Total and scramble siRNA-transfected 3T3-L1 preadipocytes were cultured in the presence of different percentages of (G) conditioned medium (Cond. med.) from proliferating 3T3-L1 preadipocytes or (H) purified human DLK1 (ng/mL). Cell numbers were then examined by Coulter counting at 72 h. Significance was tested by Student t test (A–E), a one-way ANOVA followed by a Bonferroni multiple comparisons test within each time point against scramble control (F), and two-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.0001) (G, H). qRT-PCR raw data in (F) were normalized against multiple stably expressed reference genes (data not shown).