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. Author manuscript; available in PMC: 2014 Nov 15.
Published in final edited form as: N Engl J Med. 2014 May 15;370(20):1909–1919. doi: 10.1056/NEJMoa1301824

Figure 3. Enhanced Adipogenic Effect Associated with DYRK1B R102C.

Figure 3

Permanent 3T3-L1 preadipocyte cell lines expressing nonmutant DYRK1B, the DYRK1B R102C mutation, DYRK1B-specific short hairpin RNA (shRNA), or vector alone were created, and the time course of adipocyte differentiation was examined. Panel A shows adipogenic transformations for days 2, 5, and 9. Staining with oil red O shows the presence of intracellular lipid, thus labeling adipocytes. Cells expressing nonmutant (NM) DYRK1B or DYRK1B R102C were transformed into adipocytes considerably earlier than were cells transfected with an empty vector. The accumulation of intracellular lipid (as judged by the intensity of staining) was highest in cells expressing DYRK1B R102C, followed by those expressing nonmutant DYRK1B, and lowest in cells transfected with an empty vector. No significant adipogenic differentiation was observed in cells expressing DYRK1B-specific shRNA (DYRK1B knockdown [KD]). Panel B shows the expression levels of CCAAT/enhancer-binding protein α (C/EBPα), peroxisome proliferator-activated receptor γ (PPARγ) isoforms 1 and 2, and PGC1α (a transcriptional coactivator that interacts with PPARγ), which were highest in adipocytes expressing the R102C variant, followed by those expressing nonmutant DYRK1B, and lowest in cells transfected with vector alone from early stages of adipogenesis. Conversely, the expression levels of Gli-2 (a mediator of sonic hedgehog signaling) and cyclin-dependent kinase inhibitor (p27Kip) were lowest in adipocytes expressing the R102C variant, followed by cells expressing nonmutant DYRK1B, and highest in those transfected with vector alone. β-actin was used as a loading control. Panel C shows that the expression levels of C/EBPα, PPARγ isoforms 1 and 2, and PGC1α were significantly lower in DYRK1B knockdown cells than in cells transfected with vector alone. Panel D shows that cells expressing the R102C variant had substantial adipogenic transformation without the addition of adipogenic medium, as compared with those expressing nonmutant DYRK1B or those transfected with vector alone.