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. 2012 May 31;8(5):761–777. doi: 10.7150/ijbs.4036

Figure 4.

Figure 4

Knockdown of TTP family proteins affects Mkp-1 mRNA stability during 3T3-L1 differentiation. (A) Expression profiles of Mkp-1 mRNA (upper panel) and protein (lower panel) during the early differentiation stage of adipogenesis. Two-day post-confluent 3T3-L1 preadipocytes were induced with FMDI for the indicated times. RNA was isolated for quantitative PCR analysis. The graph shows the mean ± SD of relative Mkp1 mRNA levels normalized to β-actin mRNA levels from three independent samples. WCEs were immunoblotted with anti-MKP-1 anti-β-tubulin. (B) Analysis of Mkp-1 mRNA half-life after differentiation induction for 0.5 h, 1 h, and 2 h. Actinomycin D (10 µg/ml) was added for 0, 10 min, 20 min, and 30 min to stop transcription. RNA was isolated for quantitative PCR analysis. The half-life of Mkp-1 mRNA (t1/2) was determined and indicated. *P<0.05, **P<0.01 and *** P<0.001. (C) and (D) 3T3-L1 cells at 50% confluency were transfected with TTP-specific siRNA or with control non-targeting siRNA (siCtrl). After 48 h, cells were induced with FMDI for 0, 3, and 6 h. WCEs were isolated and then immunoblotted with anti-TTP, anti-MKP-1, and anti-β-tubulin (C). (D) Mkp-1 mRNA half-life analysis following TTP knockdown. After the addition of FMDI for 1 h, cells were exposed to 10 µg/ml actinomycin D for the indicated times, and then Mkp-1 mRNA was analyzed using quantitative RT-PCR. The relative amounts of Mkp-1 mRNA are presented after normalizing to β-actin mRNA values. The mRNA half-life (t1/2) was calculated and indicated.*P < 0.05.