Journal: The EMBO Journal

Article Title: Fine-tuning BMP7 signalling in adipogenesis by UBE2O/E2-230K-mediated monoubiquitination of SMAD6

doi: 10.1038/emboj.2013.38

Figure Lengend Snippet: Monoubiquitination of SMAD6 potentiates BMP7-induced signalling. (A) Lysine 174-mutated SMAD6 increases the inhibitory ability of SMAD6 on BMP7-induced signalling. Analysis of BMP7-induced signalling activation in HEPG2 cells with co-transfection of SMAD6 (wt), lysine 182 or 174 to arginine (K182R or K174R, left panel) or increase amounts of lysine 174 to arginine (K174R, right panel). Data from triplicates are presented as the mean ±s.d. of a representative experiment. (B) Lysine 174-mutated SMAD6 increases the inhibitory ability of SMAD6 on BMP7-induced SMAD1 phosphorylation. C3H10T1/2 cells stably expressing empty vector, SMAD6, or K174R were stimulated with BMP7 at the indicated time points, and western blots were used to analyse phosphorylated SMAD1 level (pSMAD1). (C) Lysine 174-mutated SMAD6 increases the inhibitory ability of SMAD6 on BMP7-induced adipocyte differentiation. C3H10T1/2 cells stably expressing empty vector, SMAD6, or K174R were pre-treated with BMP7 for 3 days and differentiated into adipocytes for another 8 days. Cells were fixed and stained with Oil Red O as described in Materials and Methods. Whole well and higher magnification are shown. (D) Lysine 174-mutated SMAD6 increases the inhibitory ability of SMAD6 on BMP7-induced adipocyte-related genes expression. C3H10T1/2 cells stably expressing empty vector, SMAD6, or K174R were pre-treated with BMP7 for 3 days and differentiated into adipocytes for another 8 days. Adi(day) represents adipocyte differentiation days. Adipocyte cell markers AP2, CEBP/α, PGC1β, and PPARγ were analysed by real-time PCR. *indicates P<0.05 and **indicates P<0.01 (wild-type SMAD6 was compared with empty vector and K174R mutated SMAD6 was compared with wild-type SMAD6). (E) SMAD6 and UBE2O are upregulated in BMP7-induced commitment of mesenchymal stem cells to proliferating pre-adipocytes. C3H10T1/2 cells were pre-treated with 100 ng/μl BMP7 in DMEM supplemented with 10% FBS for 3 days to reach confluence, cells were induced to adipocytes as described in Materials and Methods. Cells were harvested for western blot at the indicated time points. Adi(day) represents adipocyte differentiation days. (F) The relative protein level of UBE2O or SMAD6 (compared with protein level of UBE2O or SMAD6 without BMP7 treatment) was quantified. *indicates P<0.05.

Article Snippet: Antibodies used in this study were c-Myc (a-14, sc-789, Santa Cruz Biotechnology), HA (Y-11, sc-805, Santa Cruz Biotechnology), Flag (F3165, Sigma), β-actin (A5441, Sigma), UBE2O (NBP1-03336, Novus Biologicals), SMAD6 (9519, Cell Signaling and sc-13048, Santa Cruz Biotechnology), SMAD1 (sc-7965, Santa Cruz Biotechnology, 6944, Cell Signaling and 060653, Upstate), pSMAD1 (9511, Cell Signaling), and FK2 (BML-PW8810, Enzo Life Sciences).

Techniques: Activation Assay, Cotransfection, Phospho-proteomics, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Staining, Real-time Polymerase Chain Reaction

Journal: The EMBO Journal

Article Title: Fine-tuning BMP7 signalling in adipogenesis by UBE2O/E2-230K-mediated monoubiquitination of SMAD6

doi: 10.1038/emboj.2013.38

Figure Lengend Snippet: UBE2O potentiates BMP7-induced SMAD signalling. (A, C) UBE2O positively regulates BMP7-induced signalling activation. Analysis of BMP7-induced signalling activation in HEPG2 cells with knockdown of UBE2O (A), or with increased amounts of UBE2O or C885S-mutated UBE2O (C). Data from triplicates are presented as the mean ±s.d. of a representative experiment. NS represents non-specific shRNA. (B, D) UBE2O potentiates BMP7-induced SMAD1 phosphorylation. C3H10T1/2 cells stably expressing UBE2O shRNA (shUBE2O-1, -2) (B) or stably expressing UBE2O or C885S-mutated UBE2O (D) were stimulated with BMP7 at the indicated time points. Western blots were used to analyse phosphorylated SMAD1 level with indicated antibodies. (E, F) UBE2O potentiates BMP7-induced adipocyte differentiation. C3H10T1/2 cells stably expressing empty vector, UBE2O, or C885- mutated UBE2O were pre-treated with BMP7 for 3 days and differentiated into adipocytes for another 8 days. Cells were fixed and stained with Oil Red O as described in the Materials and Methods (E) or analysed by real-time PCR for adipocyte markers AP2, CEBP/α, PGC1β, and PPARγ (F). Adi(day) represents adipocyte differentiation days. *indicates P<0.05 and **indicates P<0.01(wild-type UBE2O was compared with empty vector and C885S-mutated UBE2O was compared with wild-type UBE2O). (G) UBE2O potentiates BMP6-induced SMAD1 phosphorylation. C2C12 cells stably expressing empty vector or UBE2O-Myc were stimulated with 50 ng/ml BMP6 at the indicated time points. Western blots were used to analyse phosphorylated SMAD1 level with indicated antibodies. (H) UBE2O potentiates BMP6-induced osteoblast-like cell differentiation. C2C12 cells stably expressing empty vector or UBE2O-Myc were treated with 50 ng/ml BMP6 for 3 days, and then cells were harvested for histochemical staining to determine ALP activity. Whole well and higher magnification are shown. (I) The potentiated BMP7-induced signalling by UBE2O depends on SMAD6. Immortalised SMAD6-knockout MEF cells were infected with empty vector or UBE2O-Myc expression viruses. Cells were treated with BMP7 at the indicated time points. Phosphorylated SMAD1 level was analysed by western blot. (J) The relative BMP7-induced SMAD1 phosphorylation level in Figure 7G (normalised to SMAD1) was quantified. **indicates P<0.01, n.s. indicates no significant difference.

Article Snippet: Antibodies used in this study were c-Myc (a-14, sc-789, Santa Cruz Biotechnology), HA (Y-11, sc-805, Santa Cruz Biotechnology), Flag (F3165, Sigma), β-actin (A5441, Sigma), UBE2O (NBP1-03336, Novus Biologicals), SMAD6 (9519, Cell Signaling and sc-13048, Santa Cruz Biotechnology), SMAD1 (sc-7965, Santa Cruz Biotechnology, 6944, Cell Signaling and 060653, Upstate), pSMAD1 (9511, Cell Signaling), and FK2 (BML-PW8810, Enzo Life Sciences).

Techniques: Activation Assay, Knockdown, shRNA, Phospho-proteomics, Stable Transfection, Expressing, Western Blot, Plasmid Preparation, Staining, Real-time Polymerase Chain Reaction, Cell Differentiation, Activity Assay, Knock-Out, Infection