Figure 7.

Smad4 is required for the differentiations of preosteoblasts and preadipocyes cells. (A): C3H10T1/2, preosteoblastic MC3T3‐E1 and preadipogenic 3T3‐L1 cells were fractionated into cytoplasm and nuclei. Expression of endogenous Smad4 and Taz were observed by immunoblotting with the indicated antibodies. (B): Smad4 depletion by lentiviruses expressing two independent short hairpin RNAs (shRNAs) specific to Smad4 was confirmed by immunoblotting in Smad4‐knockdown and shGFP‐expressing MC3T3‐E1 cells (upper panel). These cells were incubated with osteogenic medium, and osteogenic differentiation was confirmed by ALP and ARS staining. (C): Smad4 depletion by lentiviruses expressing two independent shRNAs specific to Smad4 was confirmed by immunoblotting in Smad4‐knockdown and shGFP‐expressing 3T3‐L1 cells (upper panel). Adipogenic differentiation of these cells was measured by ORO staining at day 7. (D): Smad4‐knockdown and shGFP‐expressing MC3T3‐E1 cells were incubated in OM for 3 days. Cells were fractionated into cytoplasm and nuclear extracts at the indicated times and both extracts were immunoblotted with the indicated antibodies. (E): Smad4‐knockdown and shGFP‐expressing 3T3‐L1 cells were differentiated into adipocytes for 3 days. Cells were fractionated into cytoplasm and nuclear extracts at the indicated times and both extracts were immunoblotted with the indicated antibodies. Expression of α‐tubulin and α‐Lamin B1 in (A), (D), and (E) was used as cytoplasmic and nuclear markers and loading controls. Images shown in this figure are representative of three independent experiments. Abbreviations: ALP, alkaline phosphatase; AM, adipogenic differentiation medium; ARS, alizarin red S; IB, immunoblot; OM, osteogenic differentiation medium; ORO, ORO, oil red O; Runx2, runt‐related transcription factor 2; shGFP, green fluorescent protein‐specific short hairpin RNA.