Journal: Cytokine

Article Title: Bmp4 expressed in preadipocytes is required for the onset of adipocyte differentiation.

doi: 10.1016/j.cyto.2013.07.011

Figure Lengend Snippet: Fig. 3. Endogenous Bmp activity prior to differentiation induction is required for efficient lipid accumulation in 3T3-L1 cells. (A) Smad phosphorylation in response to the Tgf-b family in 3T3-L1 cells treated with dorsomorphin or LDN-193189. 3T3- L1 cells at 70% confluence were pre-treated with dorsomorphin (DM: 5 lM), LDN- 193189 (LDN: 100 nM) or DMSO as a vehicle control (V) for 20 min, followed by treatment with Tgf-b1 (200 pM), Activin A (4 nM) or Bmp2 (4 nM) for 1 h. Phosphorylated Smad1/5/8 (upper panel) and Smad2 (third panel) were examined by Western blotting, and the membranes were then re-blotted for total Smad1 (second panel) and Smad2 (fourth panel). (B) Effects of dorsomorphin and LDN-193189 on Bmp-mediated transcription. 3T3-L1 cells were transiently transfected with BRE- luc and Renilla-luc and treated with dorsomorphin (DM: 5 lM), LDN-193189 (LDN: 100 nM) or DMSO as a vehicle control (V) for 20 min, followed by treatment with or without Bmp4 (4 nM) for 16 h. Firefly luciferase activity was normalized to Renilla luciferase activity, and the luciferase activity in cell lysates in the absence of Bmp4 and inhibitors was set to 1. Data were expressed as the mean ± SE of triplicates from a representative experiment. (C) Suppression of lipid accumulation by the inhibi- tion of Bmp activity in 3T3-L1 cells. Dorsomorphin (DM: 5 lM), LDN-193189 (LDN: 100 nM) or DMSO as a vehicle control (V) was added to the culture medium from day 2 to day 0, and lipid accumulation on day 8 was examined by Oil Red O staining (upper panel). After staining, the dye was extracted with 2-propanol, and the amount was quantified by measuring the absorbance at 510 nm. The absorbance of the extracted dye from cells treated with vehicle was set to 100. Data are expressed as the mean ± SE of quadruplicates. P < 0.01 vs. vehicle. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following reagents were purchased: dorsomorphin (compound C: 6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a] pyrimidine) was from Calbiochem (La Jolla, CA, USA);LDN-193189 was from Stemgent (San Diego, CA, USA);purified Tgf-b1 was from Becton Dickinson (Franklin Lakes, NJ, USA);recombinant Activin A, Bmp2 and Bmp4 were from R & D Systems (Minneapolis, MN, USA);rabbit polyclonal antibody against phospho-Smad1 (Ser463/Ser465)/Smad5 (Ser463/Ser465)/Smad8 (Ser426/Ser428) (#9511) and rabbit polyclonal antibody against phospho-Smad2 (Ser465/Ser467) were from Cell Signaling Technology (Danvers, MA, USA);and rabbit monoclonal antibody against Smad1 (ab33902), rabbit polyclonal antibody against Smad2 (ab63576), and mouse monoclonal antibody against and b-actin (AC-15) were from Abcam (Cambridge, MA, USA).

Techniques: Activity Assay, Phospho-proteomics, Control, Western Blot, Transfection, Luciferase, Staining

Journal: Methods in Molecular Biology

Article Title: Molecular Dermatology

doi: 10.1007/978-1-62703-227-8

Figure Lengend Snippet: Fig. 2. The stages of iPSC differentiation during keratinocyte derivation. iPSCs generated from an ICR mouse were differ- entiated into keratinocytes using differentiation Protocol I. ( a ) EBs treated with RA for 2 days in suspension culture. ( b ) EB outgrown on a ColIV-coated plate in the presence of BMP4 on day 10 of differentiation. ( c ) EB outgrown on a ColIV-coated plate on day 17 of differentiation before rapid attachment plating. ( d ) iPSC-derived keratinocytes at passage 2 post-rapid attachment plating (day 25 of differentiation). All images were taken with 10× objectives.

Article Snippet: 100 ng/ m l stock solution of mouse BMP4 (R&D Systems) reconstituted in sterile 4 mM HCl containing 0.1% bovine serum albumin (BSA).

Techniques: Generated, Suspension, Derivative Assay