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S. Yang et al.
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Figure 3. GDF15 activates the downstream genes PI3K and pAKT in bone marrow adipocyte remodeling. (A) Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis of GDF15-related receptors in bone marrow (BM) adipocytes. (B) Western blot analysis of the expression of TRPV4 protein in BM adipocytes induced by recombinant human GDF15 (rhGDF15) after treatment with TGFβRI inhibitor (RepSox) or TGFβRII inhibitor (ITD1) for 4 days. (C) Oil red O stain- ing analysis of BM adipocytes induced by rhGDF15 after treatment with RepSox and ITD1 for 6 days. All images were at a magnification of 200×. (D) The number and average area of BM adipocytes from the indicated groups were measured using Image-Pro-Plus 5.1. (E, F) BM adipocytes were infected with TGFβRII-targeted shRNA (shTGFβRII) lentivirus for 48 h and then cultured with rhGDF15 for 6 days. Adipocytes were stained with Alexa Fluor 493/503-conjugated BODIPY. 4′,6- diamidino-2-phenylindole (DAPI) stained blue and lipid droplets showed green fluorescence. The number and average area of adipocytes from the indicated groups were measured using Image-Pro-Plus 5.1. The scale bar represents 50 μm. (G, H) BM adipocytes were infected with shTGFβRII lentivirus for 48 h and then cultured with rhGDF15 for 4 days. The levels of TRPV4, Smad2 and pSmad2 proteins were detected using western blot analysis. (I) BM adipocytes were treated with or without rhGDF15 and PI3K inhibitor (PI3K-IN-1, 2 mM) for 4 days. The levels of PI3K, AKT and pAKT proteins were detected using western blot analysis. β-actin protein was used as an internal control for the western blot analysis. Three independent experiments were performed. **P<0.01, *P<0.05.
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haematologica | 2020; 105(11)