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S. Yang et al.
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Figure 1. Downregulated TRPV4 contributes to increased bone marrow adipocyte lipolysis. (A) Reverse transcriptase quantitative polymerase chain reaction (RT- qPCR) verification of the expression of transient receptor potential vanilloid (TRPV) channel genes in bone marrow (BM) adipocytes. (B) Western blot analysis of TRPV4 protein in BM adipocytes from three patients. (C) BM adipocytes treated with dimethylsulfoxide (Ctr), RN1734 (5 mM) or 4a-phorbol 12,13-didecanoate (4aPDD, 0.25 mg/mL) for 4 days. Adipocytes were stained by oil red O (ORO). 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) The content of lipid droplets in BM adipocytes from the indicated groups was detected by optical density values after ORO staining. (F) RT-qPCR was used to analyze adipose triglyceride lipase (ATGL) and hormone-sensitive triglyceride lipase (HSL) mRNA in BM adipocytes from the indicated groups. (G) The content of free fatty acids (FFA) in the supernatant of BM adipocytes treated with dimethylsulfoxide (Ctr), RN1734 or 4aPDD was detected using the colorimetric method. (H) BM adipocytes were infected with TRPV4-targeted shRNA (shTRPV4) lentivirus 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 fluores- cence. The scale bar represents 50 mm. (I) The number and area of BM adipocytes infected with shTRPV4 lentivirus, quantitatively analyzed by Image-Pro-Plus 5.1. (J) The mRNA level of HSL and ATGL in BM adipocytes infected with shTRPV4 lentivirus on the fourth day, detected by RT-qPCR. β-actin protein was used as an internal control for the western blot analysis. Three independent experiments were performed. ***P<0.001, **P<0.01, *P<0.05.
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haematologica | 2020; 105(11)