Fig. 5.

Extracellular vimentin modulates expression of molecules for lipogenesis and lipolysis. (A) L-Lactate was measured in the culture media of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours. Data were normalized to the protein amount (mg) of the adipocytes used in the assay (n=2). (B) Intracellular adenosine triphosphate (ATP) was measured in 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours. Data were normalized to the protein amount (mg) of the adipocytes used in the assay (n=4). (C) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses for peroxisome proliferator-activated receptor γ 2 (PPARγ2), sterol regulatory element-binding protein 1 (SREBP1), diacylglycerol O-acyltransferase 1 (DGAT1), DGAT2, and Lipin1 were performed using RNA from 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control (n=4, n=3, n=3, n=3). (D) Western blot analysis for PPARγ, fatty acid synthase (FASN), SREBP1, Lipin1, adipose triglyceride lipase (ATGL), and DGAT1 were performed using lysates of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours. Band quantification was performed using GAPDH and α-tubulin as internal controls (n=3). (E) Western blot analysis for phosphor-hormone-sensitive lipase (P-HSL; Serine 564) was performed using lysates of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 30 minutes. Band quantification was performed using total HSL and GAPDH as internal controls (n=3). ^aP<0.05, ^bP<0.01, ^cP<0.001.