CMTM3 is a positive regulator during adipocyte differentiation. (IMAGE)
Caption
CMTM3 is a positive regulator during adipocyte differentiation. (A) Pre-adipocyte 3T3-L1 cells were transiently transfected with increasing amounts of CMTM3 (0.125, 0.25, 0.5 μg) and differentiated with MDI (0.5 mM of 3-isobutyl-1-methylxantine, 1 μM of dexamethasone, and 10 μg/mL of insulin) until day 8. An empty vector was used as the transfection control. Oil red O staining was used to visualize differentiated adipocytes. Scale bar = 100 μm. Quantitative reverse transcription PCR was used to determine the mRNA levels of Pparg, Cebpa, and Cebpb. (B) Pre-adipocyte 3T3-L1 cells were transiently transfected with shCMTM3 (0.25, 0.5 μg) and differentiated with MDI until day 8. A pSuper empty vector was used as the transfection control. Oil red O staining was used to visualize differentiated adipocytes. Scale bar = 100 μm. Quantitative reverse transcription PCR was used to determine the mRNA levels of Pparg. (C) HEK 293 cells were transfected with PPARγ (0.125 μg) and with or without CMTM3 (0.25, 0.5 μg). The aP2- and PPRE-promoter activity was assessed using the luciferase assay. (D) The schematic diagram of PPARγ deletion. HEK 293 cells were transfected with an indicated combination of CMTM3 and the deleted-PPARγ form. Co-immunoprecipitation was performed using anti-Myc, and immunoblotting (IB) was conducted using anti-Myc and anti-HA. F, PPARγ full-length; AF1, activation function 1; DBD, DNA binding domain; LBD, ligand-binding domain. (E) The schematic diagram of the CMTM3 point mutation. M1, mutation of LEFLL, located in the MARVEL domain; M2, mutation of LRALL, located in the N-terminal domain. HEK 293 cells were transfected with an indicated combination of PPARγ (0.125 μg) and CMTM3 (WT, M1, and M2) (0.25 μg). The PPRE promoter activity was performed using the luciferase assay. (F, G) HEK 293 cells were transfected with an indicated combination of HA-PPARγ and Myc-CMTM3 (WT, M1) for 24 h, and then treated with cycloheximide (40 μg/mL) for the indicated time (0, 2, 4, 8 h). The protein intensities (PPARγ) were quantified using Image J Version 1.52 k. Data were expressed as mean ± standard error of the mean of at least three experiments. ns, not significant. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.
Credit
Genes & Diseases
Usage Restrictions
Credit must be given to the creator.
License
CC BY