Fig 1.
PPM1D inhibitor SL-176 suppresses cellular lipid droplet accumulation and adipocyte differentiation.
(A) PPM1D inhibitor SL-176. (B, C) Quantification of lipid droplets in 3T3-L1 cells treated with the indicated concentrations of SL-176. Differentiated 3T3-L1 cells on Day 8 were stained with Oil Red O. (C) Absorbance of Oil Red O extract was measured at 490 nm. Data are mean ± S.D. values and obtained by three independent samples in each conditions (*P<0.05 **P<0.01 respectively, paired Student's t-test) (D) mRNA expression of the adipocyte marker by RT-qPCR. Cells were treated with differentiation medium (MDI) with or without 10 μM of SL-176. Blue, with MDI; red, 10 μM of SL-176 with MDI. The data were normalized by actin and expressed as fold change. Values are the mean ± range of duplicates. Representative data from one of at least three independent experiments are shown.
Fig 2.
Cell viability of 3T3-L1 preadipocytes after treatment with the indicated concentrations of SL-176 for 24 h was measured by MTS assay.
The data represent the mean ± S.D. of triplicate samples.
Fig 3.
SL-176 significantly reduced the size of lipid droplets in 3T3-L1 cells.
After treatment of SL-176 during adipocyte differentiation (from Day0 to Day8), lipid droplets in differentiated 3T3-L1 cells on Day 8 were stained by Monodansylpentane. Scale bar = 10 μm.
Table 1.
LD size distribution in SL-176 treated cells.
Fig 4.
Expression of adipocyte markers were reduced by SL-176.
(A) mRNA expression of the indicated genes was measured by RT-qPCR. Cells were treated with differentiation medium (MDI) with or without 10 μM of SL-176. Black line, without MDI (control); blue line, with MDI; red line, 10 μM of SL-176 with MDI. The data were normalized by actin and expressed as fold change. Values are the mean ± range of duplicates. Representative data from one of at least three independent experiments are shown. (B) Protein expression levels of PPARγ1/2 and C/EBPα in 3T3-L1 cells on Day 8 in the presence of 0, 5, 10, 15 μM SL-176 were analyzed by Western blotting. The intensity of the protein was shown below the western panel as fold change compared with intensity of 0 μM of SL-176. The values were normalized by of actin values. (C) C/EBPβ protein expression in 3T3-L1 cells on Day1-differentiated in the presence of 10 μM SL-176.