Table 1.
Gene expression change of KLF family members during 3T3-L1 differentiation.
Figure 1.
KLF8 expression during 3T3-L1 adipocyte differentiation and in mouse adipose tissue.
(A) Total RNA was extracted from 3T3-L1 cells at the indicated times before and after induction of differentiation. The expression levels of KLF5, KLF8, KLF12, and KLF17 were determined by RT-PCR. (B) Anti-rabbit polyclonal KLF8 antibody was generated and tested on 293T cells that were transfected with either pcDNA3.0 or pcDNA3.0-KLF8-FLAG. Whole-cell lysates were immunoblotted (IB) using anti-KLF8 or anti-FLAG, which verified the specific antigen-antibody interaction. (C) Western blot analysis of KLF8 expression was performed at the indicated time points during 3T3-L1 cell differentiation. (D) KLF5, KLF8, and fatty acid synthase (FASN) mRNA levels were measured in the stromal vascular fraction (SVF) or the fat fraction of mouse epididymal adipose tissue using real-time qPCR. Data represent the mean ± SD.
Figure 2.
KLF8 knockdown blocks 3T3-L1 adipocyte differentiation.
Preadipocyte 3T3-L1 cells were treated with KLF8 siRNA at about 70% confluence using Lipofectamine RNAi/MAX reagent. After 24 h, the cells were trypsinized and replated at confluent cell density. After an additional 24 h, the cells were induced to differentiate. (A) Twenty hours after induction, cell extracts were prepared, and the effect on the expression of KLF8 was analyzed by real-time RT-PCR. (B) Oil red-O staining on day 8. The low panel represents spectrophotometric count of staining from 3 independent experiments. (C) Cells were harvested at the indicated times, and cell lysates were separated by SDS-PAGE and immunoblotted with antibody against C/EBPβ, PPARγ, and C/EBPα. LAP, liver-activating protein, LIP, liver-inhibitory protein, PPARγ1, PPARγ2, and 43 or 30 kDa of C/EBPα proteins were indicated. (D) To investigate the effect of siRNA on mitotic clonal expansion, cell numbers were determined at day 0 or day 2 after induction. Fold increases compared to the cell number in D0 were marked. Data in (A), (B), and (D) represent the mean ± SD. **P<0.01.
Figure 3.
Overexpression of KLF8 results in enhanced differentiation.
Preadipocyte 3T3-L1 cells were transiently transfected using electroporation with pcDNA3.0-KLF8-FLAG or empty vector and differentiated with standard hormonal cocktail as described in Materials and Methods. (A) After 5 days of differentiation, lipid accumulation was detected by oil red-O staining. (B) Western blot analysis of whole-cell extracts prepared at the indicated times during differentiation. LAP, liver-activating protein, LIP, liver-inhibitory protein, PPARγ1, PPARγ2, and 43 or 30 kDa of C/EBPα proteins were indicated.
Figure 4.
KLF8 regulates C/EBPα and PPARγ2 promoters to induce transcription.
(A) A series of C/EBPα promoter constructs cloned into the pGL3-Basic vector was used in this study. NIH3T3 cells were transfected with the luciferase constructs using Lipofectamine, and the luciferase activities were determined after 2 days. The plasmids pcDNA3.0, pCMV-C/EBPβ, or pCMV-KLF8-FLAG were cotransfected. (B) Similarly, the PPARγ2 promoter (−350 counted from transcription initiation site) was investigated by luciferase assay. (C) Mutation analysis of the C/EBPα promoter was performed. The −205 construct was used for the site directed mutagenesis in order to introduce KLF and/or C/EBP regulatory element between −191 and −178 region. The KLF8 binding site was indicated in blue, whereas the C/EBP site was marked in red. The mutated region was underlined. NIH3T3 cells were transfected with these constructs along with KLF8 and/or C/EBPβ overexpression plasmids. (D) Mutation analysis of the PPARγ2 promoter was performed. The KLF site was mutated by site-specific mutagenesis, and the resulting construct was analysed by luciferase assay. Data represent the mean ± SD. **P<0.01.
Figure 5.
KLF8 directly binds to C/EBPα and PPARγ2 promoter.
(A) EMSA experiment using KLF binding sites of β-globin (positive control), C/EBPα, or PPARγ2 promoter as probes. A FLAG-tagged KLF8 protein was in vitro translated using TNT T7 quick master mix. (B) EMSA experiment using wild type or mutated probe of the C/EBPα promoter. FLAG-tagged C/EBPβ-LAP or KLF8 protein was in vitro translated using TNT T7 quick master mix. (C) Western blot analysis of the in vitro translated proteins. n.s, non-specific. (D) ChIP was performed on 3T3-L1 cell chromatin at the indicated time points after induction of differentiation, using control IgG, anti-C/EBPβ, or anti-KLF8 antibody. The immunoprecipitated DNA was used as a PCR template to detect the PPARγ or C/EBPα promoter regions.