Supplemental data
Supplementary Figure 1 Subcellular localizationof KLF9 changes duringadipogenesis
(a) Immunofluorescence analysis of KLF9 in3T3-L1 cells undifferentiated (upper panel) or days 4 (middle panel) and days8 postinduction(lower panel).
(b)Cytoplasmic(Cytosol) and nuclear (Nuclei) fractions of 3T3-L1 cells undifferentiated or induced to undergo adipogenesis for 4days and 8 days were extracted and the proteins from the two fractions were analyzed by SDS–PAGE and immunoblotted with antibodies for the indicated proteins. The purity ofthe cytosol and the nuclear fraction was determined with antibodies against Akt and laminB.
Supplementary Figure 2 Overexpression of KLF9 at early stage of adipogenesis has no effect on PPARγ expression.
(a-b) KLF9 was forced-expressed in 3T3-L1 preadipocytes through electroporation. 36hrsafter transfection, the cells were induced for differentiation. Western blot was performed to detect the protein level ofKLF9. CDK4 was blotted as loading control (a).Total RNA was extracted at indicated time points and the mRNA level of PPARγand C/EBPαwas analyzed by real-time PCR. Error bars indicate S.D. (n=4) (b).
Supplementary Figure 3Inhibition of KLF9 has no effect on the expression of early adipogenic transcription factors.
Two stable cell line infected with retroviruses encoding KLF9-specific siRNA(Ri-1 and Ri-2) and a stable cell line infected with control siRNA(shuffle), and a parallel passaged normal 3T3-L1 cell (normal 3T3-L1)were induced hormally and total RNA was extracted at indicated times, then real-time PCR wasused to quantify the transcription factor C/EBPβ, C/EBPδ, KLF5 and Krox20. The expression was normalized to PPIA mRNA level. Data shown are from a single experiment,representative of three.
Supplementary Figure 4 Loss of KLF9 does not affect the mRNA stability of C/EBPα andPPARγ.
Normal and KLF9 knock down 3T3-L1 cellsinduced for differentiation for 4 days were treated with 5 μg/ml of actinomycin Dfor indicated periods, and the mRNA level of PPARγand C/EBPαwas analyzedby real-time PCR. Values were normalized to the amount of actin in each sample. Resultsare expressed asmeans±SD(n=3).
Supplementary Figure 5Transactivation of PPARγ2 promoter by KLF9 is dependent on two specific sites in 0.6Kb upstream PPARγ2 promoter.
(a)A deletion series derived from a luciferase reporter constructof PPARγ2(3.2Kb) was cotransfected with KLF9 and pRL-TK(renilla) into NIH3T3 cell.Results are expressed as firefly luciferase activity normalized to renilla luciferase activity, then further normalized to the quantity of basal state(promoter activation by empty vector), respectively. Error bars indicate S.D.(n=4).
(B)NIH3T3 cellswere cotransfected with pGL3-PPARγ2 promoter(0.6Kb), or with reporterconstructs containing mutations within thepotential KLF binding sites in the 0.6Kb of PPARγ2promoter and KLF9, pRL-TK(renilla). Results are expressed as firefly luciferase activity normalized to renilla luciferase activity. Error bars indicate S.D.(n=4).
Supplementary Figure 6 Expression of C/EBPα was inhibited by chemically-synthetic siRNA oligo.
3T3-L1 cells were transfected with 100nM siRNA targeting mouse C/EBPα or control siRNA (Table S2). Total RNA and protein were extracted to analyze the expression level of C/EBPα.
Supplementary Figure 7 The competition between KLF9 and KLF2 on PPARγ2 promoter.
(a)NIH3T3cells were cotransfected with pGL3-PPARγ2 promoter (0.6kb), KLF9 and increasing amounts of KLF2expression constructs, respectively. Results are expressed as firefly luciferase activity normalized to renilla luciferase activity. Data shown are mean±SD of triplicates from a representative experiment.
(b) NIH3T3 cells were cotransfected with pGL3-PPARγ2 promoter (0.6kb), KLF2 and increasing amounts of KLF9 constructs, respectively. Results are expressed as firefly luciferase activity normalized to renilla luciferase activity. Data shown are mean±SD of triplicates from a representative experiment.
Supplemental Materials and Methods
Materials
Anti-KLF9 antibody (H-130) and anti-LaminB (C-20) were from Santa Cruz Biotechnology. Anti-Akt ( 9272) was from Cell Signaling Technology. FITC conjugated anti-rabbitIgG was from Invitrogen. Actinomycin D was from Sigma.
Immunofluorescence
For KLF9 immunofluorescence staining, 3T3-L1 cells werecultured on glass coverslips. After rinsed with phosphate-buffered saline solution, the coverslips were fixed with 4% paraformaldehyde for 20 min and permeabilized with permeabilization solution (0.2% TritonX-100 in PBS).Thenthe fixedcells were incubated in blocking buffer (3% BSA in0.2% PBST) for 30 min at room temperature,and stained by primary antibodies, rabbit anti-KLF9 antibody (H-130, Santa cruz) followed by incubating withcorresponding secondary antibodies, FITC conjugated anti-rabbitIgG . Thecells were visualized by confocal microscope (Leica).
Subcellular fractionation
Cells were rinsed once with phosphate-buffered saline solution and twice with homogenate buffer (250mM sucrose, 20mM HEPES PH7.4, 1mM EDTA). Then cells was scraped into homogenate buffer and homogenized until the cells were completely disrupted, followed by low-speed centrifugation (750 g) for 5 min. The supernatant was then centrifuged at 20000 g for 20 min at 4℃.
Actinomycin D Treatment for Determination of mRNA Stability
Normal and KLF9 knock down 3T3-L1 cells were induced for differentiation and treated with 5 μg/mlactinomycin D at day 4 of differentiation. Then total RNA was extracted at 0, 30, 60, 120, 180, 240minutes post–actinomycinD treatment, followed by real-time PCR analysis as described above.
Table S1 Primers Used in Real-time PCR and ChIP Assays
For real-time PCR analysisPrimer name / Primer sequence
KLF9 for / 5’-GTTTGCCCCTGTAAGTAGTAAGTG
KLF9 re / 5’-GGTTCAGGCCATTGTGTAGAC
PPARγ for / 5’-AGCCCTTTGGTGACTTTATGGAG
PPARγ re / 5’-GCAGCAGGTTGTCTTGGATGTC
C/EBPα for / 5’-CCCAGCGGTGCCTTGTGC
C/EBPα re / 5’-TCCTTCCCCCAGCCGTTAGTG
C/EBPβ for / 5’-TGGACACGGGACTGACGCAACACA
C/EBPβ re / 5’-TCAACAACCCCGCAGGAACATCTT
C/EBPδ for / 5’-GCAGCCCCAAAAGCCAGTAA
C/EBPδ re / 5’-GCCGTGCAGATCAGGGAAGG
aP2 for / 5’-TGCTGCAGCCTTTCTCACCT
aP2 re / 5’-AGCCCACTCCCACTTCTTTCA
Adiponectin for / 5’-CTCTCCTGTTCCTCTTAATCCT
Adiponectin re / 5’-ACCAAGAAGACCTGCATCTC
KLF5 for / 5’-CATGCCAAGTCAGTTTCTTCC
KLF5 re / 5’-TGTGCAACCATTATAATCGCAG
Krox-20 for / 5’-CTTTGACCAGATGAACGGAG
Krox-20 re / 5’-GAGAATTTGCCCATGTAAGTG
PPIA for / 5’-GGCGGCAGGTCCATCTACG
PPIA re / 5’-CTTGCCATCCAGCCATTCAGTCT
For Chip assay
PPARγ(-2000) for / 5’-CTTTTTAATTTAGAAGACACAGGTATTATC
PPARγ(-2000) re / 5’-TCAATATATCACTTGGTTCCCTTATTCCTG
PPARγ for / 5’-TACGTTTATCTGGTGTTTCAT
PPARγ re / 5’-TCTCGCCAGTGACCCACAC
*for: forward primer, re: reverse primer
Table S2 siRNA sense sequence for C/EBPα
C/EBPα / Position / Sequencenc / - / 5’-UUCUCCGAACGUGUCACGUTT
Si-348 / 348 / 5’-CGACGAGUUCCUGGCCGACTT