Knockdown of Chst11 leads to decreased Lpl activity.

<p>3T3-L1 cells were treated with siRNA oligonucleotides targeting Chst11, PPARy or control (scrambled) oligonucleotides and Lpl activity was analysed. The error bars display SEM and significance is shown by the asterisks (p<0.05), n = 3.</p

Chst11 is expressed in mature 3T3-L1 adipocytes.

<p>(A) and (B): mRNA expression profiles of Chst11 and PPARy in adipogenesis of 3T3-L1 cells during different days. Data are represented as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064284#pone-0064284-g001" target="_blank">Figure 1E</a>. (C) Representative confocal microscopy images of 3T3-L1 (pre)adipocytes. Endogenous Chst11 (red) was visualized utilizing specific antibodies, differentiated cells were identified with Nile Red (green), Hoechst was used to visualize the nuclei (blue).</p

Knockdown of Chst11 leads to decreased lipid accumulation but not adipogenesis.

<p>(A) 3T3-L1 cells were treated with siRNA oligonucleotides targeting Chst11 or control (scrambled) oligonucleotides and Chst11 mRNA and protein expression levels were determined at day 5 after differentiation by qPCR (upper panel) and Western blotting (lower panel), respectively. The error bars display SEM and significance is shown by the asterisks (p<0.05), n = 3 (B) 3T3-L1 cells were treated with siRNA oligonucleotides targeting PPARy, Chst11, Lpl, or control (scrambled) oligonucleotides, fixed and stained for lipid accumulation using Oil-red-O. Pictures are representative of 3 independent experiments. (C) 3T3-L1 cells were treated with siRNA oligonucleotides targeting Chst11, PPARy or control (scrambled) oligonucleotides and mRNA expression levels of PPARγ, C/EBPα, Lpl, adiponectin (adipoq) and Fabp4 were determined after 5 days of differentiation. Relative mRNA expression levels were related to control siRNA treated cells and normalized for the TFIIb reference gene. Samples were also generated for a western blot in which PPARγ expression was shown upon Chst11 and PPARγ knockdown. The error bars display SEM and significance is shown by the asterisks (p<0.05), n = 3.</p

Chst11 is a novel PPARy target gene.

<p>(A) Chip-Seq data of PPARγ and RXRα occupancy on the Chst11 gene according to Nielssen et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064284#pone.0064284-Nielsen1" target="_blank">[3]</a>. UCSC Genome Browser tracks at day 0, 1, 2 4 and 6 of differentiation are shown. Please note differences in y-axes. Two intronic PPARy-RXRα binding sites were designated site 1 and 2. (B) ChIP-PCR on 3T3-L1 preadipocytes and adipocytes. Chromatin was prepared on day 0 and day 6 of differentiation and subjected to immunoprecipitation using antibodies against PPARγ. Enriched DNA was analysed using quantitative PCR with primers located at site 1 and 2 in the Chst11 gene (dark gray and blacks bars, respectively). As a positive control, primers located at the Fabp4 PPREs (∼5500 bp from transcription start site; light gray bars) were used, primers located in the globin locus were used as a negative control (white bars). Results are shown relative to normalized ChIP recovery data of day 0 and results are representative of at least 3 independent experiments. (C and D) U2OS cells were cotransfected with a reporter construct (TKpGL3) containing Chst11 site 1 or site 2 sequences, or the parental reporter construct, together with empty (pCDNA) or PPARγ encoding expression vectors. Activation of the luciferase reporter in the absence or presence of 1 µM rosiglitazone is expressed as fold induction over that with empty reporter cotransfected with pCDNA in the absence of rosiglitazone after normalisation for Renilla luciferase activity. The error bars display SEM and significance is shown by the asterisks (p<0.05), n = 3 (E) Chst11 mRNA expression in 3T3-L1 adipocytes that had been treated with control or PPARy siRNA from the start of differentiation and analyzed at day 5. Relative mRNA expression levels were related to control siRNA treated cells and normalized for the TFIIb reference gene. The error bars display SEM and significance is shown by the asterisks (p<0.05), n = 3 (F) Chst11 mRNA expression in 3T3-L1 adipocytes and the effect of rosiglitazone treatment (1 µM, 24 h). Relative mRNA expression levels were normalized for the TFIIb reference gene. (G) As in panel F, but after treatment with the PPARγ antagonist GW9662 (10 µM, 24 h).</p

A, Small siRNA DUB screen perfomed in U2OS 3×PPRE miRNA FF2 cells.

<p>A partial siRNA library against 24 different deubiquitinating enzymes was tested for the ability to rescue the miRNA FF2 induced loss of Blasticidin resistance. Knock down of UCHL3 and UCHL5, but not UCHL1, resulted in colony formation. B. UCHL3 expression increases during 3T3-L1 adipocyte differentiation. Mouse 3T3L1 preadipocytes were differentiated into mature adipocytes and samples were taken at different time points during differentiation. Protein expression levels of UCHL3 were determined by Western blot analysis. As control for differentiation Fabp4 protein levels were analysed. C. UCHL3 activity in 3T3-L1 adipocytes. Cell lysates of differentiated 3T3-L1 cells (day 6) were incubated with or without HA-Ub probe, DUB activities were immunprecipitated (anti-HA agarose) and UCHL3 was detected by Western blotting. Note the difference in mobility between unmodified UCHL3 (input lane) and UCHL3 covalently bound to the DUB probe. An aspecific band is indicated (*). D. Localization of UCHL3 in differentiated 3T3-L1 cells. Nuclei were stained with DAPI, UCHL3 was visualized by indirect immunofluorescence. Merged pictures demonstrate the predominant cytoplasmic localization of UCHL3. As a control, the primary antibody was ommited. Bar, 10 µm. E. 3T3-L1 cells, stably transduced with lentiviral constructs expressing short hairpin RNAs directed against UCHL3 or control shRNA, were subjected to differentiation conditions. At day 10 of differentiation, cells were fixed and stained for triglycerides using Oil-red-O. Pictures are representative for three independent experiments. F. 3T3-L1 cells were stably transduced with either control or UCHL3 shRNA and differentiated as described under E. Cell lysates were subjected to Western blot analysis, using antibodies against UCHL3, PPARγ, FABP4 and tubulin.</p

A siRNA-mediated knock down of PPARγ rescues miRNA FF2 mediated loss of blasticidin resistance.

<p>Different PPARγ specific siRNA vectors were generated and tested for their ability to rescue the loss of blasticidin resistance. The functional siRNA vector #4 rescues the miRNA FF2 induced loss of resistance. B. The U2OS 3×PPRE miRNA FF2 cells were partially rescued with PPAR#4 siRNA expressing GFP virus. The percentage of GFP positive cells was determined using FACS analysis after 1 week of blasticidin selection at various concentrations.</p

A RNA polymerase II and RNA polymerase III driven miRNA FF2 expression results in loss of Blasticidine resistance.

<p>U2OS cells expressing the Blasticidin 3×FF2 expression cassette were transduced with miRNA FF2 expressing virus and selected (50 µg/ml Blasticidin S) for 1 week. As a control either empty virus or not FF2 specific miRNA FF3 virus was used, the latter expressing miRNA not targetting the modified 3′UTR of the blasticidin S deaminase gene. B. PPARγ2 expression compared to empty vector control as detected in the stably transfected U2OS cell line also expressing the blasticidin 3×FF2 expression cassette C. U2OS cells stably transduced with 3×PPRE miRNA FF2 virus were retransduced with blasticidin 3×FF2 virus and PPARγ2 virus at a ration of 1∶10 or 1∶25 respectively. Cells transduced at a ratio of 1∶25 show loss of resistance when incubated in 1 µM rosiglitazone and increasing amounts of Blasticidin S.</p

Schematic model of miRNA mediated siRNA screening.

<p>The upper panel illustrates the situation of cells with active PPARγ2-mediated gene expression. PPARγ drives miRNA FF2 expression, resulting in repression of the Blasticidin S deaminase expression cassette via the target repeat FF2 sequences present in the 3′ UTR. The lower panel depicts the situation of cells with an siRNA mediated knock-down of a PPARγ2 activating protein. Expression of the FF2 miRNA is diminished and Blasticidin S deaminase expression is no longer repressed, resulting in blasticidin resistance.</p

Tip60+/− mice display normal bodyweight with higher daily caloric intake.

<p>A, WT and Tip60+/− mice were weighed each week during 19 weeks of LFD or HFD feeding. Error bars represent means ± s.e.m. All groups contained 8 animals, except the Tip60+/− HFD group (n = 6). Please note slight reduction in weight around week 22 due to IP-GTT. B, Daily food intake of WT and Tip60+/− mice on LFD and HFD regimens. ** p<0.01, n.s. non-significant.</p

Tip60+/− mice display normal eWAT weight and morphology.

<p>A, Epididymal fat pad (eWAT) weights of WT and Tip60+/− mice as percentage of total body weight (BW) on a LFD and HFD regimen, measured after termination. * p<0.05, ** p<0.01. B, Plasma free fatty acid (FFA) levels. * p<0.05. C, H&E staining of representative eWAT sections from WT and Tip60+/− mice after 19 weeks of LFD or HFD feeding. Scale bars indicate 100 µm.</p