Figure 8. ShRNA-mediated suppression of PQBP1 inhibits lipid storage in white adipocytes. A) Western blot shows that PQBP1- shRNA-IRES-ZsGreen plasmid suppresses PQBP1 protein in primary mouse white adipocytes. Non-silencing shRNA (ns-ShRNA) was used as a control. The band intensities are corrected with GAPDH and standardized with non-transfected cells (NT). B) Primary white adipocytes were stained by Oil Red after transfection with PQBP1- shRNA-ZsGreen or non-silencing shRNA-ZsGreen plasmid. Non-trans- fected cells (arrowheads) possess large lipid droplets while PQBP1- shRNA-transfected cells (green) show small granules (arrows). Non- silencing shRNA-transfection did not affect the size of lipid granules (arrows in lower panels). doi:10.1371/journal.pone.0004104.g008 anti-sense siRNA sequences (59-GGATCCGGAGGAAGAGAT- TATTGCTGAATTCAAGAGATTCAGCAATAATCTCTTCC- TCCTTTTTTCTCGAGGAATTC-39) were inserted into the References 1. Waragai M, Lammers CH, Takeuchi S, Imafuku I, Udagawa Y, et al. (1999) PQBP-1, a novel polyglutamine tract-binding protein, inhibits transcription activation by Brn-2 and affects cell survival. Hum Mol Genet 8: 977–987. 2. Okazawa H, Rich T, Chang A, Lin X, Waragai M, et al. (2002) Interaction between mutant ataxin-1 and PQBP-1 affects transcription and cell death. Neuron 34: 701–713. 3. Komuro A, Saeki M, Kato S (1999) Association of two nuclear proteins, Npw38 and NpwBP, via the interaction between the WW domain and a novel proline- rich motif containing glycine and arginine. J Biol Chem 274: 36513–36519. 4. Waragai M, Junn E, Kajikawa M, Takeuchi S, Kanazawa I, et al. (2000) PQBP- 1/Npw38, a nuclear protein binding to the polyglutamine tract, interacts with PQBP1 Affects Lipid Storage BamH I and EcoR I sites of the shRNA expression vectors at the 59- and 39-ends, respectively. A control non-silencing shRNA (GGATCCGTGCGTTGCTAGTACCAACTTCAAGAGATT- TTTTACGGCGTGAATTC) was synthesized and inserted into the shRNA expression vector. Primary culture of white adipocytes and shRNA transfection White adipocytic precursor cells were obtained from Primary Cell Co., Ltd (Hokkaido, Japan). The cells were grown in growth medium (Dulbecco’s Modified Eagle Medium, 10% foetal bovine serum, 10unit/ml penicillin, 10 mg/ml streptomycin, 17 mM pantothenic acid, 33 mM (+)-biotin, 100 mM ascorbic acid, 1 mM octanoic acid, and 50 nM Triiodothyronine) at 37uC and 5% CO2 for 24 hours. The cells were transferred to 10 cm dish pre-coated with collagen at a density of 26104cells/cm2 and incubated for 12 hours. Thereafter, the cells were transfected with PQBP1 shRNA-ZsGreen plasmid using Effecten (Qiagen) and incubated for 12 hours. Transfection with a non-sense shRNA plasmid and the mock treatment were performed in parallel. Subsequently, the medium was changed to Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum, 10unit/ml penicillin, 10 mg/ml streptomycin, 17 mM pantothenic acid, 33 mM (+)-biotin, 100 mM ascorbic acid, 1 mM octanoic acid, 50 nM Triiodothyronine, 10 mg/ml Insulin, and 2.5 mM Dexa- methasone for 48 hours to differentiate the cells to white adipocyte. Finally, the cells were kept for 7 days in maintenance medium (Dulbecco’s Modified Eagle Medium, 10% fetal bovine serum, 10unit/ml penicillin, 10 mg/ml streptomycin, 17 mM pantothenic acid, 33 mM (+)-biotin, 100 mM ascorbic acid, 1 mM octanoic acid, 50 nM Triiodothyronine, and 10 mg/ml Insulin) before image acquisition. Oil Red staining of white adipocytes White adipocytes were fixed two times with 4% paraformalde- hyde in phosphate buffer, pH 7.4, for 15 min and 10 min, washed with PBS, and treated with 60% isopropanol for 1 min. Next, the cells were incubated with Oil Red for 10 min at 37uC. After Oil Red staining, cells were treated with 60% isopropanol for 2 min and washed three times with PBS. Western blot analysis of white adipocytes Western blots were performed with anti-PQBP1 rabbit antibody against human 1–265 a.a. that cross-reacts with mouse PQBP1 (sc- 32910, Santa Cruz). Western blot with the antibody showed a single band confirming the specificity (data not shown). Author Contributions Conceived and designed the experiments: HA SM HO. Performed the experiments: KT SY MM Wi TI SM. Analyzed the data: HS SM HO. Contributed reagents/materials/analysis tools: HO. Wrote the paper: HA SM HO. U5-15kD/dim1p via the carboxyl-terminal domain. Biochem Biophys Res Commun 273: 592–595. 5. Zhang Y, Lindblom T, Chang A, Sudol M, Sluder AE, et al. (2000) Evidence that dim1 associates with proteins involved in pre-mRNA splicing, and delineation of residues essential for dim1 interactions with hnRNP F and Npw38/PQBP-1. Gene 257: 33–43. 6. Kalscheuer VM, Freude K, Musante L, Jensen LR, Yntema HG, et al. (2003) Mutations in the polyglutamine binding protein 1 gene cause X-linked mental retardation. Nat Genet 35: 313–315. 7. Lenski C, Abidi F, Meindl A, Gibson A, Platzer M, et al. (2004) Novel truncating mutations in the polyglutamine tract binding protein 1 gene (PQBP1) cause PLoS ONE | www.plosone.org 8 January 2009 | Volume 4 | Issue 1 | e4104