PQBP1 Affects Lipid Storage Figure 7. Abnormal lipid metabolism in the PQBP1 homologue mutant. A) Uptake of exogenous radio-labeled fatty acid (16:0) into various lipids in normal (N2), homozygous tm3004 mutant, and homozygous tm3045 mutant nematodes. TLC was exposed to for 10 hours. B) Quantitative analysis of the results in Fig. 7A. C) Triglyceride contents in nematodes. doi:10.1371/journal.pone.0004104.g007 After screening of the mutants, we obtained a line for TD21D12.3 (tm3004) and another line for T07A9.1 (tm3045). Deleted regions in the genome of these mutants were determined by sequencing of the PCR fragments from their genomes. The primers for TD21D12.3 were 59-CCGGCGGCGATACCCAT- TAA and 59-CCTAATCAAACGACACCGTC. The primers for T07A9.1 were 59-CTGTACATATCTCTCGGGCA and 59- AGGTCGTGCGTTGAATGAGA. Nile Red staining of intestinal cells and morphological observation Nile Red (5H-benzo[a] phenoxazine-5-one, 9-doethylamino) was solved in acetone at 500 mg/ml and diluted to 2.5 mg/ml with phosphate-buffered saline. 3 ml of the solution was layered on a 35 mm NGM plate and dried at room temperature. Starved worms were cultured for 3 days before observation. Microscopic images were obtained by Olympus BX-50 and Olympus SenSys CCD camera. Fluorescence of Nile Red was observed with a Rhodamine filter (580 nm). Nile Red staining images were obtained at the same magnification (206) and same exposure time (260 msec). Signal intensities in the images were quantified using Aquacosmos (HAMAMATSU). The arbitrary signals of intestinal cells were subtracted by those of body cavity to standardize the intensities. More than 100 cells were quantified in each type of nematode. Lipid uptake and content analyses with nematodes Incorporation of exogenous [1-14C] stearic acid into C. elegans was analyzed as described previously [28]. For triglyceride analysis, lipids of synchronized young adult worms were extracted by the method of Bligh and Dyer [29], and the triglyceride contents were measured using enzymatic kit (Triglyceride E-test, Wako Pure Chemical Ltd.) according to the manufacturer’s protocol. PQBP1-shRNA plasmid construction The shRNA expression plasmids against mouse PQBP1 was derived from the RNAi-Ready pSIREN-RetroQ-ZsGreen (Clon- tech) shRNA expression vector. The double-stranded DNA oligonucleotides encoding shRNA against PQBP1 were composed of a sense strand, a 9-nucleotide hairpin loop, the complementary anti-sense strand, a poly(A) termination signal and an Xho I site. The sequence was derived from mouse PQBP1 specific siRNAs (Qiagen):GAGGAAGAGATTATTGCTGAA (SI01387120). The synthesized double strand oligonuclotides containing the sense and PLoS ONE | www.plosone.org 7 January 2009 | Volume 4 | Issue 1 | e4104