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. Author manuscript; available in PMC: 2007 Nov 28.
Published in final edited form as: Dev Biol. 2007 Mar 3;305(2):551–563. doi: 10.1016/j.ydbio.2007.02.040

Figure 5.

Figure 5

A wild type copy of puf-9 is required for hbl-1 3’UTR reporter down-regulation in the hypodermis and the neurons. (A) A diagram of the heterologous reporter gene (pFS1038) used to assay hbl-1 3’UTR-directed regulation in the hypodermis, consisting of the wild type hbl-1 3’UTR fused downstream of the Escherichia coli lacZ gene and driven by the hypodermally expressed col-10 promoter (not to scale). (B, C) puf-9(ok1136) mutant animals carrying the pFS1038 reporter showed maintenance of β-galactosidase activity in the adult stage (17%), while the same construct in wild type animals showed expected levels of reporter gene down-regulation (2%) at the adult stage. Three independent staining trials were performed. The number of lines scored for each strain are, wild type: 1 line and puf-9(ok1136): 5 lines, n > 270 for all stages scored. For statistical analysis, an unpaired Student’s t test was performed between corresponding stages for the wild type and puf-9(ok1136) backgrounds. Error bars indicate standard deviations. (C) The correct down-regulation for each construct was compared to wild type and assigned values as follows: (++++) 0%−5% of animals with hypodermal expression at the adult stage; (+++) 5%−10%; (++) 10%−20%; (+) 20%−60%; (−) >60%. (D, E) A wild type copy of puf-9 is required for adult stage gfp::hbl-1 3’UTR reporter down-regulation in the VNC. (D) Late L4 (n=18) and adult stage (n=67) puf-9(ok1136) mutants showed an increase in the number of animals with bright HBL-1/GFP in the VNC compared to the same stages in wild type animals (L4, n=21 and adult, n=63). Wild type and puf-9(ok1136) animals were scored in three independent trials for adult GFP expression, and one trial for L4 GFP expression. (E) Quantification of wild type and puf-9(ok1136) GFP levels in the VNC was performed for a sample of L1 and adult stage animals. puf-9(ok1136) mutants showed a significant up-regulation of GFP at the L1 (n=8) and adult stages (n=12) compared to wild type animals (L1, n=4 and adult, n=10). The levels of GFP expression were calculated by normalizing the quantity of average GFP signal in puf-9(ok1136) mutants against the average GFP signal in wild type animals (for L1 and adult stages). Statistics were performed using an unpaired Student’s t test.