File S1 - Characterization of PUD-1 and PUD-2, Two Proteins Up-Regulated in a Long-Lived daf-2 Mutant

Figure S1., Increase of the PUD-1 and PUD-2 protein abundance in the daf-2 mutant is mostly due to post-transcriptional regulation independent of daf-16. (a) Quantitative RT-PCR of pud-1 and pud-2 in WT, daf-16(mu86), daf-2(e1370), and daf-16(mu86); daf-2(e1370) worms. Data are shown as mean ± s.e.m. of three independent experiments, each with duplicate measurements. (b) Anti-PUD-1 and anti-PUD-2 western blots showing the protein levels in whole-worm lysates of the indicated strains. The anti-tubulin signals control for loading. (c) Summary of the densitometry measurements of three independent experiments including the one shown in (b), expressed as mean ± standard deviation. * p vs. the wild type N2. Figure S2. PUD-1::GFP and PUD-2::GFP expressed from transgene arrays under the control of their native promoters. (a) GFP expression constructs of PUD-1 (pYG2) and PUD-2 (pWX1). (b-c) Both are strongly expressed in the intestine and less strongly in the hypodermis. The inset shows that PUD-1::GFP or PUD-2::GFP is expressed in the nucleoplasm of intestinal cells, largely excluded from the nucleolus except for one or more puncta. (d-e) A temperature shift from 20 °C to 27 °C stimulated the expression of PUD-1::GFP (d) and PUD-2::GFP (e). Figure S3. hqIs28 and hqIs60, but not hqIs24, extended daf-2 lifespan. hqIs28 and hqIs60 extended the lifespan of daf-2(e1370) (a) or daf-2(RNAi) (b) mutants, whereas hqIs24 (c) did not. Figure S4. The distribution of the PUD gene family members in the C. elegans genome and the location of niDf209. (a) pud-1.2, pud-2.2, pud-3, pud-4, pud-1.1 and pud-2.1 are next to each other on Chromosome V. pud-1.2 is a perfect duplicate of pud-1.1 and pud-2.2 is a perfect duplicate of pud-2.1. The boundary of niDf209 as annotated in the wormbase.org is not precise. The actual endpoints of niDf209 are indicated. The flanking sequences are GTACTGTAGGCC [15979 bp deletion] [1667 bp insertion] TGTAATTCCACG. The 1667 bp insertion consists of a 67-bp sequence“TACTGTAGGATTACTGTAGTTTAAAAAAGGGATTTCAGCTTTCGAAAAGGTATTGAACGAAGATTAG” (indicated by a very short blue segment right before an orange arrow) followed by an inverted duplicate of a 1600-bp fragment from Y19D10B.5 “TGTAGAATTCT C….AACGGAACGGTT” (orange arrows). (b) pudl-1 and pudl-2 are adjacent genes on Chromosome IV. Figure S5. Transplanting niDf209 from JU258 to the N2 background by homologous recombination. Figure S6. pYG17, an RNAi construct for knocking down multiple members of the PUD gene family. Figure S7. Sequence alignment of the PUD gene family proteins. Sequences were aligned by MUSCLE program and slightly adjusted. The abbreviation of species name and Genebank ID are indicated for each sequence. The secondary structures of PUD-2 are indicated above the alignment. Shading of black, grey and light grey represent 100%, 80% and 60% conservation, respectively. Table S1. Data collection and refinement statistics. Table S2. Endogenous promoter-driven expression of GFP translational fusion proteins. Table S3. mRNA levels of pud family genes. Table S4. Constructs. Table S5. C. elegans Strains.