Figure 6. MST-FOXO Signaling Promotes Longevity in C. elegans. (A) In vitro kinase assays were carried out by incubating recombinant MST1 (Upstate) together with recombinant His-DAF-16 followed by immunoblotting with the phospho-FOXO or His antibody.(B) Lysates of 293T cells transfected with an expression plasmid encoding HA-DAF-16 or HA-DAF-16 S196A together with the FLAG-MST1 expression vector or control plasmid were immunoblotted with the phospho-FOXO, HA, or FLAG antibody.(Ca and Cb) Pcst1::gfp expression in wild-type nematodes. Fluorescent (Ca) and Nomarski (Cb) views of an L4 nematode are shown and are representative of more than five independent transgenic lines at all postembryonic stages examined (L1-adult; data not shown).(D) Left panel: CST knockdown shortens nematode life span. For statistical analysis, see Table S2. Right panels: RT-PCR from synchronized L4 nematodes maintained on bacteria harboring the
cst-1 RNAi plasmid or control bacteria for two generations.(E and F) CST knockdown reduced body movement when compared to control (log rank, p < 0.0001; animals observed/initial number examined: N2 = 42/53,
cst-1(RNAi) = 42/50). CST knockdown also reduced pharyngeal pumping when compared to control (log rank, p < 0.01; animals observed/number examined: N2 = 39/53,
cst-1(RNAi) = 38/50).(G) CST knockdown in Pmyo-3::
myo-3::gfp nematodes promoted advanced muscle deterioration, including sarcomere disorganization, bending, and fragmentation. Images are representative of day 14 adult nematodes.(H) Age-associated oily droplets between the pharyngeal bulbs developed earlier in response to CST knockdown when compared to control N2 nematodes (days 5 and 6; ANOVA; p < 0.05). Increased
cst-1 expression delayed the onset and progression of oily droplets (day 7; ANOVA; p < 0.01). Data are represented as mean ± SEM. Arrowheads in middle panel indicate representative oily droplets. Right panels: Increased
cst-1 gene dosage in Ex1050 nematodes is shown by RT-PCR from young adult nematodes.(I) C. elegans movement was scored by their ability to move during a 20 s time interval in response to gentle tapping of plates. Increased gene dosage of
cst-1 delays the onset of age-related physiological processes (log rank, p < 0.01; n: Ex1060 = 26/50, Ex1050 = 39/55).