Figure S1. Metformin Response Genes Identified from an RNAi Screen in C. elegans, Related to Figure 1(A) Larval distribution of wild-type animals 3 days post exposure to metformin. The drug treatment to synchronized wild-type animals was begun at the L1 stage,and the counting to determine proportion of worms at different larval stages was carried out on day 3 of drug treatment. Three independent experiments wereconducted (n = 45-92 animals are counted in each condition). Data represent means of three experiments and SEM.(B) Representative DIC images of scores from the RNAi screen for genes rendering metformin resistance at 150 uM (highlighted with green boxes) and metforminsensitivity at 25 uM (highlighted with red boxes). The scoring was conducted 3 days after synchronous L1 C. elegans were dropped on RNAi plates containingmetformin or not, when the animals fed bacteria carrying vector RNAi reached egg laying adult stage on no drug plates. In the metformin resistance screen with150 uM metformin, colored in green, scores were assigned based on C. elegans stage (indicated with example images from animals experienced different RNAitreatment, i.e., L1 (0), L1-L2 mixed stage (1), L2-L3 mixed (2), L3-L4 mixed (3), L4 (4), and egg laying adults (5)). In the metformin sensitivity gene screen with 25 mMmetformin colored in red, the scores also corresponded to C. elegans stages but with egg laying adults (0) -L1 (5).(C and D) Sample images of the hits with significant scores from the RNAi screen, including metformin resistance gene F37H8.3 (CeACAD10) (C) and metforminsensitive mitochondrial genes (D). The C. elegans gene names are followed by names of their human orthologs.(E) Animals carrying mutations in mitochondrial components
gas-1/complex I and
mev-1/complex II are hypersensitive to 25 mM metformin. n = 40-78 wormsanalyzed; ##p < 0.01 and **p < 0.01, by two-way ANOVA.(F) CeACAD10 expression in the head and body of C. elegans exhibits a mainly cytoplasmic distribution in the intestine (large areas of red fluorescence). Neuronsin the ventral nerve cord also express CeACAD10 (chain of bright dots) but this expression is not sensitive to biguanides (not shown).