Supplemental Data S 1 DAF-16 / FOXO Regulates Transcription of cki-1 / Cip / Kip and Repression of lin-4 during C . elegans L 1 Arrest
Supplemental Data S 1 DAF-16 / FOXO Regulates Transcription of cki-1 / Cip / Kip and Repression of lin-4 during C . elegans L 1 Arrest
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L. Baugh;P. Sternberg
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作者：
L. Baugh;P. Sternberg
对于本文所述的所有实验，将在20 ℃下生长的幼蠕虫（daf-2（e979）除外，其在15 ℃下生长）溶解，并通过所述的次氯酸钠处理收集胚胎[S1]。在接种有大肠杆菌的培养皿中，在标准线虫生长培养基（NGM）加1.7%琼脂上使胚发育和孵化。coli OP 50或5 ml S基础缓冲液[S1]中。我们发现，孵化幼虫在水性缓冲液（S基础）的转盘上的结果在最直接的，可重复的逮捕可能。最初的实验是用在无菌NGM平板（无OP 50）上孵化的幼虫进行的，但在这些条件下，L1发育的数量在停滞之前发生变化（相当于在20 ℃下发育6-8小时）。目前尚不清楚NGM平板上这种可变的、延迟的L1停滞是否是由于培养基的气味、从培养基中获得的营养或琼脂培养基上尸体、鸡蛋壳等的可及性。与提供营养的无菌培养基一致，在NGM琼脂中打洞的幼虫通常发育超过L1阶段。我们还观察到罕见的，nonarresting蠕虫孵化在S basal时，管包含S basal被允许坐着不动，而不是被旋转的转盘上，大概是由于在管底部的同类相食。
Preparation of Arrested L1s For all experiments described here, young adult worms grown at 20oC (except for daf-2(e979), which were grown at 15oC) were dissolved and embryos were collected by sodium-hypochlorite treatment as described [S1]. Embryos were allowed to develop and hatch either on standard nematode growth medium (NGM) plus 1.7% agar in Petri plates seeded with E. coli OP50 or in 5 ml S basal buffer [S1] in a test tube on a carousel. We found that hatching larvae in aqueous buffer (S basal) on a carousel results in the most immediate, reproducible arrest possible. Initial experiments were done with larvae hatched on sterile NGM plates (no OP50), but variable amounts of L1 development occur before arrest under these conditions (equivalent to as much as 6–8 hr development at 20oC). It is unclear whether this variable, delayed L1 arrest on NGM plates is due to the smell of the medium, nutrition acquired from the medium, or the accessibility of carcasses, egg cases, etc. on the agar medium. Consistent with sterile medium providing nutrition, larvae that burrow in the NGM agar often develop beyond the L1 stage. We also observed rare, nonarresting worms hatched in S basal when the tube containing the S basal was allowed to sit still as opposed to being rotated on a carousel, presumably resulting from cannibalism at the bottom of the tube.