Synchronous larvae were washed twice in M9 buffer, counted and adjusted to 10 larvae per 10 μl. Nematodes were raised in cell culture flasks (Sarstedt, Nürmbrecht, Germany) or OP50 spread NGM plates. OP50-NGM was given as a standardized food source with a volume 4.4-fold of the larvae containing M9 solution used. L1 larvae were maintained under continuous shaking at 20°C, reaching adulthood within 3 days.
PX627 were treated with 1 mM indole-3-acetic acid (auxin, Alfa Aesar, Haverhill, MA, USA) to induce infertility during the L3 stage [12 (link), 13 (link)]. Wild-type N2 nematodes were treated with 100 μM 5-fluoro-2´-deoxyuridine (FUdR) or M9 control after reaching young adulthood. PX627 were also treated with M9 control 48 h prior to the assessment on day-2. The day upon which the nematodes reached young adulthood was defined as day 1. To ensure ad libitum food supply until day 10, at day-2 and day-6, old OP50-NGM was discarded after sedimentation of gravid nematodes and fresh OP50-NGM, with effectors incorporated, was given. For the measurements on day-2, FUdR- and auxin-treated young controls and untreated nematodes were co-assessed, whereas at day-10, only sterilized and unfertilized animals could be assessed.
PX627 were treated with 1 mM indole-3-acetic acid (auxin, Alfa Aesar, Haverhill, MA, USA) to induce infertility during the L3 stage [12 (link), 13 (link)]. Wild-type N2 nematodes were treated with 100 μM 5-fluoro-2´-deoxyuridine (FUdR) or M9 control after reaching young adulthood. PX627 were also treated with M9 control 48 h prior to the assessment on day-2. The day upon which the nematodes reached young adulthood was defined as day 1. To ensure ad libitum food supply until day 10, at day-2 and day-6, old OP50-NGM was discarded after sedimentation of gravid nematodes and fresh OP50-NGM, with effectors incorporated, was given. For the measurements on day-2, FUdR- and auxin-treated young controls and untreated nematodes were co-assessed, whereas at day-10, only sterilized and unfertilized animals could be assessed.
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