Table 1.
Commercial sources of compounds screened.
Fig 1.
Characteristics of the tra-2(ar221);xol-1(y9) mig-24p::Venus screening strain.
(A) Percentage of animals with either a single fluorescent linker cell, two fluorescent distal tip cells, or a combination of fluorescent linker and distal tip cells is shown for three growth conditions. For the 15°C and 25°C conditions, gravid adults were placed at the indicated temperature and their progeny scored at the L4 stage. For the assay protocol condition, gravid adults were bleached and their eggs allowed to hatch in M9 overnight at 20°C. Synchronized L1s were then grown at 25°C and scored at the L4 stage. (B) Comparison of linker cell survival in screening strain and standard linker cell death scoring strain. Animals were grown at 25°C and scored for linker cell survival 2–4 hours after the L4-adult transition [12] (C) Percentage of linker cells remaining in tra-2(ar221);xol-1(y9) mig-24p::Venus animals over time after an initial growth period of 32 hours on agar plates with food, washing in S Basal, and a change to new growth conditions for 12 additional hours. X-axis numbers indicate hours after media change (no parentheses) and total hours after L1-arrest (parentheses). (D, E) tra-2(ar221);xol-1(y9) mig-24p::Venus in migrating (D) and dying (E) linker cells. Arrowheads, linker cell.
Fig 2.
(A) Assay workflow: male tra-2;xol-1 mig-24p::Venus animals grown on agar plates with OP50 at 25°C for 32 hours are resuspended in S-Basal medium and transferred to plates with screening compounds in most wells (grey squares), positive (blue) and negative (red) controls. Plates are incubated for 12 hours and scanned using a fluorescence cytometer. (B) Image of Tyrphostin A9-treated well. (C) Same as (B), except counted objects marked (yellow). Large debris (red arrow) is not counted by the software, though some smaller fluorescent shapes that are not cells are erroneously labeled as cells (white arrow). “Count” indicates object count output from the MetaXpress software.
Fig 3.
Tyrphostin A9 causes linker cell persistence.
(A) Screen of the LOPAC library using Tyrphostin A9 as a positive control. Compounds are plotted with an arbitrary index (X-axis). Percent inhibition (Y-axis) is normalized to negative control and positive control (Tyrphostin A9 treated) counts for each plate screened. The Tyrphostin A9 data point is indicated (arrow). (B) Effects of Tyrphostin A9 on animal viability, development, and linker cell presence are compared to DMSO exposure alone. LC, linker cell. (C) Image of a tra-2(ar221);xol-1(y9) mig-24p::Venus animal, treated with Tyrphostin A9 for 12 hours, showing persistent fluorescent linker cell (arrowhead) and undeveloped tail (arrow). (D) Effect on linker cell persistence after 12 hours of treatment with varying concentrations of Tyrphostin A9. Negative control mean is defined as 0% inhibition and maximum linker cell count in Tyrphostin A9 treated animals is defined as 100% inhibition. The EC50 for Tyrphostin A9 is calculated to be 45.6 nM.
Table 2.
Assay positives from pilot screen.
Fig 4.
Primary screen results and attrition in secondary analyses.
(A) Compounds screened ranked by percent inhibition and normalized to negative and positive control values for each plate screened. All screened compounds are depicted except those with normalized inhibition >160% (23/24298 events). Solid line indicates cutoff for additional testing. Dashed lines indicate percent inhibition of negative controls (0%) and positive controls (100%). (B) Secondary screening resulted in 11 compounds that were further examined.
Table 3.
Compounds reproducibly resulting in persistent linker cells.
Fig 5.
Chemical structures of compounds identified from pilot screen (A, B) and primary screen (B-M). (A) Tyrphostin A9, (B) Tyrphostin AG 879, (C) CB8776, (D) CB0146, (E) EN9834, (F) EN5065, (G) EN1918, (H) EN7212, (I) Leflunomide, (J) EN2416, (K) CB0736, (L) EN1123.
Fig 6.
Effects of compound treatment on viability and development.
(A) Toxicity following 12-hour incubation with Leflunomide (black), EN5065 (blue), or EN1918 (red) on agar plates with OP50 E. coli, starting 32 hours after L1 arrest. Animals classified as dead did not move and exhibited no pharyngeal pumping. Error bars, SD. (B) Toxicity of CB0146 (squares) and Tyrphostin AG 879 (circles) as in (A). LD50 values calculated at 2.8 μM for CB0146 and 260 nM for Tyrphostin AG 879. (C) Surviving LCs scored in animals after 12-hour treatment with Leflunomide (black), EN5065 (blue), or EN1918 (red) across a range of compound concentrations. Error bars, SD. (D) Surviving LCs scored in animals after 12-hour treatment with CB0146 (squares) and Tyrphostin AG 879 (circles) across a range of compound concentrations. Error bars, SD. (E) Effect of compounds on male tail development in liquid assay. Error bars, SD. *, p<0.01, student’s t-test.