(A) Cartoon of C. elegans germ line (B) Wild-type, mat-2(ts), and zyg-1(ts) germ lines stained with MAD-1, MAD-2, CENPA or P-CHK-1(Ser344) (red), α-tubulin (green), and counterstained with DAPI (blue) following growth at 25°. Scale bars = 5μM. (C) Quantification of H3S10P-positive nuclei per germ line in wild-type and zyg-1(ts) worms treated with atr, chk-1, mad-1 or control (L4440) RNAi at 25° (n ≥ 20). zyg-1(ts) mean = 9.0 ±0.5 SEM vs. WT = 5.0 ±0.4, zyg-1(ts); mad-1(RNAi) = 4.4 ±0.3, zyg-1(ts); atl-1(RNAi) = 6.2 ±0.4, zyg-1(ts); chk-1(RNAi) = 9.1±0.5 p = 0.88; ***p<0.0001. (D) Quantification of H3S10P positive nuclei per germ line in mat-2(ts) worms grown at 25° treated with control, mad-1, mad-2, mad-3, bub-3, atr, or chk-1 RNAi (n≥48). H3S10P counts between mat-2(ts) and RNAi depletions were not significant except for mad-2(RNAi), which had more H3S10P than control RNAi, p = 0.02, indicating efficient arrest.