C RNAi feeding library [88]. Cultures had been plated onto NGM plates containing 25g/ml Carbenicillin and 1mM IPTG and had been applied inside two weeks.Assessment of SAC RNAi efficiencyFollowing depletion of SAC, L4 worms have been fed cyb-3 RNAi for 24 hrs. Worms had been dissected and embryos had been placed on a 3 agarose pad and imaged by DIC at 40X on a Ziess compound microscope to monitor arrest.Irradiation experimentsYoung adult worms had been irradiated with 30Gy (3000 rad) from a Cs-137 supply. Worms had been dissected 8 hrs post irradiation for MAD-2 and CENPA localization studies and 24 hrs post irradiation for Abc Inhibitors products recovery experiments.Hydroxyurea experimentsFor high dose experiments, L4s were placed on NGM plates containing 25mM hydroxyurea (HU) (Sigma Aldrich) for 16 hrs before either dissection, transfer for recovery, or progeny viability assays. Cell cycle arrest was assayed by counting DAPI stained nuclei for chk-1 and atr RNAi efficiency. For low dose HU experiments, staged young adults were exposed to 5mM HU for 2 hrs ahead of being moved to a–HU plate for dissection, recovery, or progeny viability assays. HU was permitted to dissipate into plates for no less than three hrs before worms were introduced. For low dose HU exposure, cell cycle kinetics were assayed by counting H3S10P.Metaphase arrest and delayFor antibody staining after MAT-2 or ZYG-1 inactivation, mat-2(ax102) and zyg-1(b1) L4s had been transferred for the restrictive temperature of 25 for 16 or 48 hrs just before dissection, respectively. To decide metaphase delay/arrest, zyg-1(b1) and mat-2(ts) L4s were transferred to the restrictive temperature of 25 for 24 hrs just before dissection and staining with H3S10P.WesternWorm lysates were generated from unmated fog-2(q71) worms to eliminate contribution from embryos and had been resolved on 12 SDS-PAGE gels and transferred to Millipore Immobilon-P PVDF membranes. Membranes had been blocked with 5 BSA and probed with rabbit antiCENPA (1:250, Novus), and anti-Mortalin/Grp75 mouse monoclonal antibody N52A/42 (1:20, AB_10674108; UC Davis/NIH NeuroMab Facility, Davis, CA) as loading control., followed by IRDye680LT- and IRDye800-conjugated anti-rabbit and anti-mouse IgG secondary antibodies obtained from LI-COR Bioscience (Lincoln, NE). 0.01 SDS was added to antirabbit secondary.PLOS Genetics | DOI:10.1371/journal.pgen.April 21,21 /DNA Harm Response and Spindle Assembly CheckpointCell linesHuman U2OS osteosarcoma cells and COS monkey kidney cells were, obtained in the ATCC. U2OS cells have been grown in McCoy’s 5A modified medium, COS cells were grown in DMEM and each were supplemented with ten fetal bovine serum and were cultured at 37 in five CO2.Immunofluorescence in cell linesCells were grown on glass coverslips and treated with 5mM HU for 24 hrs or 1g/mL colchicine (Sigma) for 16 hrs. Cells were fixed with 4 paraformaldehyde and 0.1 triton, then blocked with 5 BSA for 1 hr prior to key antibodies were added and incubated at area temperature overnight. Secondary antibodies had been incubated for two hrs at area temperature. To determine fluorescence intensity, integrated density was identified for 2 equal regions in each the nucleus along with the cytoplasm. The ratio of nuclear to cytoplasmic signal was calculated dividing the averages from the 2 measurements. For each and every situation, n!50 cells. Main antibodies have been applied in the following dilutions: rabbit anti-H3S10P (1:500) (Millipore), rabbit anti-MAD2L1 (1:500) and mouse anti-nuclear pore complicated (MAb414) (1:500) (Abcam), mouse.
