Extended Data Fig. 3: APC/C and WDR5 are required for human ES cell survival.

a, Mass spectrometry analysis of Flag–WDR5 purified from mitotic HEK293T cells. Values listed in brackets are total spectral counts of tryptic peptides of indicated proteins. b, Depletion of WDR5 phenocopies the depletion of APC2 in H1 human ES cells. H1 cells were depleted with the indicated siRNAs for 72 h before collection for western blot analysis. This experiment was performed once. c, A cumulative fraction curve measuring the length of each metaphase-to-anaphase transition. n = 112 cells for control siRNA; n = 105 cells for siRNA against APC2; n = 106 cells for siRNA against WDR5; and n = 217 cells for siRNAs against both APC2 and WDR5. d, Depletion of APC2 or WDR5 causes cell death in H1 human ES cells. Cell death was measured by trypan blue staining of dead cells (mean of n = 4 independent experiments ± s.d.). e, Quantifying cell survival using chromosome catastrophe as a proxy for cell death. H1 human ES cells virally expressing H2B–mCherry were transfected with the indicated siRNAs for 24 h before imaging by confocal microscopy. n = 97 cells for control siRNA; n = 104 cells for siRNA against APC2; n = 90 cells for siRNA against WDR5; and n = 213 cells for siRNAs against both APC2 and WDR5. f, Sister cells die immediately following mitotic exit when depleted of APC2 and WDR5. H1 human ES cells virally expressing H2B–mCherry were transfected with siRNA against APC2 and/or siRNA against WDR5 for 24 h before imaging by confocal microscopy. The time of death, as defined by cells undergoing chromosome catastrophe, was measured for each sister (mean of n = 57 pairs of cells ± s.d.). g, Representative frames of live-cell imaging from four independent experiments (in minutes) tracking the nuclei of siRNA-depleted H1 human ES cells virally expressing H2B–mCherry. Arrows mark individual sister cells upon mitotic exit. Chromosome catastrophe was used a proxy for cell death (time points 198 and 342). h, Flag–WDR5 associates with APC/C in mitotic H1 human ES cells. Flag–WDR5 immunoprecipitations were performed on asynchronous H1 human ES cells (A) or H1 human ES cells arrested in mitosis (M). Bound proteins were determined by SDS–PAGE and western blotting. This experiment was performed two independent times with similar results. i, Overexpressed haemagglutinin (HA)-tagged USP44 associates with Flag–WDR5 in both asynchronous and mitotic HEK293T cells. MYC–WDR5 was used as the control vector. This experiment was performed three independent times with similar results.