Fig. 4: DONSON regulates the intrinsic S/G2 checkpoint by suppressing unscheduled ETAA1 degradation. | Nature Communications

Fig. 4: DONSON regulates the intrinsic S/G2 checkpoint by suppressing unscheduled ETAA1 degradation.

From: The DNA replication machinery transmits dual signals to prevent unscheduled licensing and execution of centrosome duplication

Fig. 4

a A schematic of the intrinsic S/G2 checkpoint based on previous studies19,20,73. b The subcellular localization pattern of CyclinB1 of WT cells in G1, S, and G2 phases. c The expression pattern of CyclinB1 in S phase upon Chk1- or ATR-inhibition. HeLa cells were treated with 250 nM CHIR-124 (Chk1i) or 2 μM VE-821 (ATRi) for 6 h and EdU for 30 min to classify cells in S phase. d Histograms represent the frequency of the S phase cells with the indicated phenotypes observed in (c). n  =  3 independent experiments, 30 cells for each. e The expression pattern of CyclinB1 in S phase after siRNA treatment against the indicated proteins. f Histograms represent the frequency of the S phase cells with the indicated phenotypes observed in (e). n  =  3 independent experiments, 30 cells for each. g Representative immunoblot analysis of whole cell extracts for the indicated proteins from HeLa cells treated with siControl or siDONSON for 48 h. HSP90 was used as a loading control. h Representative immunoblot analysis of whole cell extracts for ETAA1 upon treatment with a proteasome inhibitor in DONSON-depleted cells. HeLa cells were treated with siControl or siDONSON for 48 h and 20 μM MG132 for 6 h. α-Tubulin was used as a loading control. i Ubiquitination of ETAA1 upon DONSON depletion. HeLa-Tet3G mNG-ETAA1 cells were treated with siControl or siDONSON and doxycycline (Dox) for 48 h, transfected with a HA-ubiquitin expressing plasmid for 24 h, and treated with 20 μM MG132 for 6 h. mNG-ETAA1 was immunoprecipitated with mNG-beads. Immunoprecipitates were blotted with HA antibody to detect ubiquitylated ETAA1. IP, immunoprecipitation; WCE, whole cell extract. j, l Inducible expression of ETAA1 in DONSON-depleted cells. HeLa-Tet3G mNG-ETAA1 cells were treated with siControl or siDONSON following Dox treatment for 24 h. Arrowheads indicate centrosomes. k, m Histograms represent the frequency of S-phase cells with the indicated phenotypes observed in (j) and (l), respectively. n  =  3 independent experiments, 30 cells for each. All scale bars, 5 μm. Values are mean percentages ± s.d. Tukey’s multiple comparison test was used in (f), and a two-tailed, unpaired Student’s t-test was used in (k) and (m) to obtain P-value. Source data are provided as a Source Data file.

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