Fig. 9: S112 residue and Tpx2-like motifs are essential for Bora function during the mammalian cell cycle.

a Schematic for the structure–function analysis of Bora during the mammalian cell cycle. Endogenous Bora located on chromosome (Chr) XIII was C-terminally tagged with Neongreen (NG) and the Auxin-Inducible degron (AID) in DLD1 cells constitutively expressing the Tir1 subunit of the SCF^TIR1 E3-ligase. A wild-type or a mutant copy of untagged Bora driven by the doxycycline promoter (pTRE3G) was inserted on Chr XIX of the same cells using CRISPR/Cas9 genome editing. Doxycycline promotes expression of untagged wild-type or mutant Bora, while Auxin induces SCF^TIR1-mediated proteasome degradation of Bora-NG-AID resulting in a Bora knockdown. b Workflow used to follow the progression of the cells through the cell cycle. Expression of Bora^WT or the indicated mutants were induced with doxycycline during the second thymidine block. Bora-NG-AID was then depleted at the time of release from DTB using Auxin. Cells were collected 10 and 13 h after DTB release for Western blot and FACS analyses. c Histogram plots showing the cell cycle distributions (percentage of cells in G1, S, and G2/M) for DLD1-Bora-NG-AID cell lines, expressing either Bora^WT or Bora^{[S41A S112A S137A]} and treated as outlined in b. AS: Asynchronous cells; Ctrl: non-treated; A: Auxin-treated cells; A/D: Doxycyclin + Auxin-treated cells. d Box plots indicate median (middle line) and min max (Wiskers) showing the quantified rescue index (RI) for each DLD1 Bora-NG-AID cell line expressing Bora^WT (in dark green) or the indicated mutants: Bora^{[S41A S112A S137A]} (in red), Bora^S41A (in blue), Bora^S112A (in orange), Bora^S137A (in green), Bora^∆35 (in magenta), Bora^{[F25D Y31D]} (in pink), and Bora^{[F103D F104D]} (in purple) assessed 10 (first column) and 13 (second column) hours after DTB release; n indicates the number of replicates. e Western blot analysis of protein lysates from a DLD1-Bora-NG-AID cell line expressing Bora^WT or the indicated mutants 10 h after DTB release as depicted in b, c, and d. Bora, pT210-Plk1, and panPlk1 antibodies were used for these analyses (from top to bottom). (−) Asynchronous cells.