Extended Data Figure 6. Evidence that CEP192 is the target of TRIM37 that accounts for enhanced sensitivity to PLK4 inhibition.

(a) Schematic for partial CEP192 inhibition using a short-term inducible knockout, followed by live imaging of mitosis. (b) Evidence in CHP134 cells that CEP192 is a functionally significant target of TRIM37. A CHP134 clonal cell line with reduced TRIM37 expression (~12% relative to parental CHP134 cells) was stably transduced with a CEP192 shRNA that reduced expression by ~75% (immunoblot and quantification below). Live imaging of mitosis showed that while reduction of CEP192 levels had no significant effect on the duration of mitosis in DMSO-treated cells, it significantly extended mitotic duration in centrinone-treated cells. (c) Evidence that the C-terminus of CEP192 is ubiquitinated in a TRIM37-dependent manner. The experiment shown in Fig. 4d included co-transfection of HA-tagged ubiquitin. Shown here is the HA-ubiquitin blot (together with FLAG and Myc blots) of the immunoprecipitated C-terminal fragment that binds TRIM37. Ubiquitination of this fragment was enhanced in the presence of WT relative to ligase-mutant TRIM37. The FLAG blot shown is the same as in Fig. 4d; the Myc blot is a different exposure of the blot shown in Fig. 4d. The other CEP192 fragments are not shown because their stability was affected by co-expression with WT but not ligase-mutant TRIM37, which makes comparisons of ubiquitination profiles difficult. For gel source data see Supplementary Figure 1.