Fig 2: Characterization of higher molecular weight (HMW) TRIM37 species. (related to Figure 4)(A) Immunoblot showing expression levels of wild-type (WT) TRIM37 and indicated mutants in RPE-1 tet-on TRIM37 cells. Higher molecular weight (HMW) TRIM37 species are prominently formed in the C18R mutant and indicated with an arrow. β-Actin, loading control. Representative data; n = 3 biological replicates.(B) Same as in (A) but with MG132 (10 μM) treatment to achieve proteasomal inhibition and stabilization of WT TRIM37. β-Actin, loading control. Representative data; n = 3 biological replicates.(C) Top, immunoblot showing detection of various higher molecular weight (HMW) species of TRIM37 upon treatment with increasing concentrations of DSS crosslinker. Vinculin is used as a loading and oligomerization control. Dotted lines indicate separate cropped sections of the same immunoblot. Representative data; n = 3 biological replicates. Bottom, Densitometric analysis of immunoblot with a graph depicting normalized HMW TRIM37 intensity upon increasing DSS concentrations relative to DMSO control (−DSS). Mean ± s.e.m.(D) Representative Sanger sequencing traces of the TRIM37 locus in parental and CRISPR–Cas9 edited RPE-1 TRIM37C18R cells, highlighting the mutation (TGT>CGT) responsible for the biallelic C18R residue substitution, denoted by an asterisk.(E) Left, immunoblot showing endogenous TRIM37 protein levels across the indicated cellular fractions from RPE-1 TRIM37C18R cells. Validation markers include CEP192, Centrobin, and SAS6 for centrosomal proteins, and Lamin A/C for the nuclear fraction. Ponceau-stained blot indicates loading. Representative data; n = 3 biological replicates. WCE, whole-cell extract; exp, exposure. Right, Densitometric analysis of immunoblot in with a graph depicting endogenous TRIM37 enrichment in indicated fractions relative to WCE. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate enrichment of TRIM37 in each cellular fraction relative to WCE. Mean ± s.e.m.(F) Left, immunoblot showing detection of various higher molecular weight (HMW) species of endogenous TRIM37 upon treatment of RPE-1 TRIM37C18R cells with increasing concentrations of DSG crosslinker. Vinculin is used as a loading and oligomerization control. Representative data; n = 3 biological replicates. Right, Densitometric analysis of immunoblot with a graph depicting normalized HMW TRIM37 intensity upon increasing DSG concentrations relative to DMSO control (−DSG). Mean ± s.e.m.

Fig 3: Substrate-independent clustering is sufficient to activate TRIM37. (related to Figure 6)(A) Top, schematic of the TRIM37G322V-mNeonGreen-CRY2 optogenetic fusion construct. The star denotes the TRAF domain mutation (G322V). Bottom, illustration of the blue light (BL)-activated optogenetic system enabling TRIM37 clustering independent of binding to a centrosome substrate.(B) Representative time-lapse images of RPE-1 cells expressing the optogenetic construct detailed in (A) incubated in the presence or absence of MG132. Timestamps indicate minutes post blue light exposure. Scale bar = 10 μm.(C) Quantification of mNeonGreen fluorescence intensity from (B), with each condition comprising >30 cells. Mean ± s.d.(D) RPE-1 cells expressing optogenetic constructs detailed in (A) were incubated with or without doxycycline (Dox) and MG132 (10 μM) in the absence or presence blue light for 3 h before immunoblotting for the indicated proteins. Higher molecular weight (HMW) TRIM37 species were prominently formed only in MG132 and BL-stimulated conditions and are indicated with an arrow. Ponceau-stained blot indicates loading. Representative data; n = 3 biological replicates. exp, exposure

Fig 4: Impairment of TRIM37 oligomerization attenuates synthetic lethality in 17q23-amplified cells with PLK4 inhibition.(A) Immunoblot showing TRIM37 protein levels in TP53−/− MCF-7 cells. TRIM37 wild-type (WT), TRIM37 knockdown (KD) via shRNA, and cells harboring the C109S mutation in approximately half of the TRIM37 alleles present (TRIM37C109S) were used. Vinculin, loading control. Representative data; n = 3 biological replicates.(B) Left, Representative data of a 10-d clonogenic survival of indicated MCF-7 cell lines from (A) treated with DMSO (control) or PLK4 inhibitor (PLK4i) (125 nM). Right, Quantification of relative growth in the presence PLK4i relative to DMSO. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate differences between each experimental condition (KD and C109S) and WT. Mean ± s.e.m(C) Quantification of mitotic CEP192 foci in PLK4i-treated TP53−/− MCF-7 cells that lack centrosomes. n = 3, biological replicates, each comprising >30 cells. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate differences between each experimental condition (KD and C109S) and WT. Mean ± s.e.m(D) Representative images for (C). Scale bars, 5 μm.(E) Representative Sanger sequencing traces for the TRIM37 locus in parental TP53−/− MCF-7 cells subjected to TRIM37 knockdown (KD) via shRNA, and CRISPR–Cas9 edited TRIM37C109S KI cells. The mutation (TGT>TCT) leading to the C109S residue substitution is denoted by an asterisk. Silent mutations introduced to prevent re-editing are highlighted.

Fig 5: Chimeric TRIM5 bearing the TRIM37 TRAF domain regulates MTOCs.(A) Schematic overview of the domain swap strategy, which replaces the TRIM5 SPRY domain with the TRIM37 TRAF domain to generate a chimeric TRIM5-TRAF protein.(B) Immunoblot showing total protein expression levels of indicated HA-tagged TRIM5 constructs in RPE-1 tet-on TRIM5 cells. Actin, loading control. Representative data; n = 3 biological replicates.(C) Representative images of the localization and effect of indicated HA-tagged TRIM5 constructs on centrosomal CEP192 levels in RPE-1 tet-on TRIM5 cells. Representative data; n = 3 biological replicates. Scale bars, 5 μm.(D) Quantification of centrosomal CEP192 signal upon doxycycline-induced expression of indicated constructs in RPE-1 tet-on TRIM5 cells from (C), with TRIM37 included as a benchmark. n = 3 biological replicates, each with >100 cells. P values, one-way ANOVA with post hoc Tukey’s multiple comparisons test. Mean ± s.e.m.(E) Representative images of RPE-1 TRIM37−/− cells expressing the indicated HA-tagged TRIM5 constructs. Inset #1 denotes the centrosome, marked by CEP192, and inset #2 denotes the Centrobin assembly, identified by intense Centrobin staining that is non-centrosome localized. Representative data; n = 3 biological replicates. Scale bars, 5 μm.(F) Quantification of Centrobin assembly occurrence in RPE-1 TRIM37−/− cells expressing the indicated HA-tagged TRIM5 constructs from (E). n = 3 biological replicates, each with >100 cells.