Systematic MS-based analysis of TCL1A interacting partners identifies mitotic checkpoint regulators. (A) Experimental setup: TCL1A-bound protein complexes were isolated from JVM3 CLL-like cells ± transfected TCL1A; at baseline (untreated culture), under genotoxic stress (5 μM etoposide for 4.5 hours) or synchronized in mitosis (100 ng/mL nocodazole for 18 hours, release for 1 hour), followed by LFQ LC-MS/MS MS. (B) MS identified in total 962 significantly TCL1A-interacting proteins (cutoffs per group: FDR q < 0.05, FCh >2; Student t test). Most proteins differentially interact with TCL1A in JVM3^TCL1A B cells only at baseline (blue), under genotoxic stress (yellow), or during mitosis (green). (C) Overview of enriched pathway clusters within the TCL1A interactome identified by ORA using Cytoscape plug-in ClueGO (version 2.5.8). Enriched pathways highly depend on the condition. (D) Overview of selected enriched pathways (Reactome pathway database) within the TCL1A interactome identified in panel C. (E) STRING network of TCL1A interactors involved in the cell cycle checkpoint of JVM3 cells under genotoxic stress (left) and synchronized in mitosis (right) identified by ClueGO. Colors indicate the involvement in the particular cell cycle checkpoint (green indicated G1/S transition; blue, G2/M checkpoint; red, separation of sister chromatids; yellow, inhibition of APC/C via direct inhibition of the APC/C complex). Note the marked network expansion upon mitosis induction. Mitotic checkpoint proteins (eg, CDC20, MAD2, and CDC27, a subunit of the APC/C E3 ubiquitin-ligase complex) appear centrally involved in TCL1A signaling in mitosis only. ER, endoplasmic reticulum; TCA, trichloroacetic acid; tRNA, transfer RNA.