Having proven the selectivity and the sensitivity of the optimized PLA protocol, we next addressed the question of the detectability of fusion proteins (i.e. ETV6-RUNX1) using our optimized PLA protocol. To that aim, we overexpressed ETV6-RUNX1 transcript and protein (Fig. 4a and 4b) in Nalm6 cells (Nalm6+ETV6-RUNX1 cells) and carried out PLA (Fig. 4c). The negative control (RUNX1 antibodies alone) displayed 0.7 (± 1.8, n = 296) dots demonstrating a very low background. The positive control (detection of RUNX1 protein using two anti-RUNX1 antibodies) showed 23.6 (± 27.6, n = 302) dots per nucleus. In those Nalm6+ETV6-RUNX1 cells, protein proximity between RUNX1 and CBFB was maintained (10.2 dots per nucleus) as observed in Nalm6 cells. Detection of ETV6-RUNX1 fusion protein showed 23.3 (± 25.0, n = 419) dots per nucleus in Nalm6+ETV6-RUNX1 cells whilst the same mix of antibodies detected less than 1.2 dots per cells in Nalm6 cells (Fig. 2b and 4c). This result convincingly demonstrates that PLA can be a powerful tool to detect overexpressed fusion proteins. We wanted further to detect the endogenous ETV6-RUNX1 fusion protein. For that purpose, we used REH cells which harbor the chromosomal translocation t(12;21)(p13;q22) that generates the fusion gene and protein ETV6-RUNX1 (Fig. 2a). PLA using single-species RUNX1 antibodies showed no background (0.4 dots per nucleus; negative control) whereas incubation with two species-different antibodies directed against RUNX1 displayed on average 29.8 dots (± 17.6, n = 426) (positive control) (Fig. 4d) confirming the efficiency of the protocol on REH cells. Importantly, PLA using a mix of anti-RUNX1 and anti-ETV6 antibodies showed 21.6 ± 12.0 (n = 415) dots per cells. We observed that the majority of ETV6 and RUNX1 proximities were localized within the nucleus. Because the non-translocated allele of ETV6 is absent in REH cells, this result of ETV6 and RUNX1 proximity means the presence of ETV6-RUNX1 fusion protein in those cells. We were next interested in assaying the molecular proximity between the ETV6-RUNX1 fusion protein and CBFB. To the best of our knowledge, even if this interaction was suspected , it has never been formally demonstrated so far. PLA using a mix of anti-CBFB and anti-RUNX1 antibodies, as well as PLA using a mix of anti-CBFB and anti-ETV6 antibodies revealed a molecular proximity between ETV6-RUNX1 and CBFB (Fig. 4d). Those data demonstrate that PLA can be a powerful tool to detect overexpressed as well as endogenous fusion proteins in cell lines.Fig. 4Our optimized PLA protocol effectively detects overexpressed or endogenous fusion proteins in pre-B lymphoblasts. The Nalm6 cell line overexpressing ETV6-RUNX1 (Nalm6+ETV6-RUNX1) was validated using RT-qPCR (a) and western blot (b). a Results are presented in terms of a fold change after normalizing ETV6-RUNX1 mRNA levels with GAPDH mRNA. Each value represents the mean of ± S.D. of three independent transduced cells. b Representative images of western blot showing expression of RUNX1 or HSC70 proteins in both cell lines are represented. c Pictures and quantification of PLA signals on Nalm6 cells overexpressing ETV6-RUNX1 protein (Nalm6+ETV6-RUNX1 cells) (a) or on REH cells that expressed endogenous ETV6-RUNX1 protein (b). Nuclei were stained with DAPI. RUNX1 ab110035 antibodies were incubated alone (−) or with RUNX1 ab23980, CBFB ab133600 or ETV6 sc11382 antibodies. Each picture (upper panel) is representative of a typical cell staining observed in 5 fields chosen at random. The quantification of the number of PLA dots per nucleus is presented with the mean values ± S.D