They coupled the PLA assay with a conventional immunofluorescence technique, which allowed for multiparametric fluorescent and morphological analysis. G4 in cells. The first strategy utilizes small quadruplex-binding ligands [15,16], while the second is based on antibodies recognizing G-quadruplexes [17,18]. Both methods were successfully applied for the visualization of cellular G4. Importantly, emerging pieces of evidence suggest that the formation of G-quadruplexes is regulated through interactions with different proteins. Particular attention has been paid to the helicases, which are enzymes involved in resolving G4. Among them, the best characterized are BLM (Bloom syndrome RecQ like helicase), BRIP1 (BRCA1 interacting protein C-terminal helicase-1, also known as FANCJ), PIF1 (PIF1 5-to-3 DNA helicase), and WRN (Werner syndrome RecQ like helicase) [19]. PIF1 is a helicase that is active in the nucleus (mainly at telomeres) and in mitochondria. It binds to G4, especially in the S phase [20,21]. BRIP1 forms a complex with BRCA1 and shows a greater affinity for G4 structures than for single-stranded or double-stranded DNA [22]. Both PIF1 and BRIP1 play an important role in the suppression of DNA instability at G4 motifs [20,22,23]. Besides helicases, a wide range of G4-binding proteins have also been identified so far (for a review, see reference [23]). The majority of evidence comes from in vitro studies, yet far less is known about their role in G4 unwinding or stabilization in cells. In general, G4 toxicity stems from replication stress. During DNA synthesis, the replication forks can stall as a result of encounters between the replication complex and template modifications, such as the presence of G-quadruplexes. These stalled forks are a major source of genome instability [24]. An important mechanism that contributes to DNA damage tolerance AKBA is a direct bypass of template lesions via translesion DNA synthesis (TLS), which is mediated mainly by polymerase theta and encoded by gene [20]. Furthermore, BRIP1 helicase is particularly active in TLS [22]. Interestingly, administration of the G4 stabilizing small molecule compounds slows down the replication and stops the replication forks [24]. One of the G4-stabilizing ligands is heme a ubiquitous cellular cofactor, known to control gene expression by regulating the activity of heme-dependent transcription activators or repressors [25]. A large fraction of cellular heme is associated with hemoproteins and remains exchange inert. A labile heme pool, which is available for heme signaling, is far less abundant and buffered at a concentration of below 1 mol/L [26]. The labile fraction may increase after extracellular heme overload, enhanced heme synthesis, accelerated hemoprotein breakdown under oxidative conditions, or impaired heme degradation [25,27]. Free heme excess is known to enhance the generation of reactive oxygen species (ROS) and induces the oxidative stress that may cause damage primarily to lipid membranes, but also to proteins and nucleic acids [25]. Plenty of physicochemical studies showed that ferrous and ferric heme (Fe(II)-protoporphyrin IX and Fe(III)-protoporphyrin IX) binds tightly to various RNA and especially DNA G-quadruplexes [28,29,30,31,32]. Intramolecular parallel G4Cheme structures or mixed-type G4Cheme hybrids show significant oxidative activity (both one-electron and two-electron oxidation), with kinetics that is comparable to those of heme-utilizing protein enzymes, including peroxidases, peroxygenases, and monooxygenases [29,30,33]. One can suppose that the oxidative activity of G4Cheme complexes DNM2 may imply a potential mechanism for heme-mediated DNA oxidation. The availability of free heme depends on its cellular uptake, synthesis, and degradation. The latter process is directly regulated by heme oxygenases, namely, constitutively expressed and transcriptionally induced deficiency AKBA will enhance the accumulation of heme-stabilized G-quadruplexes. In order to fill the gap in our knowledge of the role of protein partners in the maintenance of G-quadruplexes in vivo, we propose a proximity ligation assay (PLA) as a useful method in such studies. Initially, this technique was designed to detect the localization of specific proteins in cells/tissues and to observe the dynamics of interactions between proteins of interest [35,36]. Using a G4-specific antibody, we adapted PLA for the in-cell visualization of interactions between G4 structures and different proteins. To the best of our knowledge, AKBA we showed for the first time in human cells that BRIP1 protein is located in the vicinity of G-quadruplexes. We also detected HMOX1 as the.