Sequence homology of Suppressor of IKKepsilon and Wound Inducible Transcript 3.0 confound co‐localization studies

Abstract

Wounds created in the oral cavity heal at a rapid rate and leave minimal to no scarring. During the healing process of these wounds, expression of a gene, wound inducible transcript 3.0 (WIT3), was activated. The mechanism of how WIT3 affects the healing process is not known, but may involve an interaction between WIT3 and cytoskeleton structures. WIT3 shares 51% sequence identity with Suppressor of IKKe (SIKE). The function of SIKE is also unknown, but this protein was first identified as part of the innate immune response to viral infection. In this response, SIKE was shown to be phosphorylated following a viral infection and form an association with cytoskeletal proteins. As WIT3 shares a high degree of sequence identity and potential interactions with the cytoskeleton, the primary goal of this work was to determine if WIT3 is associated with the same cytoskeletal proteins as SIKE and/or even directly with SIKE. To assess localization and colocalization, immunofluorescence assays in four cell lines, epithelial ovarian cancer (DOV13), chronic myelogenous leukemia (HAP1), HAP1 cells with CRISPR/Cas9 knockouts of SIKE (SIKE‐CR), or WIT3 (WIT3‐CR), were completed using commercial SIKE or WIT3 specific antibodies. For SIKE in DOV13 cells, fluorescence was observed in actin‐like structures at the leading edge of the cells, in cytosolic puncta, and at the plasma membrane, similar to previously published data. The HAP1 cells formed cell clusters without apparent leading cell edges. Without lamellipodia, no staining of actin‐like or stress fiber‐like structures were observed, but staining at the plasma membrane and cytosolic puncta were identified in HAP1 and WIT3‐CR cell lines. For WIT3, two antibodies were tested, A and B. Antibody A showed that WIT3 was in the cytoplasm, whereas Antibody B showed that WIT3 was in mitochondrial‐like networks surrounding the nucleus and extending throughout the cytoplasm. The differences between the WIT3 antibodies in the same cell lines suggested that one or other of the antibodies was not specific to WIT3. As a control, staining for SIKE or WIT3 in their respective knockout cell lines was completed. Surprisingly, these negative controls yielded positive results. Knockout status of the cell line was confirmed via immunoblot. Cross reactivity was tested via immunoblot with purified SIKE and WIT3 recombinant protein. Together, these data were consistent with cross reactivity of the antibodies for SIKE and WIT3. To overcome this issue, stable cell lines introducing fluorescently tagged SIKE or WIT3 into their respective knockout cell lines were created. N‐terminal mTurquoise or C‐terminal sYFP2 tagged SIKE or WIT3 was transfected into their respective knockout cells and stable transfectants selected via geneticin. Colocalization using IFA and FRET (e.g mTurquoise‐SIKE to WIT3‐sYFP2) should resolve the issue of cross reactivity between SIKE and WIT3 antibodies observed in standard IFA assays. Our studies highlight the potential limitations of tools developed for highly homologous proteins and alternative approaches.