The aim of our study was to investigate the tudor domain-containing protein 7 (TDRD7) subcellular localization, which could be linked to diverse functions of this protein within the cell. Methods. In this study we employed cell imaging technique for detecting TDRD7 subcellular localization, Western blot analysis of HEK293 cell fractions with anti-TDRD7 monoclonal antibodies and bioinformatical search of possible TDRD7 isoforms in Uniprot, Ensemble, UCSC databases. Results. We have observed specific TDRD7-containing structures in cytoplasm as well as in the nucleus in HEK293 cells. The Western blot analysis of subcellular fractions (cytoplasm, mitochondria, nucleus) allowed us to detect three lower immunoreactive bands, with the aproximate molecular weight of 130, 110 and 60 kDa (we termed them as TDRD7b, TDRD7g and TDRD7d) and specific subcellular localization. The bioinformatical analysis of TDRD7 primary structure allowed us to determine two alternative transcripts from TDRD7 gene coding for proteins with calculated molecular weight of 130 and 60 kDa. Conclusion. The presented data demonstrate the existence at protein level of potential TDRD7 isoforms: TDRD7b, TDRD7g and TDRD7d. The expression profile of these splice variants and their role in cells remains to be elucidated.

To generate and characterize MCF-7 cell lines with altered expression of p85, p70 and p60 S6K1 isoforms: p85-/p70-/p60-MCF-7 and p85-/p70-/p60+MCF-7. Methods. CRISPR/ Cas9 gene editing, western blot analysis, immunofluorescence analysis, scratch assay. Results. Modified MCF-7 cells with knocked down expression of p85, p70, p60 or only p85 and p70 S6K1 isoforms were generated. Selective inhibition of only p85 and p70 isoforms in p85-/p70-/p60+MCF-7 cells was accompanied by actin cytoskeleton rearrangements, appearance of fibroblast-like cell morphology and significantly increased cell locomotor activity. Downregulation of all three S6K1 isoforms in p85-/p70-/p60 -MCF-7 cells inhibited cell migration with no changes in the cell morphology. Alterations in had a different impact on the ribosomal protein S6 phosphorylation and Akt signaling. Conclusion. Analysis of the modified MCF-7 cell lines revealed different impact of expression of S6K1 isoforms on MCF7 cell locomotor activity and the S6K1-and Akt-dependent signaling. Our data suggest that p60-S6K1 could be involved in regulation of the cell migration. The generated cells can be used for further analysis of functional activity of the S6K1 isoforms. K e y w o r d s: mTOR/S6K1 signaling, MCF-7, S6K1, CRISPR/Cas9, breast cancer К л ю ч е в ы е с л о в а: mTOR/S6K1 сигналинг, MCF-7, S6K1, CRISPR/Cas9, рак молочной железы.

TDRD7 is a scaffold protein whose specific function is unknown. It has been identified in complexes with proteins that regulate cytoskeleton dynamics and centrosomal movements, mRNA transport, and protein translation apparatus. Here we report the generation and characterization of monoclonal antibodies against TDRD7 protein. Bacterially expressed His-tagged fragments of TDRD7 were used as antigens. Spleen cells from immunized mice were collected and fused with SP2/0 myeloma cells using PEG 2000. High titer anti-TDRD7 antibody-producing hybridoma cell lines were identified by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution. Antibodies produced by E6 clone were further tested for their reactivity with the TDRD7 recombinant proteins. The results obtained clearly indicate that E6 anti-TDRD7 antibodies recognize specifically recombinant 6His-tagged TDRD7 proteins and endogenous TDRD7 in Western blotting, immunoprecipitation, and immunocytochemistry. In summary, these antibodies will be useful for researchers investigating TDRD7 and molecular complexes involving this protein.

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Ribosomal protein S6 kinase 2 (S6K2) is a serine/threonine kinase that belongs to the family of AGC kinases, which includes PKB/Akt, PKC, PDK1, and SGK1. Mammalian cells express two isoforms of S6K, termed S6K1 and S6K2. Each of these has nuclear and cytoplasmic spicing variants, which originate from different initiation start codons. Nuclear isoforms of S6K1 and S6K2 are slightly longer, as they possess additional sequences at the N-terminus with nuclear localization signals. Biochemical and genetic studies implicated S6Ks in the regulation of cell size, growth, and energy metabolism. Deregulation of S6K signaling has been linked to various human pathologies, making them excellent targets for drug discovery. The aim of this study was to produce monoclonal antibodies directed at the N-terminal regulatory region of S6K2, which shows very low homology to S6K1 or other members of the AGC family. To achieve this goal, two S6K2 fragments covering 1-64aa and 14-64aa Nterminal sequences were expressed in bacteria as GST/6His fusion proteins. Affinity purified recombinant proteins were used as antigens for immunization, hybridoma screening, and analysis of generated clones. We produced a panel of S6K2-specific antibodies, which recognized recombinant S6K2 proteins in ELISA and Western blot analysis. Further analysis of selected clones revealed that three clones, termed B1, B2, and B4, specifically recognized not only recombinant, but also endogenous S6K2 in Western blot analysis of HEK293 cell lysates. Specificity of B2 clone has been confirmed in additional commonly used immunoassays, including immunoprecipitation and immunocytochemistry. These properties make B2 MAb particularly valuable for elucidating signal transduction pathways involving S6K2 signaling under physiological conditions and in human pathologies.