Fig 2: GJIC documented by the SL-DT technique in different cell lines. (A) Principle of the SL-DT assay. Fluorescent gap junction permeable dye (e.g., Lucifer Yellow) is loaded into the cell monolayer by a clean cut with a sharp scalpel blade (I–II). Subsequent dye transfer through the cell monolayer depends on the number of gap junctions and their functional state, and it is proportional to the levels of GJIC (IIIA-B). (B) Illustrative microphotographs of different permanent cell lines where the levels of GJIC were evaluated by the SL-DT technique, either in nontreated cells or following treatment with a model GJIC inhibitor, such as 12-O-tetra-decanoylphorbol-13-acetate (TPA) or 1-methylanthracene (1-MeA). Beas-2B, human bronchial epithelial cell line (ATCC CRL-9609); Caco-2, human epithelial colorectal adenocarcinoma cells (ATCC HTB-37™); C10, murine pulmonary epithelial cell line; HaCaT, an aneuploid immortal keratinocyte cell line from adult human skin (RRID:CVCL_0038); HBE1, immortalized human bronchial epithelial cell line (RRID:CVCL_0287; Kerafast #ENC002); HL1-1, adult human liver stem cells; RGC, rat glial cells; TM3, murine immortalized immature Leydig cell line (ATCC CRL-1714TM); TM4, murine immortalized immature Sertoli cell line (ATCC CRL-1715TM); WB-F344, normal rat liver epithelial cell line (RRID:CVCL_9806; JCRB0193). Prepared according to information from [27].

Fig 3: Scheme of method of cancer cells transformation into other types of cells. Treatment of different cancer cell lines with complex of the DDMC vector with mix of separated sncRNAs with or without cell differentiation factor due to transformation of cancer cells in different types of cells. Cells and cell lines: 1. A-431 (ATCC® CRL-1555™) is human epidermoid carcinoma; 2. A-172 (ATCC® CRL-1620™) is human glioblastoma cell line; 3. IMR-32 (ATCC® CCL-127™) is human neuroblastoma cell line; 4. CaCo2 (ATCC HTB-37 TM) is human colorectal adenocarcinoma cell line; 5. Stem-cell-like cell is intermediate form of cells, which was obtained before full cancer cell transformation; 6. CD117 positive cells [15]; 7. Epithelization-like process (not published); 8. Dendritic-like cell (not published); 9. CD4 positive cells [14], [15]; 10. Platelet-like cells [13]; 11. Paneth-like cells (not published); 12. Neuron-like cells (not published). (Author made all microscopic photos. Magnification of fluorescent and light microscopy was ×600).

Fig 4: Caco-2-NF-κB-hrGFP C3 and HT-29-NF-κB-hrGFP E5 and F6 clones showed different NF-κB activation (a) and IL-8 production (b) in response to LAB. Caco-2-NF-κB-hrGFP C3 cells were seeded at 1 × 105 cell/well, while HT-29-NF-κB-hrGFP E5 and F6 clones were seeded at 5 × 104 cell/well, cultured ON, and treated for 2 h with L. reuteri ATCC 23272 or L. plantarum ATCC 8014. Then, gentamycin and TNF-α (1 ng/mL) were added; 48 h or 18–24 h later (for Caco-2-NF-κB-hrGFP C3 and HT-29-NF-κB-hrGFP E5 and F6, resp.), cells were trypsinized and NF-κB activation (measured by the % GFP+ cells) was analyzed by flow cytometry. Cell viability was over 90% for all tested conditions. Data was normalized against TNF-α control (considered as 100%). IL-8 quantification was performed in the harvested cell culture supernatant by flow cytometry. Results were expressed as the mean ± SD of triplicates of a representative experiment. * P < 0.05 using One-Way ANOVA with Dunnett's posttest.

Fig 5: Serial passage of Modified vaccinia virus Ankara (MVA) (ATCC VR-1508) in human cell line. Caco-2 (ATCC HTB-37) cells were infected with purified MVA at a multiplicity of infection (m.o.i.) of 0.01 and the virus was blindly passaged in Caco-2 40 times. At each passage (three days post infection) infected cells were harvested, freeze-thawed three times and the virus titer was determined [52].