Fig 2: Compound 22 exhibits some cytotoxic, but no genotoxic activities in TK6 cells. (A) – Quantification of %MNi induction in TK6 cells treated with three doses of compound 22 (0.5, 2 and 4 µM) compared to the negative (1% DMSO; vehicle) and positive (carbendazim; Crbz, 8.4 µM) controls, after 1.5–2 cell-cycles (24 h). MNi were scored from the mononucleated images collected on the ImageStream Mark II using the DeepFlow neural network algorithm as described in Wills et al. [77]. Graph shows the mean value of %MNi ± SD (n = 3, except for Crbz and the highest concentration tested of compound 22; n = 2). Relative Cell Growth (RCG, as %) is also displayed. (B) - ?H2AX observed in TK6 cells co-cultivated with either 0.5, 2 or 4 µM compound 22 compared to the positive (45.4 µM MMS and 8.4 µM Crbz) and negative (1% DMSO; vehicle) controls. The dotted line represents the fold change cut off value of 1.5. (C) - p53 measured in the same six TK6 cell/compound co-cultures described in (B). The dotted line represents the fold change cut off value (1.5). For (B) and (C), each experimental point is shown (n = 3, except for Crbz and the highest concentration tested of compound 22; n = 2). Error bars represent SD. For (A), (B) and (C) any significant difference compared to the DMSO control was quantified by a one tailed Dunnett's test (*, ** and **** representing p < 0.0332, p < 0.0021 and p < 0.0001, respectively). (D) Cell-cycle analyses conducted with compound 22. The percentage of TK6 cells in each cell-cycle phase (G1, S and G2/M) is shown for compound 22 treated cells as well as the positive and negative controls (n = 3). For each phase, any significant difference compared to the DMSO control was indicated as *, ***, **** representing p < 0.0332, p < 0.0002, p < 0.0001, respectively (by two tailed Dunnett's test).