Fig. 3

Effects of MI130110 on cell division. a Anti-proliferative assay showing the in vitro potency of MI130110. The assay was performed as described in the “Materials and methods” section with CD13-positive U-937 (solid circles), NB-4 (solid squares) and HT1080 (solid triangles) cell lines and CD13-negative RPMI 8226 (hollow circles) and Raji (hollow squares) cell lines. b HT1080 and EA.hy926 were cultured in the presence of different concentration of MI130110 (0, 1, and 10 μg/mL) for 24 and 48 h. Then, 2E06 cells were harvested, fixed in ethanol and their nuclei stained with PI, and analyzed by cytofluorimetry. The percentage values shown correspond to the percentage of cells in G2 phase. c HT1080 cells were incubated with either anti-CD13 TEA1/8 mAb (5 μg/mL) or MI130110 (5 μg/mL) for 24 h at 37 °C and processed as described in the “Materials and methods” section. The expression of β tubulin, DAPI-staining of DNA, and a merged composition of representative fields of cells treated with anti-CD13 mAb and ADC is shown. Scale bars are shown. d Quantification of cells in interphase and mitosis after 24 h treatment with anti-CD13 TEA1/8 mAb (5 μg/mL) or MI130110 (5 μg/mL). Cells were processed as described in c. Cells in interphase or undergoing mitosis from a total of 7 representative fields (24 x optical magnification) for each condition were counted. A total of 539 (93.5%) cells were in interphase and 20 (6.5%) in mitosis in the treatment with anti-CD13 TEA1/8 mAb. A total of 215 (54%) cells were in interphase and 185 (45%) in mitosis in the treatment with MI130110. e MI130110 treatment causes mitotic catastrophe. Cells were treated and processed for immunofluorescence as described in the “Materials and methods” section. Figure shows representative cells undergoing mitosis (128 x optical magnification) treated either with MI130110 or with naked anti-CD13 TEA1/8 mAb. Staining of CD13 (red), α-tubulin and acetylated α-tubulin (purple), β-tubulin (green) and chromosomes (blue), and a fluorescence merged image (merge) is shown. Scale bars are shown