Figure 1

αCD22 Ab-MXD3 ASO conjugate demonstrates MXD3 knockdown leading to cell apoptosis in preB ALL cells. (A) Reh cells treated with αCD22 Ab-MXD3 or αCD22 Ab-control ASO conjugate, or left untreated, were measured for MXD3 protein expression by fluorescent immunocytochemistry 4 h after treatment. The concentration of the conjugate in each well was 0.5 umol/L. Images were acquired at 40× magnification/1.4 numerical aperture at room temperature using a Nikon Ti-U inverted microscope and NIS-Elements BR software. Scale bar indicates 50 µm. The overlay pictures show a composite image of both MXD3 protein (with Alexa488) and nuclei (with DAPI). The images shown are from one representative experiment out of three experiments. (B) Mean fluorescence intensity (MFI) was used to quantify MXD3 protein expression. Each bar represents the average MFI of all measured cells per treatment type from three independent experiments. MXD3 αCD22 Ab conjugate versus untreated (**p = 0.008) versus αCD22 Ab-control ASO conjugate (*p = 0.032). (C) Cell apoptosis measured by annexin V using flow cytometry. Dot plots shown are from one representative experiment out of three experiments. (D) Quantification of annexin V positive cells. Data are means from three independent experiments. Error bars represent SEM (n = 3 for each time point). The cells treated with αCD22 Ab-MXD3 ASO conjugate showed significantly more annexin V positive cells than either the untreated cells (***p < 0.001) or those treated with the αCD22 Ab-control ASO conjugate (***p < 0.001), at both 2 and 4 h. (E) Cell apoptosis measured by caspase 3 and 7. Cells were treated the same way as in (D) with the same control. Histograms represent caspase measured by luminescence signal at 2 and 4 h after treatment. Data are an average of three independent experiments in triplicate. Error bars represent SEM (n = 9 for each time point). Cells treated with the αCD22 Ab-MXD3 ASO conjugate showed significantly higher average caspase than either the untreated cells (***p < 0.001) or those treated with the αCD22 Ab-control ASO conjugate (***p < 0.001), at both 2 and 4 h.