Aza/pano disrupts ALL cell adhesion by downregulating CD81 surface expression via a decreased rate of exocytosis. (A) Bar graph showing the percentage of bound untreated Nalm6 cells and aza/pano-treated cells. Error bars denote standard deviation from the Mean of values generated from 3 independent experiments. (B) Flow cytometry plot overlays showing the surface expression of CD81 in untreated Nalm6 cells or cells treated with aza (500 nM)/pano (1.5 nM) for 48 hours. Isotype control antibody plots are included for reference. Representative plots from 3 independent experiments are shown. (C) CD81KO Nalm6 cells were pretreated with aza/pano for 48 hours before transfer onto Saos-2 monolayers and treatment with Ara-C. Viability of ALL cells was determined using flow cytometry. Error bars denote standard deviation from the mean. NS stands for nonsignificant P > .05. (D) Flow cytometry plot overlays showing the total expression of CD81 in untreated or aza/pano-treated Nalm6 cells. Isotype control antibody plots are included for reference. Representative plots from 3 independent experiments are shown. (E) Nalm6 cells treated with aza/pano for 24 hours were blocked with unconjugated CD81 antibody. Newly appearing CD81 protein at the cell surface was captured at indicated time intervals using an APC-conjugated CD81 antibody by flow cytometry. The rate of CD81 exocytosis was calculated based on the best fit line (control rate: 106.3 MFI/min; aza/pano rate: 60.23 MFI/min). Error bars denote standard deviation from the mean of 2 independent experiments. (F) Nalm6 cells treated with aza/pano for 24 hours were incubated with unconjugated CD81 antibody. Cy5-tagged anti-mouse secondary antibody staining was performed to determine the percentage of CD81 protein internalized at a given time point. Time to 50% internalization was calculated based on the nonlinear regression curve (control, 46.1 minutes; aza/pano, 44.4 minutes). Error bars denote standard deviation from the mean calculated from 2 independent experiments.