The kinetics of changes in CD20 expression on Daudi cells following exposure to 0.5 Gy γ-radiation.

<p>(A and B) Total CD20 levels: The changes in CD20 expression were measured using anti-CD20 antibodies. Results are expressed as ratio of levels of CD20 (CD20/β-Actin) with respect to 0 hr control. (C) CD20 expression on cell surface: The levels of CD20 at cell surface levels were calculated using quantiBRITE beads and expressed as numbers of CD20 molecules/cell. The index histogram is showing levels of CD20 at 20 hr (0.5Gy) with respect to 0 hr. Statistical analysis was done using Student's t-test (***<i>p</i><0.001).</p

The changes in ROS and MMP (ΔΨm).

<p>(A, B and C) To measure changes ROS levels, fluorescence of CM-DCF was acquired at Ex λ of 488±10 nm and Em λ 517–527 nm and the results are expressed as the mean fluorescence ±SD of three independent experiments and statistical analysis was performed using ONE way ANOVA. Significant values are represented as; ***<i>p</i><0.001 for Rtx <i>vs</i> 0.5Gy+Rtx or 1.5Gy+Rtx, ***<i>p</i><0.001 for Tst <i>vs</i> 0.5Gy+Tst or 1.5Gy+Tst, ##<i>p</i><0.01 for sham irradiated control <i>vs</i> 0.5Gy, ##<i>p</i><0.001 for sham irradiated control <i>vs</i> 1.5Gy. Corresponding Isotype controls antibodies were used to measure changes in ROS levels. (D and E) The changes in ΔΨm were expressed from the mean fluorescence ±SD of three independent experiments and statistical analysis was performed using ONE way ANOVA. Significant values represents as; #p<0.05 for control <i>vs</i> 1.5Gy, *p<0.05 for control <i>vs</i> Rtx or 0.5Gy+RTX or 0.5Gy+Tst or 1.5Gy+Tst, **p<0.01 for control <i>vs</i> 1.5Gy+Rtx. Corresponding Isotypic controls were used to measure changes in ΔΨm levels [Human IgG1 (for Rtx) and Mouse IGG2a (Tst)].</p

Anti-CD20 mAbs-induced programed cell death.

<p>Schematic diagram illustrating the kinetics of changes in CD20 expression followed 0.5Gy γ-radiation and sequence of events in the proposed possible cell death pathway evoked by type I anti-CD20 mAbs (Rtx) and type I anti-CD20 mAbs (Tst). Anti-CD20 mAbs ligation results in cross-linking and homotypic adhesions followed by generation of ROS and create genotoxic stress which ultimately culminates in apoptotic and non-apoptotic cell death. *SB =  Strand Break.</p

Cross-linking or homotypic adhesions (aggregations) and cell death.

<p>(A–B) The binding of Fab regions of Anti-CD20 mAbs (Rtx and Tst) on CD20 and Fc region binding to FcγRIIB1 thereby induction of cross-linking or homotypic adhesions (aggregations) as well as extra cross-linking induced by corresponding secondary antibodies were observed microscopically (20x). Isotype control antibodies were taken separately to measure non-specific cross-linking. (C, D and E) Anti-CD20 mAbs induced cell death was measured using PI uptake by flowcytometrically. PI is membrane impermeable, generally excluded from viable cells and therefore, commonly used for identifying alterations in biological membrane and thereby death of cells in a population. The fold induction cell death was measured from mean fluorescence ±SD and statistical analysis was performed using ONE way ANOVA. Significant values represented as; ***<i>p</i><0.001 for Rtx <i>vs</i> 0.5Gy+Rtx and 1.5Gy+Rtx. ***<i>p</i><0.001 for Tst <i>vs</i> 1.5Gy+Tst, **<i>p</i><0.01 Tst <i>vs</i> 0.5Gy+Tst. Further, ###<i>p</i><0.001 for sham irradiated control <i>vs</i> 1.5Gy, and $$$<i>p</i><0.001 for sham irradiated control <i>vs</i> Tst. Isotype control antibodies were taken separately to measure non-specific induction of cell death. (F) Induction of cell death by extra cross-linking using secondary antibodies: Cells expressing different levels of CD20 and treated with monoclonal anti-CD20 antibodies or isotypes (separately) were further incubated with corresponding secondary antibodies. PI uptake was also measured flowcytometrically to measure extra crosslinking induced cell death. (G) Cell cycle analysis: For cell cycle analysis, binding of PI with DNA were used as a marker of DNA content which depicts phase of cells in cell cycle.</p